Why is angiotensin converting enzyme localized in the lungs

Why is angiotensin converting enzyme localized in the lungs

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I understand that it's also found elsewhere, such as in renal capillaries, but I can't see logic behind it being located in the lungs. Isn't ACE's function, through making more angiotensin II, causing increased fluid and sodium retention and increasing peripheral resistance? What benefit does that purpose get from ACE being mainly in the lungs?

ACE is present on the luminal surface of vascular endothelia throughout the body and is abundantly present in the endothelium-rich lungs.

ACE in the kidney-particularly in the endothelial cells of the afferent and efferent arterioles- can produce enough ANG II to exert local vascular effects.

Thus, the kidney receives ANG II from two sources:

✓ Systemic ANG II comes from the general circulation and originates largely from the pulmonary region, and

✓ Local ANG II forms from the renal conversion of systemic ANG I.

• In addition, the proximal tubule secretes ANG II into its lumen and thus achieves intraluminal concentrations in excess of those in the general circulation. ANG II in the circulation has a short half-life (~2 min).

Tissue ACE phenotyping in lung cancer

Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Affiliations Department of Medicine, Division of Pulmonary, Critical Care, Sleep and Allergy, University of Illinois at Chicago, IL, United States of America, Department of Medicine, University of Arizona Health Sciences, Tucson, AZ, United States of America, Medical Center, Moscow University, Moscow, Russia

Roles Data curation, Investigation, Methodology, Project administration, Visualization

Affiliation Department of Pediatric and Adolescent Surgery, Paracelsus Medical University, Salzburg, Austria

Roles Data curation, Formal analysis, Investigation, Methodology, Visualization

Affiliation Institute of Pathology, Paracelsus Medical University, University Hospital Salzburg, Salzburg, Austria

Roles Data curation, Formal analysis, Investigation, Methodology, Visualization

Affiliation Institute of Pathology, Paracelsus Medical University, University Hospital Salzburg, Salzburg, Austria

Roles Formal analysis, Methodology, Resources, Writing – review & editing

Affiliation Department of Medicine, Columbia University, New York, NY, United States of America

Roles Funding acquisition, Resources, Writing – review & editing

Affiliation Department of Medicine, University of Arizona Health Sciences, Tucson, AZ, United States of America

Morphological Analysis Of SARS-CoV-2

We will be taking a look in this article at the structure of the corona virus responsible for the current pandemic and how its structure causes its clinical manifestation. Coronaviruses are a large family of common viruses which are found in humans and animals many cases of the common cold are due to a corona virus.

They have caused two large-scale outbreaks in the past two decades the SARS virus in 2002 and the MERS virus in 2012. It's generally been considered that these corona viruses could cause future disease outbreaks. Because they're known to be able to evolve within animals and then jump to humans fire an intermediate host in SARS palm civets and raccoon dogs were identified as the intermediate covid-19 is an example of this which is believed to have jumped from bats to pangolins to humans in a local seafood market in Wuhan China during 2019.

Covid-19 refers to the corona virus infectious disease found in 2019 the actual disease itself is referred to as covid-19. But the virus is called the SARS cough - which stands for severe acute respiratory syndrome coronavirus - and was named because its structure very closely resembles that of the SARS virus from 2002. This is the seventh known corona virus to infect humans two of which were similarly highly pathogenic MERS and SARS. The other four are of low pathogenicity and endemic in humans.

Let's now take a look at the structure of the SARS cough - virus so looking at this virus we can see that it has a series of protein spikes on its surface which when viewed under a microscope appear like a crown which gives rise to the name corona. Which is Latin for Crown and is therefore common to all the corona viruses.

There are four structural proteins which is similar to other corona viruses the S the E the M and the N proteins.

  1. The S stands for spike.
  2. The E stands for envelope.
  3. The M stands for membrane and
  4. The N stands for nucleo capsid.

So let's take a look at these different structural proteins in turn beginning with this crown-like structure which is the S or spike protein this protein is responsible for allowing the virus to attach to the membrane of the host cell. It contains a receptor binding domain which recognizes a specific receptor the angiotensin converting enzyme receptor 2 which is expressed in their lungs heart kidneys and intestines it has been shown that this protein binds to the ACE 2 receptor with at least the same affinity and potentially as much as 20 times greater affinity than the SARS virus this could be one of the explanations for the reasons, why it's spreading so easily the spike protein itself has two functional subunits s1 binds to the host cell receptor and s2 mediates the fusion of the viral and cellular membranes, because of the critical role that protein plays in binding to target cells and cellular entry. It is a particular focus in the design of vaccinations and medical treatments for covid-19.

Let's take a look at the next protein the M or membrane protein. The membrane protein is the most abundant on the viral surface and defines the shape of the viral envelope. It can be thought of as the central organizer for corona virus assembly and interacts with the other structural proteins.

Moving on to the E or envelope protein. This is the smallest of the major structural proteins on the viable membrane. Which appears to have several roles we know that it is integral in the assembly and release of the virus from host cells and during viral replication it is largely localized at the site of intracellular trafficking more specifically at the endoplasmic reticulum and the Golgi apparatus so essentially the M and E proteins play a critical role in turning the host cell apparatus into workshops where the virus and our own cells work together to make new viral particles underneath the surface proteins.We have the viral envelope. This is the viruses out that is derived from the hosts cell membrane so ourselves or the animals it's a fatty layer and worth noting that in contact with soap it will break down killing the virus and this is the reason why hand-washing with soap is so important to prevent the spread of this virus underneath this layer is what's called the capsid this is a protein shell that encloses the genetic material of the virus inside this capsid.

We have the nuclear capsid or N protein. This protein is bound to the virus's single strand of RNA which is where all its Dometic information is held to allow itself to replicate the N protein appears to be multifunctional in particular it essentially inhibits a lot of the host cells defense mechanisms and assists the viral RNA in replicating itself and therefore in creating new viral particles.

So we've looked now at some of the important structural features of the SARS coronavirus to a lot of our understanding of the pathogenesis of covid-19 comes from work on the original SARS virus. The costly viral structures in morphology are so similar there is likely to be significant crossover in the biochemical interactions and pathogenesis.

Let's now look a little more at how the virus infects humans. So the virus is spread mainly by respiratory droplets ie a cough or sneeze which aerosol is the virus allowing it to travel into our nasal or all cavities. We also know that it can live on surfaces for hours and even up to a few days on some surfaces so if you touch an infected surface it's very easy to then touch your own face and to knock you late the mucous membranes in your eyes mouth or nose with the virus initially it can get into the upper airway, so the nasal or throat area and this is why you can get those symptoms like a common cold, stuffy nose, headache, sore throat and fever. It is within the mucosal epithelium of the upper GI tract where primary viral replication is thought to occur similar to SARS. SARS coronavirus to is able to get further into our respiratory system and into our lung epithelial cells we're further viable that location occurs.

Let's talk a little bit more about the ACE to receptor interaction the SARS coronavirus 2 binds violet spike or S protein to the ACE 2 receptor, this mechanism of binding is the same way that the SARS virus was able to bind to airway epithelial cells. The host cell has proteases which are enzymes that break down proteins and these cleave the spike protein we think that this process activates the protein in order to trigger the process of membrane fusion, before injecting the viral genome into the host cell a similar mechanism of protein cleaving facilitates.

Cell entry in influenza as well as this mechanism of direct cellular entry the virus may also enter the cell via endocytosis. This is the process by which material enters a cell after being surrounded by an area of the cell membrane which then buds off inside the cell to form a vesicle once inside the cell fire a specific RNA and proteins are synthesized within the cytoplasm further viral proteins are then assembled with the blueprint of information contained within the viral RNA using the hosts cellular machinery specifically the endoplasmic reticulum and Golgi apparatus with specific processes to form the envelope glycoproteins new variants are then assembled by fusing to the plasma membranes and released as vesicles via the cellular EXO CITIC secretory processes. So the stress is placed on our own cells by viral infection and the interaction of our own immune system with the viral antigens presented by the infected host cells lead to cell death during this process of cell death. Multiple inflammatory mediators are released which creates an inflammatory response leading to a buildup of mucus and thickening and hyperplasia of the cells within our Airways this inflammation causes irritation of the cells lining our Airways which leads to the cough.

Let's move further down into the lower respiratory tract now and see how the virus acts within the lungs. So let's take a look at the path that the virus might take, so looking at the track here are the windpipe this branch is into left and right main bronchi. These bronchi branch into lobar bronchi we have three on the right and two on the left and these then branch into segmental bronchi. The segmental bronchi branch into bronchial which terminate as respiratory bronchioles at the end of which are the alveoli the alveoli are the tiny air filled pockets responsible for gas exchange. We have around 600 million of these alveoli and they are responsible for exchanging oxygen and carbon dioxide between the blood and the air we breathe in due to the direct action of the virus and also due to our own immune systems response to viral infection the alveolar walls can become inflamed and thickened and fill the alveolus with fluid, which can impair their ability to exchange gases and this can lead to the symptom of shortness of breath in some people this process of cellular infection by the SARS cough - virus can lead to an exaggerated immune response with a huge release of pro-inflammatory mediators causing what is known as a cytokine storm or cytokine release syndrome.

Cytokines are small proteins involved in cell signaling and are crucial in mediating immune responses this cascade of inflammatory mediators causes an uncontrolled systemic inflammatory response, which leads to acute respiratory distress syndrome or a RDS. This is the rapid and widespread inflammation in the lungs which causes the epithelial and endothelial cells of the lungs to secrete inflammatory mediators which fill the alveoli in addition these inflammatory signaling cells recruit other cells of the immune system into the alveoli, which further contributes to and amplifies the problem further the systemic inflammatory state causes increased capillary permeability which results in even more fluid entering the alveoli. So essentially this is non cardiogenic pulmonary edema compounding the problem over all this pathological process severely impairs the ability of the lungs to exchange oxygen and carbon dioxide as it's now become filled with fluid and inflammatory infiltrate in cases of severe a RDS invasive mechanical ventilation is required to adequately oxygenate the body.

So that's what underpins the pathology at the extreme end of the spectrum in the large majority of cases of covid-19 infection the disease follows a mild course as the viruses eliminated via normal immune processes. on CT as seen on these axial and coronal slices typical findings are lower lobe predominant bilateral subfloor ground glass density opacities while these findings are frequently seen in covid positive patients. These are not specific the differential for these appearances includes other viral pneumonias interstitial lung diseases such as cryptogenic organizing pneumonia and atypical bacterial pneumonias so that completes this article with some covid-19 pictures. ❤️✌️.

Physiological Reviews Summary

(1) The airway surface liquid (ASL) plays a pivotal role in lung defense. Diabetes is related with higher ASL glucose concentration, ASL volume accumulation in alveolar space, imbalance of reactive oxidative species (ROS), and inflammatory chemokine production.

(2) The COVID-19 infection promotes injuries in type I and type II pneumocytes and lung endothelial lesions, with subsequent additional secretion of protein-rich exudate in the alveolar space and intravascular coagulation in lung vessel, which leads to a reduction in surfactant and gas exchange.

(3) The association between diabetes and SARS-CoV-2 increases the glucose and protein concentration in ASL, leading to increase the risk of pneumonia.

(4) The prevalence and severity of hypoxemia and severe hyperinflammation is higher in COVID-19 diabetic patients.

(5) The harmful clinical outcomes and mortality rate of COVID-19 are higher in diabetic subjects.


To understand the age-related pattern of ACE2 abundance in the human lung we utilized the large, harmonized RNAseq dataset available from the GTEx consortium [30]. GTEx version 8 contains data from 578 unique deceased donor lungs, binned by decade of age at death [33]. In unadjusted analysis, ACE2 expression was not significantly associated with age (β = 0.082 TPM/decade, p = 0.227, linear regression) or sex (p = 0.303, Wilcoxon rank-sum test) (Fig 1A and 1B). The dataset also describes the mode of death, summarized by the Hardy scale [37], which categorizes clinical circumstances proximate to the end of life. The strongest category associated with ACE2 expression was ventilation at the time of death showing a 103% increase in median ACE2 levels (0.715 TPM without vs. 1.452 TPM with ventilation, p < 1x10 -15 , Wilcoxon rank-sum test). Lung ACE2 expression, when stratified by Hardy score, revealed a significant relationship with age expression of ACE2 increased by 0.208 TPM for every decade of life (p = 0.003, multivariable regression), or a 14% increase each decade when compared to individuals in their 20’s (Fig 1C). Transcript abundance from deceased donors can be affected by the tissue ischemic time that transpires prior to sample stabilization [38]. In order to assess whether this variable contributes to ACE2 abundance from deceased donor lungs, we incorporated this feature as a covariate in our model (S3 Fig in S1 File). The contribution of tissue ischemic time to ACE2 expression was not significant (β = -0.0003 TPM/minute, p = 0.248) and did not change the effect of age on ACE2 expression or its significance (β = 0.212 TPM/decade, p = 0.002). We also compared ACE2 expression across the human body. Interestingly, when controlling for the manner of death, the lung exhibited the most significant increase in ACE2 expression with age over all tissues (S1 Table in S1 File).

A) Human lung ACE2 expression is colored by decade of life when the individual died. In unadjusted linear regression, there was no significant effect of age on ACE2 expression (p = 0.227, n = 578 individuals). B) ACE2 expression in lung stratified by sex. Analysis adjusted for age and sex, revealed no significant effect for either variable (p = 0.228 for age, p = 0.920 for sex, multivariate regression, n = 395 males, n = 183 females). There was also no significant difference when compared by sex alone (p = 0.303, Wilcoxon rank-sum test). C) ACE2 expression in lung stratified by the Hardy scale. This scale indicates the length of time spent in the terminal phase before death, which is depicted above each grouping of data points. Within each Hardy scale group, the data are sub-stratified by age. (n = 26 for a score of 1 representing a violent and fast death lasting <10 minutes, n = 156 for a score of 2 representing a fast death by natural causes lasting 10 minutes– 1 hour, n = 31 for a score of 3 representing an intermediate rate of death lasting 1 hour– 24 hours, n = 64 for a score of 4 representing a slow death with a terminal phase lasting > 24 hours, n = 299 for a score of 0 representing donors supported by a ventilator preceding death, n = 2 with an unknown score). For all panels, each point represents a sample from a unique individual. Box plots indicate quartiles. A linear model fit to the data is inset, indicating the estimated coefficient for age (β1) and its significance.

In order to characterize ACE2 protein abundance in the lung, we validated a commercial antibody directed against this key viral receptor (S4A and S4B Fig in S1 File). Prior analysis of ACE2 localization by IHC revealed strong enrichment within the luminal surface of cortical tubule cells of the kidney and enterocytes of the small intestine [39]. Our ACE2 IHC staining on healthy control tissue revealed a strikingly similar pattern, which highlighted the kidney cortex and specifically the apical membrane of brush border cells (S4A Fig in S1 File). We also detected strong ACE2 expression along villi and the apical membranes of small intestine enterocytes (S4B Fig in S1 File). Within the lung, ACE2 has been shown to localize to epithelial cells of the alveolus [39]. Data from murine scRNAseq reveal detectable expression of Ace2 within type II pneumocytes (or alveolar type II / AT2 cells), ciliated columnar cells, alveolar macrophages, and to a lesser extent, stromal cells (S5 Fig in S1 File). Consistent with these data, our IHC revealed strong ACE2 staining within AT2 cells in normal alveoli, concentrated within the membrane, as well as alveolar macrophages (Fig 2). Taken together, these data indicate that our staining protocol accurately identifies ACE2 expressing cells within human tissue, including the lung. Therefore, we investigated whether the distribution of ACE2 in the lung is associated with ventilation and age.

In normal human lung from a 23-year-old female using a 1x objective (left, scale bar 3mm), strong ACE2 expression is observed within bronchioles and alveoli. The region outlined by the red dashed box is shown magnified at low power (middle, scale bar 200μm) which highlights prominent ACE2 expression in AT2 cells (red arrowheads) along the alveolar septum and in alveolar macrophages (green arrowhead). At high power (right, scale bar 50μm), ACE2 staining can be seen concentrated along the membrane of AT2 cells (red arrowheads) and within the cytoplasm of an alveolar macrophage (green arrowhead). Sections were stained for ACE2 using DAB and counterstained with hematoxylin.

We identified 12 lung samples from 11 patients requiring mechanical ventilation for AHRF available from our institution’s archive. All 11 patients had DAD (S2 Table in S1 File), a pathological finding consistently observed in patients with severe Covid-19 lower respiratory disease [10, 11]. All tissue was collected prior to 2019 (range 2010–2018), excluding the possibility of SARS-CoV-2 involvement. H&E control sections revealed the expected histologic findings, depending on the phase of DAD, with reactive AT2 hyperplasia, hyaline membrane formation, interstitial thickening and/or fibrosis, and exudative edema (S1B Fig in S1 File). When stained by IHC, prominent ACE2 expression within the alveolar parenchyma was observed in reactive AT2 cells with increasing ACE2 staining intensity detected with advanced age (Fig 3A and 3B). ACE2 expression was quantified either by normalizing to tissue area (Fig 3C) or cellularity (Fig 3D) which revealed a significant increase with age (p = 0.004 and p = 0.003, respectively, linear regression). As a control for precision, 1 of the 11 patients had a double lung explant with 2 samples collected contemporaneously (1 from the left lung and 1 from the right lung). In this 67 year-old-man, the pathological findings were the same for each sample (S2 Table in S1 File). Importantly, both specimens yielded similar results when quantified for ACE2, indicating that our procedure measured its expression with good intra-individual reproducibility (Fig 3C and 3D). Excluding either of these samples did not change the effect of age on ACE2 expression (p = 0.009 by area and p = 0.003 by cellularity excluding the right lung p = 0.008 by area and p = 0.006 by cellularity excluding the left lung, linear regression).

A) Representative images of lung stained for ACE2 from a 40-year-old man with acute lung injury are shown using a 1x objective (left, scale bar 3mm) with the region outlined by the red dashed box magnified at low power (middle, scale bar 200μm) and a second field at high power (right, scale bar 50μm). Clusters of reactive AT2 cells (red arrowheads) are present along the alveolar septum which exhibit low level ACE2 expression. B) Representative images of lung stained for ACE2 from a 67-year-old man with acute lung injury superimposed on fibrosing interstitial lung disease are shown using a 1x objective (left, scale bar 3mm) with the region outlined by the red dashed box magnified at low power (middle, scale bar 200μm) and a second field at high power (right, scale bar 50μm). Numerous reactive AT2 cells (red arrowheads) exhibiting nucleomegaly and abundant cytoplasm can be seen demonstrating strong ACE2 staining. C) Quantitative IHC for ACE2 was carried out on samples from ventilated patients (n = 12 samples from 11 patients). Total ACE2 expression from 5 low power fields is plotted relative to the patient’s age at the time of specimen collection. A linear fit to the data is indicated by the dashed line with the 95% confidence interval highlighted in grey. D) The same specimens quantitated in (C) were normalized by cellularity and the average ACE2 expression per cell is plotted along with a linear fit to the data and its 95% confidence interval. The red arrow in (C) and (D) indicates a patient providing 1 sample from the left lung and 1 sample from the right lung during the same procedure, utilized as a control for intra-individual reproducibility. The green and blue circle in (C) and (D) indicate the staining intensity of the samples depicted in Fig 5A and 5B, respectively. Sections were stained for ACE2 using DAB and counterstained with hematoxylin.

Alveolar macrophages comprised a minor fraction of total cells in samples from ventilated patients (median 3.4%, range 1.7% - 10.9%) and by visual inspection did not show the prominent age-related change in ACE2 expression seen in AT2 cells. To determine whether the abundance of alveolar macrophages contributes strongly to the effect of age on ACE2 expression, we quantified the number of alveolar macrophages in each sample. When normalized to either tissue area (S6A Fig in S1 File) or cellularity (S6B Fig in S1 File), the number of alveolar macrophages did not change with age (p = 0.768 and p = 0.427, respectively, linear regression).

We considered that factors other than age might be determining the intensity of ACE2 staining in lung samples from ventilated patients. Therefore, we studied a number of features including the length of time the sample was archived, the ventilation parameters used proximal to sample collection (namely FiO2 and PEEP), the severity of DAD, the presence or absence of ILD, history of smoking, and patient sex. None of these features correlated significantly with ACE2 expression when normalized to either tissue area or cellularity (Table 1 and S7A–S7D Fig in S1 File).

To compare our results in ventilated patients to samples collected from spontaneously breathing individuals, we collected an additional set of 20 lung samples from patients who underwent lung excision/biopsy for either pneumothorax repair or metastatic non-pulmonary neoplasms (S2 Table in S1 File). In all cases, care was taken to select portions of adjacent uninvolved lung parenchyma to analyze for ACE2 expression by IHC. Similar to our control sections, ACE2 staining was largely found within AT2 cells and alveolar macrophages (Fig 4A and 4B). When compared across ages, there was no significant difference either upon visual inspection or when quantified normalizing to tissue area (p = 0.231, linear regression) (Fig 4C) or cellularity (p = 0.349, linear regression) (Fig 4D).

A) Representative images of lung stained for ACE2 from a 26-year-old female who underwent blebectomy for a spontaneous pneumothorax are shown using a 1x objective (left, scale bar 3mm) with the region outlined by the red dashed box magnified at low power (middle, scale bar 200μm) and a second field at high power (right, scale bar 50μm). The normal alveolated lung parenchyma reveals AT2 cells which are positive for ACE2 (red arrowheads) scattered among largely ACE2 low type I pneumocytes. B) Representative images of lung stained for ACE2 from a 77-year-old man undergoing wedge resection for suspected metastatic angiosarcoma are shown using a 1x objective (left, scale bar 3mm) with the region outlined by the red dashed box magnified at low power (middle, scale bar 200μm) and a second field at high power (right, scale bar 50μm). Histologic findings in the normal alveolated parenchyma adjacent to the lesion were similar to those found in (A) with strong ACE2 staining in scattered AT2 cells (red arrowheads) and occasional alveolar macrophages. C) Quantitative IHC for ACE2 was carried out on samples from non-ventilated patients (n = 20 patients). Total ACE2 expression from 5 low power fields is plotted relative to the patient’s age at the time of specimen collection. A linear fit to the data is indicated by the dashed line with the 95% confidence interval highlighted in grey. D) The same specimens quantitated in (C) were normalized by cellularity and the average ACE2 expression per cell is plotted along with a linear fit to the data and its 95% confidence interval. Sections were stained for ACE2 using DAB and counterstained with hematoxylin.

During our image acquisition, we noticed 2 cases exhibiting prominent ACE2 expression within the lung vascular endothelium (Fig 5A and 5B). This feature was not observed in any of the other 29 cases we examined. Upon chart review, we discovered that the first case was from a 53-year-old man who was actively receiving inpatient lisinopril at the time of lung explant (Fig 5A). The second case was from an 83-year-old man receiving valsartan during his immediately preceding admission at an outside institution, and at the time of transfer to our hospital, where his surgical resection occurred on post transfer day 4 (Fig 5B). Both patients were on supportive mechanical ventilation for AHRF prior to lung sample collection. The prominent age-related changes in AT2 cell ACE2 expression were again evident, but in addition, there was strong staining within the endothelium, indicating increased ACE2 expression within the vasculature. None of the other 29 cases in our dataset were from patients on angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs) (S2 Table in S1 File).

A) Representative images of lung stained for ACE2 from a 53-year-old man with organizing diffuse alveolar damage superimposed on fibrosing interstitial lung disease are shown using a 1x objective (left, scale bar 3mm) with the region outlined by the red dashed box magnified at low power (middle, scale bar 200μm) and second field at high power (right, scale bar 50μm). AT2 cell hyperplasia can be seen with lower level ACE2 expression present along the alveolar septum (green arrowheads). Strongly stained endothelial cells (red arrowheads) can be seen throughout the entire specimen from this patient who was on daily lisinopril at the time of specimen collection. B) Representative images of lung stained for ACE2 from an 83-year-old man in the organizing phase of diffuse alveolar damage are shown using a 1x objective (left, scale bar 3mm) with the region outlined by the red dashed box magnified at low power (middle, scale bar 200μm) and second field at high power (right, scale bar 50μm). In addition to high ACE2 expression in reactive AT2 cells (green arrowheads), strong staining can be seen in numerous vascular endothelial cells (red arrowheads) throughout the thickened septal area in this patient on valsartan 4 days prior to specimen collection. Sections were stained for ACE2 using DAB and counterstained with hematoxylin.

Inhibition of the angiotensin-converting enzyme (ACE) protects against the progression of several cardiovascular diseases. Because of its dual role in regulating angiotensin II and bradykinin levels, the positive clinical effects of ACE inhibitors were thought to be the consequence of concomitant reductions in the production of angiotensin II and the degradation of bradykinin. Recent evidence suggests that some of the beneficial effects of ACE inhibitors on cardiovascular function and homeostasis can be attributed to novel mechanisms. These include the accumulation of the ACE substrate N-acetyl-seryl-aspartyl-lysyl-proline, which blocks collagen deposition in the injured heart, as well as the activation of an ACE signaling cascade that involves the activation of the kinase CK2 and the c-Jun N-terminal kinase in endothelial cells and leads to changes in gene expression. Moreover, at least one other ACE homologue (ACE2) is proposed to counteract the detrimental effects associated with the activation of the classical renin-angiotensin system. These data reveal hitherto unexpected levels of internal regulation of the renin-angiotensin system.

Circulating angiotensin II (Ang II) is the main effector of the renin-angiotensin system (RAS) and is involved in the global regulation of sympathetic activity and blood pressure as well as fluid and electrolyte balance. The angiotensin-converting enzyme (ACE) is an ectoenzyme that plays a role in the generation of Ang II by catalyzing the extracellular conversion of the decapeptide Ang I. In the classical scheme of things, two enzymes regulate the RAS: ACE and renin, the latter being released from the juxtaglomerular cells of the kidney into the circulation, where it converts angiotensinogen to Ang I. In addition to Ang I, ACE hydrolyzes a number of other substrates (see below) but probably the most important, at least for the regulation of vascular tone, is the potent vasodilator bradykinin. However, we clearly do not know all there is to know about the RAS, and a number of reports over the last few years have indicated that several other ACE substrates have marked effects on cardiovascular homeostasis and that ACE homologues exist that modulate the activity of the classical RAS.

Angiotensin-Converting Enzyme

Two distinct forms of ACE (dipeptidyl-carboxypeptidase I/kininase II) are expressed in humans, a somatic form that is particularly abundant on the endothelial surface of lung vessels (but that is also expressed in all other endothelial cells types as well as in some smooth muscle cells, monocytes, T lymphocytes, and adipocytes), and a smaller germinal form found exclusively in testis. Both forms of ACE exist at the cell surface as ectoenzymes, where they hydrolyze circulating peptides. A soluble form of ACE (soluble or plasma ACE), which is derived from the membrane-bound form through the action of the ACE secretase, is also present in serum and other body fluids. 1

Testicular ACE is the ancestral form of the molecule with a single active site, and somewhat surprisingly, its crystal structure was published only recently. 2 Somatic ACE arose as a consequence of gene duplication 3 and contains two active sites (termed the N and C domains). The structure of the N-terminal domain of somatic ACE is still unknown, but the C-terminal domain is expected to be identical to that of testis ACE. There appear to be differences in the function of the two sites, and Ang I conversion is reported to take place preferentially within the C domain. Indeed, selective C domain inhibition is sufficient to prevent Ang I–induced vasoconstriction, at least in small porcine coronary arteries. 4 On the other hand, both the N and C domains contribute to the degradation of bradykinin, 4 whereas Ang 1-7 is cleaved by the N-terminal active site of ACE and inhibits the enzymatic activity of the C-terminal site. 5 There is also evidence suggesting that the N domain of ACE may be functionally less relevant because the RXP407 peptide that specifically inhibits the ACE N domain active site has no effect on blood pressure. 6

More recently, testis ACE was reported to possess a glycosylphosphatidylinositol (GPI) hydrolase activity. This activity was not significantly inhibited by ACE inhibitors and not affected by substitution of core amino acid residues essential for peptidase activity, suggesting that the active site for GPI hydrolase (GPIase) activity is distinct from that of the dipeptidyl carboxypeptidase. This novel function of ACE was implicated in the cleavage of GPI-anchored proteins such as TESP5 and PH-20, two proteins involved in capacitation, from the sperm membrane, and the loss of this activity was proposed to account for the infertility of male ACE −/− mice. 7 However, location and access were determinant in bringing enzyme and substrate together, at least in F9 cells, because lipid rafts needed to be disrupted with filipin in order for ACE to access GPI-anchored proteins. 7 Although an attractive hypothesis, two groups have recently performed similar experiments and come to the conclusion that there is no overwhelming evidence to indicate that ACE possesses GPIase activity, and the infertility of male ACE −/− mice can be attributed entirely to the loss of its dipeptidyl carboxypeptidase activity. 8,9

Soluble ACE

A metalloprotease, the so-called ACE secretase, cleaves ACE between Arg 1203 and Ser 1204 on the extracellular side of the transmembrane domain 10 to generate a C-terminal truncated, soluble, or plasma form of the enzyme. 11 Soluble ACE in healthy subjects arises essentially from the endothelium, but in disease states, it can be found in other biological fluids including cerebrospinal and bronchoalveolar fluids. Plasma ACE levels have recently been suggested to represent a risk factor for coronary stent restenosis, 12 coronary artery disease, 13 and myocardial infarction. 14 Indeed, elevated plasma ACE activity, determined less than four hours after the onset of myocardial infarction in humans, has been suggested to be a significant predictor of the development of left ventricular dilation one year after infarction. 15

For a short while, it was tempting to speculate that one of the major roles of soluble ACE would be to cleave GPI-anchored proteins from the plasma membrane of blood and vascular cells because this enzyme is not bound by the rules governing the localization of the membrane-bound enzyme. However, there is no hard evidence that soluble ACE possesses GPIase activity. 8,9 Moreover, given that even small amounts of serum inhibit the GPIase activity of ACE, 16 it is highly unlikely that soluble ACE plays an important role in the release of GPI-anchored proteins in vivo.

The Importance of the Local RAS

Cells and tissues not implicated in the classical RAS are now known to possess all the molecular machinery necessary to generate Ang II. Tissues classed as expressing a functional local RAS express angiotensinogen, renin, renin-binding protein, ACE, chymase, as well as Ang II receptor 1 (AT1) or AT2 and secrete Ang II.

The Heart and the Systemic Vasculature

All the components of the RAS have been found in the cardiomyocytes as well as in endothelial cells and vascular smooth muscle cells. These cell types are able to generate Ang II, and proinflammatory/proatherosclerotic stimuli such as high cholesterol and insulin are able to activate this local RAS to increase oxygen-derived free radical production and induce oxidative stress.

There is increasing evidence that the local RAS may be involved in the maintenance of cardiovascular structure and repair. ACE levels are increased in many forms of vascular and cardiac hypertrophy, and the administration of ACE inhibitors has led to regression of hypertrophy. This effect of ACE on vascular remodeling is highlighted by the report that the in vivo gene transfer of ACE into the uninjured rat carotid artery results in the development of vascular hypertrophy independent of systemic factors and hemodynamic effects. 17 Selective overexpression of ACE in the heart also results in morphological changes in the atria, arrhythmia, and sudden death. 18


In atherosclerotic human coronary arteries, ACE immunoreactivity is associated with macrophages as well as with smooth muscle cells and T lymphocytes. 19,20 Given that Ang II activates monocytes and stimulates the expression of tissue factor as well as the release of proinflammatory cytokines, the induction of ACE in monocytes most likely exacerbates inflammatory responses. Indeed, ACE expression is reported to be higher in ruptured plaques than in fibrosclerotic plaques, and in the former, ACE is highly expressed in macrophages accumulated around the attenuated fibrous cap. Such findings indicate that the presence of ACE within lesions, atheromatous plaques, and ruptured plaques contributes greatly to the further progression of atherosclerosis. 20

Adipose Tissue

Human preadipocytes possess a complete functional RAS, and undifferentiated preadipocytes as well as immature adipocytes secrete Ang II. 21 The expression of the RAS components seems to relate to body weight because obese women are reported to have higher circulating angiotensinogen, renin, aldosterone, and ACE levels than lean women and lower angiotensinogen gene expression in adipose tissue. On the other hand, weight reduction (≈5%) reduced angiotensinogen, renin, and aldosterone levels and decreased ACE expression. 22 Although ACE −/− and AT1 −/− mice have no obvious changes in fat deposition, an ACE inhibitor and an AT1 receptor antagonist were found to reduce adipocyte size and to increase insulin sensitivity in Sprague-Dawley rats fed a fructose-rich diet. 23 The increase in insulin sensitivity also fits with a recent report that in subjects with essential hypertension and insulin resistance, RAS blockade with either an ACE inhibitor or an AT1 antagonist increased the secretion of the insulin-sensitizing adipokine adiponectin. 24

The importance of local RAS in monocytes as well as within adipose tissue and the vascular wall may well lie in the modulation of cell activation or differentiation and the subsequent release of cytokines and adipokines, which also affect the progression of cardiovascular disease. Inhibition of local tissue-specific RAS may also account for the observation that ACE inhibitors with high tissue affinity confer a greater degree of vascular RAS suppression than those with low tissue affinity despite similar suppression of the circulating RAS.


Although circulating Ang II levels decrease in response to acute ACE inhibitor treatment, 25 circulating Ang II levels tend to increase in patients taking ACE inhibitors over long periods. 26,27 This phenomenon highlights the fact that ACE is not the only enzyme implicated in the generation of Ang II and the ACE inhibitor–induced elevation in Ang I facilitates its hydrolysis by other peptidases.

Chymases are serine proteases belonging to the chymotrypsin family and are found in mast cells in multiple tissues and species. Human α-chymase is able to convert a number of substrates including Ang II from Ang I, interleukin-1β from its precursor, and endothelin-1 from big endothelin as well as to activate matrix metalloproteases. 28 Chymase is thought to be responsible for >80% of tissue Ang II formation in the human heart and >60% of that in arteries. 29,30 Therefore, it is not surprising that chymase has been implicated in the pathogenesis of cardiovascular diseases, particularly in cardiac hypertrophy, heart failure, atherosclerosis, and restenosis. 31 In mast cell-deficient (Kit w /Kit w−v ) mice, chymase cannot be detected in the vasculature, and there is no additional effect of AT1 receptor blockade on the blood pressure of animals receiving an ACE inhibitor. 32 Moreover, genetic deletion of ACE results in marked differences in circulating plasma Ang II and Ang I, but tissue (heart, kidney, and lung) concentrations of Ang II and the Ang II/Ang I ratio are not different in mice expressing different amounts of ACE (ie, ACE +/+ , ACE +/− , and ACE −/− mice). Because the latter observations were correlated with an increase in chymase activity in the kidneys and hearts of ACE −/− mice, it is tempting to speculate that chymase is important in maintaining steady-state Ang II levels in tissue, even though the contribution of chymase to total Ang II production (including that in plasma) is estimated as being <2%. 33 In humans, the chymase-specific substrate [Pro 11 D-Ala 12 ] Ang II induces a venoconstriction that is unaffected by ACE inhibition, 34 and non–ACE-dependent Ang II generation occurs in resistance arteries from patients with coronary artery disease. 29 However, differences in the contribution of chymase to the generation of Ang II in different tissues and between species makes it difficult to estimate the importance of this enzyme. 28 Although selective chymase inhibitors have been developed and promising results have been obtained in animal models of myocardial infarction, cardiomyopathy, and tachycardia-induced heart failure, these substances have yet to be tested in humans. 35

ACE Substrates

Both of the ACE isoforms hydrolyze a spectrum of circulating peptides and catalyze the hydrolysis of substance P, Ang 1-9, N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), cholecystokinin, hemopressin, and amyloid β-protein in addition to Ang I and the vasodilator peptides bradykinin and kallidin. 36


ACE is identical to kininase II, 37,38 the enzyme that metabolizes bradykinin to inactive fragments. In fact, ACE more readily hydrolyzes bradykinin than Ang I. Therefore, one consequence of ACE inhibition is that the metabolism of bradykinin is attenuated and the local concentration of this potent vasodilator in the vicinity of the endothelium is enhanced. It became clear relatively early on just how many of the actions of ACE inhibitors can be attributed to effects on the metabolism of both Ang I and bradykinin. For example, coadministration of the B2 receptor antagonist icatibant attenuated the mean arterial blood pressure response to perindoprilat 39 as well as the in vivo flow-dependent vasodilatation of human resistance and epicardial coronary arteries 40 and the radial artery. 41 The vasodilator effects of bradykinin are thought to be particularly potent because it is one of the rare stimuli that elicits the activation of the three most important endothelium-derived vasodilator autacoids (ie, NO, prostacyclin, and the endothelium-derived hyperpolarizing factor). 42 Assessing the role of bradykinin is rather difficult in rodents, particularly in mice, in which the B2 kinin receptor is expressed only in some vascular beds, and Ang II seems to be the stronger arm of the RAS in these animals. However, in a mouse model of chronic heart failure induced by myocardial infarction, ACE inhibition was associated with improved cardiac function and remodeling, and the effects of the inhibitors were attenuated in mice lacking the B2 receptor. 43 Moreover, in addition to their effects on vascular tone, ACE inhibitors improve neovascularization in the diabetic ischemic leg via a mechanism that is no longer apparent in B2 receptor knockout mice. 44 These compounds also partially prevent the development of left ventricular hypertrophy via an effect that is sensitive to icatibant and can most probably be attributed to the enhanced expression and activity of the endothelial NO synthase. 45

Angiotensin 1-7

Ang 1-7 possesses just one amino acid less than Ang II (Figure 1), and although it is cleaved by the N-terminal active site of ACE at half the rate of bradykinin, it actually inhibits the enzymatic activity of the C-terminal site. 5 Although initially thought to be without effect, Ang 1-7 is reported to potentiate the effects of bradykinin as well as those of ACE-resistant bradykinin analogues. 46,47 Somewhat intriguingly, the potentiation of bradykinin-induced vasodilation in spontaneously hypertensive rats treated short-term or long-term with ACE inhibitors was reverted by an Ang 1-7 antagonist, thus unmasking a key role for an Ang 1-7–related mechanism in mediating the effects of this class of compounds. 47

Figure 1. Peptides hydrolyzed by the ACEs.

Current interest in Ang 1-7 as a biological mediator has been stimulated by reports that it can be generated from Ang II by ACE2, a monocarboxypeptidase that shares ≈42% identity with the catalytic domain of somatic ACE. 48 Ang 1-7 is a vasodilator, and the fact that the expression of ACE2 is increased in disease and after treatment with ACE inhibitors, the ACE2/Ang 1-7 axis has been suggested to act as a natural damping mechanism for the activation of the classical RAS. 49

The actions of Ang 1-7 often functionally antagonize those of Ang II, for example, although Ang II increases blood pressure, Ang 1-7 is a vasodilator 50 it decreases blood pressure in hypertensive animals 51 and reduces vascular cell growth. 52 Ang 1-7 also participates in the regulation of cardiac function 53 and preserves cardiac function, coronary perfusion, and aortic endothelial function in a rat model for heart failure. 54 Ang 1-7 can bind to AT1 and AT2 receptors at high concentrations, as well as to its own receptor: Mas, an orphan receptor. Deletion of Mas abolishes the binding of Ang 1-7 to mouse kidneys as well as the Ang 1-7–induced relaxation of isolated aortae. 55 There is also evidence that Mas receptor activation modulates some of the effects of AT1 and AT2 receptors 56 by physically associating with them to generate hetero-oligomers and functionally antagonizing the actions of Ang II. 57


N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP or goralatide) is a potent natural inhibitor of hematopoietic stem cell proliferation that is degraded mainly by ACE. 58,59 In vitro, Ac-SDKP is a potent angiogenic factor 60 and inhibits collagen production by cardiac fibroblasts, 61 whereas in vivo, it blocks collagen deposition in the left ventricle of rats with hypertension or myocardial infarction. 62 ACE inhibitor treatment results in a significant increase in plasma Ac-SDKP levels in humans, 63 and in rats, the exogenous application of Ac-SDKP exerts many effects (including inhibition of Ang II–induced cell proliferation, left ventricular macrophage/mast cell infiltration, and collagen deposition) that are generally associated with ACE inhibitor therapy. 64,65 Thus, it is tempting to suggest that some of the beneficial effects of ACE inhibitors are not directly related to their effects on the RAS but rather are a consequence of increased circulating Ac-SDPK levels. Certainly, in rats made hypertensive by Ang II infusion, a monoclonal antibody to Ac-SDKP prevented the ACE inhibitor–induced decrease in left ventricular collagen deposition as well as monocyte infiltration, cell proliferation, and transforming growth factor-β expression. 66 Prevention of the degradation of Ac-SDKP has been celebrated as a novel mechanism of action of ACE inhibitors and clearly represents one of the most important observations in this field over the last five years. However, we still have a lot to learn about the cells targeted by Ac-SDKP and the molecular mechanisms involved in mediating its effects.

Amyloid β-Protein

An early and pathogenically important feature of Alzheimer disease is the progressive accumulation and deposition of the amyloid β-protein in brain regions serving memory and cognition. Biochemical, cell biological, animal modeling, genetic, and emerging clinical data all suggest that amyloid β-protein is an upstream initiator of the disease process and its associated neuropathology. 67 One way to undermine the accumulation of amyloid β protein and potentially delay the development of the disease could be to enhance its degradation by proteases expressed in the brain. The enzymes in question are the neutral endopeptidase, 68 the endothelin-converting enzymes, 69 and ACE. 70

There is circumstantial (not to mention controversial) evidence supporting a link between ACE and amyloid β-protein levels in the brain. Elevated levels of ACE have been reported in the temporal cortex of brains from Alzheimer disease patients, 71,72 whereas no apparent link could be detected in other clinical studies. 73,74 Enhanced cortical ACE activity was also associated with a prominent perivascular ACE and Ang II immunoreactivity surrounding some cortical vessels pointing to an underlying microvascular pathology in the process of neurodegeneration. 72 Interestingly, the ACE degradation product, a truncated 33-residue peptide, exhibited decreased aggregation and cytotoxic potential than the full-length amyloid β-protein. 70 Thus, reduced ACE activity, such as that realized after prolonged pharmacological inhibition, could be expected to accelerate amyloid β-protein accumulation and accelerate disease development just such an effect has been demonstrated in vitro. 75

ACE to B2 Kinin Receptor Cross-Talk

Over the last decade, there have been a number of reports showing that ACE inhibitors can amplify responses to bradykinin, although accumulation of the peptide cannot be assumed to occur, such as in continuously perfused systems. However, whether or not a cross-talk between these two proteins actually takes place, and if so, in which form, is still highly controversial.

The Case for …

One example of the type of experiment that led to the speculation that a cross-talk exists is the ACE-inhibitor induced Ca 2+ signal observed in bradykinin-desensitized endothelial cells (Figure 2). In these cells, superfusion with a high concentration of bradykinin elicits a biphasic Ca 2+ response but desensitizes the B2 kinin receptor so that a 10-fold higher concentration of bradykinin is unable to elicit a second response. However, under the same experimental conditions, the addition of an ACE inhibitor, in this case ramiprilat, elicits a Ca 2+ signal that is similar to the initial response. The ACE inhibitor–induced Ca 2+ signal is related to the activation of the B2 receptor because it is sensitive to the B2 kinin receptor antagonist icatibant. 76–78 However, it is highly unlikely that ramiprilat is able to immediately increase the local concentration of bradykinin to such an extent that the desensitized receptor can be reactivated. Because ACE inhibitors do not directly bind to the B2 receptor and the expression of both ACE and the B2 receptor are required for such a phenomenon to be observed, 79 we and others proposed that some form of cross-talk may occur between ACE and the B2 kinin receptor. 79–81 This hypothesis was also initially supported by the fact that ACE inhibitors can resensitize cells desensitized by ACE-insensitive bradykinin analogues. 78,82–84 However, the analogues used in these initial experiments are now known to be not as resistant as originally thought to degradation by ACE, and markedly different results have been obtained with a new generation of bradykinin analogues (see below).

Figure 2. ACE inhibitors initiate a cross-talk between ACE and the B2 kinin receptor. Human umbilical vein endothelial cells were loaded with fura-2 and mounted in a perfusion chamber on a fluorescent microscope. After stabilization of the Ca 2+ signal, the perfusion buffer was replaced with one containing bradykinin (Bk 100 nmol/L BK1) after ≈10 minutes, the buffer was again replaced with one containing either a higher concentration of Bk (1 μmol/L BK2) or the combination of Bk (100 nmol/L) and ramiprilat (100 nmol/L) and the signal monitored for an additional 10 minutes. Adapted from Benzing et al. 80

For ACE to B2 receptor cross-talk to take place, the two proteins need to communicate with each other either directly or via closely associated signaling molecules. There has been a report of a direct physical interaction between the two proteins in Chinese hamster ovary (CHO) cells overexpressing the B2 kinin receptor and ACE. 85 This interaction was assumed to occur between extracellular structures because cross-talk could still be demonstrated between the B2 receptor and an ACE mutant in which the terminal 18-aa residues were deleted from the cytosolic tail. 85 Moreover, the localization of ACE to lipid rafts by replacing the transmembrane and cytosolic portions of the molecule with a GPI anchor resulted in a loss of the ACE inhibitor–induced resensitization of the B2 receptor. 85 However, treatment of the latter cells with filipin to disperse lipid rafts restored the cross-talk. When interpreting the results of such experiments, it is important to realize that the activated B2 receptor is sequestered into caveolae, 80,86,87 whereas ACE is generally excluded from this cell compartment as well as from lipid rafts. 88,89 This means that if the two proteins do physically interact, they must do so in another cell compartment. The events involved in the de novo activation of B2 receptors may well differ from those involved in the reactivation of desensitized receptors because the latter but not the former are reportedly sensitive to pharmacological inhibitors of protein kinase C (PKC) and serine/threonine phosphatases. 90

Angiotensin peptides, specifically Ang 1-9 and Ang 1-7 have been suggested to mediate the cross-talk between ACE and the B2 receptor in the CHO overexpression system. 81,91 The Ang 1-7 and Ang 1-9–induced reactivation of the B2 kinin receptor in CHO cells (assessed by determining arachidonic acid release) was sensitive to a series of pharmacological inhibitors of PKC and phosphatase as well as to the tyrosine kinase inhibitor genistein. 91 A more detailed analysis of the molecular mechanisms underlying this phenomenon was not performed, but the effect was attributed to a potential conformational change in the ACE–B2 receptor heterodimer. No evidence has been provided to indicate that a similar interaction occurs under physiological conditions, but the time course of the events in question needs to be taken into account because the ACE inhibitor–induced resensitization of the B2 receptor is immediate. It is difficult to envisage that Ang 1-7 or Ang 1-9 levels (or bradykinin for that matter) can increase rapidly enough to account for many of the experimental findings reported to date in endothelial cells 80 or in the isolated perfused heart. 78

Little detailed biochemical work has been performed in cells that constitutively express ACE and the B2 kinin receptor. The only investigation using “native” endothelial cells (ie, directly scraped off isolated porcine aortae) reported that although bradykinin initiated the sequestration of the B2 receptor to caveolin-rich membranes, pretreatment of these cells with an ACE inhibitor significantly attenuated the recovery of B2 kinin receptors from caveolae while increasing that from membranes lacking caveolin. 80 This effect could not be attributed to the inhibition of bradykinin degradation because no effect was seen in the presence of an inhibitory concentration of the synthetic ACE substrate hippuryl- l -histidyl- l -leucine. Ramiprilat also decreased [ 3 H]bradykinin binding to caveolar membranes when applied either before or after bradykinin stimulation. These data led to the suggestion that ACE inhibitors interfere with the targeting of the B2 receptor to caveolae, implying that effects other than the inhibition of ACE activity per se may account for the effects of this class of compounds. 80

The Case Against …

The most damming evidence against the ACE–B2 cross-talk hypothesis is that although it is evident after the addition of ACE inhibitors to bradykinin-desensitized cells, it is generally not observed in cells or in blood vessels in which the B2 receptor is desensitized with the new generation of ACE-resistant B2 receptor agonists. 92–95 Such data suggest that the metabolism of bradykinin by ACE is indeed so fast in the microenvironment of the B2 receptor that concentrations of the peptide increase to micromolar levels within milliseconds of the addition of ACE inhibitors. However, little information is available regarding the effects of these ACE-resistant agonists on receptor occupancy, efficacy, or speed of inactivation and sequestration.

The Verdict

Unfortunately, in the case of ACE–B2 receptor cross-talk, we seem to have a “hung jury” with the cases for and against currently prohibiting a real conclusion. In an attempt to investigate the phenomenon of ACE–B2 receptor cross-talk from a completely different aspect, we reasoned that the binding of an ACE inhibitor to ACE should be able to elicit an intracellular event and that ACE should be capable of outside-in signaling. Moreover, the cytoplasmic tail of ACE should be able to bind soluble intracellular signal molecules or adaptor proteins that initiate a chain of events ultimately linking to effects such as the reactivation of the B2 kinin receptor. Although the so-called ACE signaling pathway (outlined in the next section) was identified as the result of studies to address this hypothesis, it has not yet been possible to find a direct link between the binding of an ACE inhibitor to ACE and the reactivation of the B2 kinin receptor (our unpublished observation, 2005).

Signal Transduction by ACE

In all of the species studied to date, the short cytoplasmic tail of somatic ACE contains between three and five serine residues, one of which (Ser 1270 human sequence) is located in a highly conserved 13-aa sequence at the extreme C-terminal end of the protein. In endothelial cells, Ser 1270 is phosphorylated by the kinase CK2, which also physically interacts with the protein. The basal phosphorylation of ACE by CK2 stabilizes its localization in the plasma membrane because the mutation of this site and the inhibition of CK2 both enhance the cleavage/secretion of the enzyme. 96 In contrast, the cytoplasmic tail of rabbit testis ACE is reported to be tyrosine phosphorylated rather than serine phosphorylated. 97 However, the latter modification is not relevant to that of the human somatic enzyme, which does not contain a tyrosine residue. 98

What Are the Components of the ACE Signaling Pathway?

CK2 is not the only protein that associates with the cytoplasmic tail of ACE in endothelial cells. Using ACE immunoprecipitated from ACE overexpressing cells as well as an affinity column composed of a peptide corresponding to the cytoplasmic tail of ACE, mitogen-activated protein kinase kinase 7 and c-Jun N-terminal kinase (JNK) were also found to associate with the intracellular domain of the human enzyme. ACE phosphorylation is an essential step in the ACE signaling cascade, and the CK2-dependent phosphorylation of Ser 1270 is required for the activation of ACE-associated JNK. The increase in JNK activity results in the translocation of phosphorylated c-Jun to the nucleus, an enhanced binding of the activator protein-1 transcription factor to DNA followed by the increased expression of genes such as ACE 99 and cyclooxygenase-2 (COX-2 100 Figure 3). Although at first glance, it seems unlikely that an ACE inhibitor–induced increase in the expression of ACE or COX-2 could be associated with improved endothelial cell function, increased ACE levels have been demonstrated in lung tissue and plasma from ACE inhibitor–treated rats 101 and in the serum from patients who distinctly benefit from ACE inhibitor therapy. 102 Moreover, prostacyclin production is significantly increased in subjects treated with ACE inhibitors, 103 and selective COX-2 inhibition diminishes the positive effects of ACE inhibitors on blood pressure. 104,105

Figure 3. The ACE signaling pathway. ACE inhibitor binding to ACE activates ACE-associated CK2, leading to phosphorylation of ACE Ser 1270 . Depending on initial phosphorylation, ACE-associated JNK becomes activated, most likely via ACE-associated MKK7 (mitogen-activated protein kinase kinase 7), leading to an accumulation of phosphorylated c-Jun in the nucleus and enhancement of the DNA binding activity of activator protein-1 (AP-1 c-Jun dimer). AP-1 activation influences endothelial gene expression (ie, increases ACE as well as COX-2 expression). At the same time, the activated ACE-associated CK2 phosphorylates the nonmuscle MYH9, which is involved in anchoring ACE to the plasma membrane/cytoskeleton.

A number of other proteins are reported to associate with the cytoplasmic domain of somatic ACE. For example, β-actin and the nonmuscle myosin heavy chain IIA (MYH9) associate with ACE in endothelial cells and seem to play a role in the cleavage/secretion of the enzyme. 106 Moreover, ACE inhibitors elicit the phosphorylation of MYH9 by CK2, which is dependent on the initial phosphorylation of ACE itself. Although the cellular consequences of these events are not entirely clear, it seems that the phosphorylation of MYH9 stabilizes its association with ACE and anchors it more firmly in the cell membrane, thus decreasing soluble ACE levels. 106

Rabbit testis ACE associates with a number of other proteins including ribophorin and the chaperone immunoglobulin-binding protein. 107 The latter interaction is not likely to affect signaling but very likely to affect protein maturation because the overexpression of immunoglobulin-binding protein inhibited ACE secretion, an effect that could be attributed to the retention of the enzyme in the endoplasmic reticulum. 107 Several PKC isoforms (PKCι, PKCγ, PKCδ, and PKCλ) can also be coimmunoprecipitated with rabbit testis ACE using an anti-ACE antibody. 107 However, it is currently unclear whether this association occurs with the mature or the immature protein and to which domain the proteins bind. There is little evidence to indicate that an association occurs between PKC and the cytoplasmic tail of the mature (plasma membrane bound) human somatic ACE, and in in vitro phosphorylation assays, a constitutively active PKC did not phosphorylate either the native human somatic ACE protein or a peptide corresponding to its cytoplasmic tail. 106 On the other hand, it does appear that PKC may modulate ACE signaling pathways (in addition to affecting ACE cleavage/secretion) by exerting a regulatory influence on the activity of ACE-associated CK2. Indeed, the pretreatment of endothelial cells with the PKC inhibitor RO 31-8220 enhanced the activity of ACE-associated CK2 as well as the phosphorylation of both ACE and MYH9. 106 One property of CK2 is that it can form heterocomplexes with other kinases, which regulates its function and substrate specificity. 108 There is at least circumstantial evidence of such an interaction between PKC-ζ and CK2 in a monoblastic cell line (U937 cells) and the PKC-ζ/CK2 complex influences the basal turnover of IκBα. 109 Calmodulin binds to the cytoplasmic domain of both rabbit testis ACE 110 as well as to that of human somatic ACE (our unpublished observation, 2004). However, whether or not this interaction can be modulated by ACE inhibitors and whether or not ACE-associated calmodulin plays a role in the ACE signaling cascade remain to be determined.

Open Questions

The ACE signaling pathway (Figure 3) has been mainly addressed in ACE-overexpressing endothelial cell lines as well as in primary cultures of endothelial cells. The evidence that such a pathway exists in vivo in humans and in mice is mainly circumstantial, and a detailed analysis of the long-term effects of the signaling pathway outlined above must await the conclusion of studies using mice carrying a mutation in Ser 1270 . An additional intriguing point relates to the existence of an endogenous ligand for the signaling pathway because although a weak effect of bradykinin has been reported on the phosphorylation of ACE on Ser 1270 , no other ACE substrate was able to activate ACE-associated CK2 or JNK. On the basis of the observations outlined above, it is tempting to suggest that ACE has three functions: (1) it generates Ang II from Ang I, (2) it degrades bradykinin to inactive peptides, and (3) after the binding of an ACE inhibitor that might mimic the function of an as yet unidentified agonist, ACE acts as a signal transduction molecule.


A few years ago, we thought we knew all about the RAS and a lot about its regulation. The identification of a novel ACE (ACE2) as well as an ACE-dependent signaling pathway and intracellular Ang II receptors implies that the RAS has still unexpected facets with clinical implications. One of the most intriguing aspects that remain to be elucidated is probably the link between the RAS and type 2 diabetes, especially in the light of the clinical effectiveness of ACE inhibitors 111,112 in delaying the onset of diabetes.

Original received December 12, 2005 revision received February 2, 2006 accepted March 1, 2006.

Work performed in the author’s laboratory is supported by the Deutsche Forschungsgemeinschaft (FL 364/1-2) and the European Vascular Genomics Network, a network of excellence supported by the European Community Sixth Framework Programme (Contract N° LSHM-CT-2003-503254).

ACE inhibitors all work in the same way by inhibiting the action of the angiotensin converting enzyme.

However, there are differences in their effectiveness at reducing blood pressure, their side effect profile, and their ability to prevent people from dying from a heart-related or other cause.

One review of 29 studies 1 concluded that trandolapril was the most effective at reducing both systolic and diastolic blood pressure, while lisinopril was the least effective and is associated with the highest incidence of all-cause mortality. Ramipril was associated with the lowest risk of all-cause mortality. Another ACE inhibitor, enalapril, rated highly for heart pumping measures such as ejection fraction and stroke volume, but was associated with the highest risk of side effects such as cough, gastrointestinal discomfort, and a reduction in kidney function.

Generic name Examples of brand names
benazepril Lotensin
captopril Capoten
enalapril Epaned, Vasotec
fosinopril Monopril
lisinopril Prinivil, Zestril
moexipril Univasc
perindopril Aceon
quinapril Accupril
ramipril Altace
trandolapril Mavik

What are ACE inhibitors? Are they a possible treatment or prophylactic for SARS-CoV-2?

Angiotensin converting enzyme (ACE, aka ACE1) is another protein, also found in tissues such as the lung and heart, where ACE2 is present. Drugs that inhibit the actions of ACE1 are called ACE inhibitors. Examples of these drugs are ramipril, lisinopril, and enalapril. These drugs block the actions of ACE1 but not ACE2. ACE1 drives the production of ANG II. In effect, ACE1 and ACE2 have a “yin-yang” relationship ACE1 increases the amount of ANG II, whereas ACE2 reduces ANG II.

By inhibiting ACE1, ACE inhibitors reduce the levels of ANG II and its ability to increase blood pressure and tissue injury. ACE inhibitors are commonly prescribed for patients with hypertension, heart failure and kidney disease.

Another commonly prescribed class of drugs, angiotensin receptor blockers (ARBs, e.g., losartan, valsartan, etc.) have similar effects to ACE inhibitors and may also be useful in treating COVID-19.

Evidence for a protective effect of ACE inhibitors and angiotensin receptor blockers in patients with COVID-19 was shown in recent work co-authored by one of us - Dr. Loomba.

No evidence exists to suggest prophylactic use of these drugs we do not advise readers to take these drugs in the hopes that they will prevent COVID-19. We wish to emphasize that patients should only take these drugs as instructed by their health care provider.

The Effect of Comorbid ACE2 Dysregulation on COVID-19–Associated Symptoms

Many diseases are associated with impaired ACE2 activity in the implicated organs. One of the major corollaries of this impairment is an elevated proinflammatory response (Kangussu et al., 2019 Patel et al., 2016b). Ultimately, tissue injury and fibrosis may result. Patients with underlying diseases such as hypertension, diabetes, and coronary heart diseases have been shown to have more severe COVID-19 symptoms (Guan et al., 2020 Huang et al., 2020). This may be attributed to the pre-existing proinflammatory state, which is further propelled by the cytokine storm induced by SARS-CoV-2. Moreover, it can also be a consequence of an interplay with RAS and thus further loss of functional ACE2 units that are shed from the cell surface or downregulated after internalization following interaction with SARS-CoV-2 (Haga et al., 2008 Services et al., 2014 Gheblawi et al., 2020).

On the other hand, extrapulmonary symptoms in infected individuals with no known underlying diseases are increasingly emerging in patients with COVID-19. Besides pneumonia, other symptoms associated with the virus span the intestinal tract, cardiovascular system, central nervous system, and others (Fang et al., 2020 Zheng et al., 2020). We propose that the emergence of such symptoms is related to the presence of ACE2 in these specific tissues. Conversely, the disruption of ACE2 in patients with underlying diseases may contribute to the severity of symptoms detected in these individuals. Therefore, it is important to highlight the distribution of ACE2 in various organs and its dysregulation in specific diseases and to monitor related symptoms in inflicted patients.

Pulmonary Disease

The Role of ACE2 in Pulmonary Diseases.

Pulmonary hypertension in rats led to an enlarged right ventricle, interstitial fibrosis, and increased pulmonary wall thickness (Ferreira et al., 2009). In tandem, renin, ACE, angiotensinogen, AT1R, and proinflammatory cytokines were also elevated. These manifestations were precluded by treatment with a synthetic ACE2 activator (Ferreira et al., 2009). In a related study, rats in which pulmonary fibrosis was induced displayed reduced levels of ACE2, augmented collagen deposits, increased expression of TGF-β and other cytokines, and elevated AT1R expression (Shenoy et al., 2010). Moreover, lung injury in ACE2-knockout mice was associated with an exacerbated reduction of exercise capacity, further impairment of lung function, and increased lung fibrosis (Li et al., 2016). This phenotype was markedly attenuated by Ang1–7 or ACE2 overexpression. These findings emphasize the integral role of ACE2 in pulmonary protection against inflammation and fibrosis. Similar findings were observed upon assessing the role of ACE2 in mouse lungs in situations of inhaled endotoxins (Sodhi et al., 2018). The loss of ACE2 expression induced an activation of des-Arg 9 bradykinin/bradykinin B1 receptor pathway, leading to an increase in chemokines, macrophage inflammatory protein-2, TNF-α, and neutrophil infiltration, thus exaggerating lung inflammation and injury (Sodhi et al., 2018). Even in chronic airway inflammatory conditions like asthma, Ang1–7 significantly reduced the number of autoinflammatory-related leukocytes, including macrophages, eosinophils, and neutrophils (El-Hashim et al., 2012). Ang1–7 also reduced immune cell infiltration, fibrosis, and goblet cell metaplasia, which suggests airway remodeling. As such, it becomes plausible to assume that suppression of ACE2 activity/expression might exacerbate lung injury after viral invasion.

ACE2 and Pulmonary Symptoms in Patients with SARS-CoV-2.

ACE2 is highly expressed in the lungs (Letko et al., 2020), in which 83% of ACE2-displaying pulmonary cells were alveolar cells, implicating the alveoli as a reservoir for viral invasion (Li et al., 2020b). Recent investigation showed that although lung inflammatory disorders including asthma and chronic obstructive pulmonary disease are not associated with changes in ACE2 expression, smokers had increased pulmonary expression levels, further confirming the susceptibility of these patient groups (Grundy et al., 2020 Li et al., 2020a). Interestingly, this is thought to occur as a consequence of nicotine-mediated stimulation of α7-nicotinic acetylcholine receptors (Leung et al., 2020). Conversely, others propose that some of the SARS-CoV-2 proteins might carry amino acid sequences similar to those of known neurotoxins that block nicotinic acetylcholine receptors (Farsalinos et al., 2020b,c). This suggested a possible role for α7-nicotinic acetylcholine receptors as potential sites of interaction with SARS-CoV-2. As such, one might speculate that this interaction drives inflammatory cytokine production, whereby infected lung macrophage will be deprived of the anti-inflammatory effect exerted by nicotine-mediated activation of α7-nicotinic acetylcholine receptors on their surface (Kloc et al., 2020). If this competitive relationship between SARS-CoV-2 and nicotine on α7-nicotinic acetylcholine receptors is proven experimentally, it could potentially offer a molecular explanation for the clinical debate that nicotine, and hence tobacco smoking, might be protective against COVID-19 (Farsalinos et al., 2020a). On the other hand, the large surface area of the lungs favors their high susceptibility to inhaled viruses (Zhang et al., 2020a). Because of ACE2 abundance on pneumocytes and pulmonary vessels, massive SARS-CoV-2 entry and subsequent alveolar wall destruction is expected, contributing to the lung damage associated with COVID-19 (Hamming et al., 2004 Zhou et al., 2020b). Contrary to other coronaviruses, SARS-CoV-1 and SARS-CoV-2 have been found to decrease cell membrane–bound ACE2, which in turn promotes severe acute respiratory complications (Kuba et al., 2005 Haga et al., 2008 Ingraham et al., 2020), further alluding to the paradoxical nature of ACE2’s role in the course of pulmonary infections. However, other work reports a rebound in pulmonary ACE2 expression 48 hours postinfection that was correlated to inflammatory cytokine production (Li et al., 2020a), raising the question of why this induced increase in expression does not lead to the same beneficial effect associated with basal ACE2 expression/function.

Another factor that accounts for the much higher SARS-CoV-2 pulmonary infectivity is that the viral entry is mediated by cellular membrane proteases (Hoffmann et al., 2020). Transmembrane protease serine 2 (TMPRSS2) is expressed in the epithelial cells in human lungs and is involved in the regulation of airway surface liquid. Previous studies have identified TMPRSS2 as a key element in SARS-CoV pathogenesis (Matsuyama et al., 2010). TMPRSS2 enhances viral entry through cleavage of ACE2 and activation of cell membrane fusion by cleaving S protein Services et al., 2014. TMPRSS2 contributes to higher levels of cytokine and chemokine production after SARS-CoV infection. In TMPRSS2-knockout mice, the levels of monocyte chemoattractant-1 (MCP-1), IL-1α, IL-1β, and IL-12 in the lungs induced by SARS-CoV infection were much lower (Iwata-Yoshikawa et al., 2019). A new study using primary human airway epithelial cells reported that SARS-CoV-2 viral entry is highly dependent on TMPRSS2, as the inhibition of TMPRSS2 by camostat mesylate totally inhibited viral infection (Hoffmann et al., 2020). Camostat mesylate, currently used in Japan as an antiviral drug for other indications, could be useful in patients with COVID-19 (Kawase et al., 2012 Zhou et al., 2015 Yamamoto et al., 2016). After the SARS-CoV-2–induced cytokine storm, patients usually suffer from signs of microvascular dilation, increase of pleural effusions and thickening, and fibrotic streaks, which could lead to subsequent progression into acute lung injury, ARDS, and respiratory failure (Zhang et al., 2020b Zhou et al., 2020c).

Cardiovascular Disease

ACE2 and Cardiovascular Diseases.

Chronic hypertension and myocardial infarction are associated with cardiac remodeling, a pathologic process leading to heart failure and mortality (Grobe et al., 2007 Maaliki et al., 2019). The remodeling occurs after prolonged and enhanced production of AngII, and inhibition of the ACE1/AngII/AT1R system confers protection from these effects. In fact, induced overexpression of ACE2 reverses cardiac remodeling (Grobe et al., 2007) in a manner mediated by Ang1–7, which specifically reduced myocyte hypertrophy and interstitial fibrosis without affecting blood pressure. Furthermore, overexpression of ACE2 prevents cardiac hypertrophy in hypertensive rats (Sriramula et al., 2011). In a related study, ACE2 mRNA expression was markedly reduced in rat models of hypertension and was associated with defects in cardiac contractility, increased AngII levels, and upregulation of hypoxia-induced genes (Alaaeddine et al., 2019). Drugs used to treat hypertension, such as the AT1R blockers, upregulate myocardial expression of ACE2 and reduce levels of inflammatory markers like MCP-1, interleukins, and nuclear factor κ-light-chain enhancer of activated B cells (Sukumaran et al., 2012). The ACE1/AngII/AT1R system also promotes atherosclerotic lesion formation, aneurysms, and proinflammatory cytokine secretion (Daugherty et al., 2000). ACE2 has been shown to counteract these effects. For example, knockout studies of ACE2 in mice led to plaque growth as well as an increase in several proinflammatory and atherogenic proteins, like adhesion molecules, IL-6, and MCP-1 (Thomas et al., 2010).

ACE2 and CVD Symptoms in Patients with COVID-19.

Cases of acute cardiovascular anomalies have been reported in patients with COVID-19 that were previously healthy. Patients show high levels of troponin, creatine kinase, arrythmias, and incidences of myocardial injuries, which lead to higher rates of intensive care unit admission and mortality (Guo et al., 2020 Inciardi et al., 2020 Wang et al., 2020a). Patients with high troponin levels also had high levels of C-reactive protein, suggesting an inflammatory pathway linked to the SARS-CoV-2–induced myocardial injury (Guo et al., 2020). Thus, the myocardial injury seen could be attributed to SARS-CoV-2 direct viral entry as well as an increased production of cytokines that can result in decreased coronary blood flow and oxygen supply (Oudit et al., 2009). In addition, similar to other respiratory infections (Milbrandt et al., 2009), patients with COVID-19 demonstrate elevated D-dimer levels, which were also associated with disseminated intravascular coagulation, severe symptoms, and higher risk of mortality (Guan et al., 2020 Zhou et al., 2020a). Contributary mechanisms could include a series of inflammatory-immunologic reactions, which could directly contribute to atherosclerotic plaque rupture, predisposing the patient to ischemia and thrombosis (Zhou et al., 2020a).

Significantly, ACE2-mediated viral endothelial entry could induce vascular disorders and an accelerated coagulation in patients with COVID-19 (Gallagher et al., 2008). Indeed, SARS-CoV-2 was shown to infect endothelial cells in different vascular beds in patients with COVID-19 (Varga et al., 2020). Treatment with clinical-grade human recombinant ACE2 precluded SARS-CoV-2 infection in blood vessel organoids, highlighting the importance of ACE2 in endothelial infection (Monteil et al., 2020). The endothelium plays a critical role in the regulation of the adherence of immune cells, capillary permeability, and clotting and platelet activation, all of which could be altered by viral infection (Dalrymple and Mackow, 2014). Under normal conditions, vascular endothelial growth factor (VEGF) induces repair of vascular damage. Hantavirus, a virus affecting the respiratory system, was shown to bind to VEGF and disengage the normal regulation of VEGF-induced permeability (Gavrilovskaya et al., 2012). A similar profile is encountered in patients with COVID-19. Indeed, serum VEGF levels were elevated in patients with COVID-19 (Huang et al., 2020), possibly triggered by hypoxia and inflammatory changes and contributing to increased vascular permeability and pulmonary edema. Interestingly, increased ACE2 expression was shown to reduce VEGF production in vitro and in vivo (Cheng et al., 2016), ameliorating increased vascular permeability during lung injury (Yu et al., 2016), which raises the question of whether SARS-CoV-2–induced endothelial ACE2 downregulation could underlie this observation. These findings highlight the importance of monitoring VEGF levels in patients with COVID-19 and also suggest a possibility of introducing treatment modalities to target VEGF responses. Bevacizumab, a humanized monoclonal anti-VEGF antibody, is currently being studied for a potential beneficial effect in reducing lung injury due to increased vascular permeability ( NCT04275414) (, 2020).

On the other hand, accumulating evidence indicates an association between existing cardiovascular and metabolic disease with further progression of severe COVID-19 complications (Liu et al., 2020a Rodriguez-Morales et al., 2020 Wehbe et al., 2020 Zhou et al., 2020a). Higher mortality rates and progression of ARDS were reported in patients with hypertension, diabetes, and coronary artery diseases (Wu et al., 2020 Wu and McGoogan, 2020). This could be attributed to stimulation of immunoinflammatory pathway, dysregulation of RAS, and most importantly, viral entry through upregulated ACE2, as previous work showed that patients with heart failure had higher ventricular ACE2 expression levels (Zisman et al., 2003). Indeed, a recent study reported that patients with COVID-19 and heart failure had higher cardiac ACE2 expression (Chen et al., 2020a). Specifically, higher ACE2 expression was observed in cardiac pericytes, raising the possibility that cardiac injury could be initiated by microvascular dysfunction. Moreover, ACE2 expression in vascular endothelial cells could facilitate localized vascular injury and subsequent viral spread. As such, in situations of altered vascular function, vascular inflammation and/or increase in VEGF, SARS-CoV-2 vascular viral entry, and viral spread could be aggravated.

Inflammatory Disease: Obesity, Diabetes, and Autoimmune Disorders

ACE2 activity has been detected in macrophages, in which its deficiency led to an increased production of AngII proinflammatory cytokines, like TNF-α, MCP-1, IL-6, and matrix metallopeptidase-9 and endothelial adhesion molecules (Thomas et al., 2010 Thatcher et al., 2011). Importantly, overexpression of ACE2 by macrophages significantly reduced their MCP-1 production (Guo et al., 2008), and Ang1–7 treatment decreased macrophage inflammatory response (Souza and Costa-Neto, 2012). These findings further support the vital role of ACE2 in relieving the proinflammatory response thus, both low- and high-grade inflammatory conditions could affect the pathogenesis of COVID-19.

Interestingly, obesity is associated with increased RAS activity in adipose tissue (Yvan-Charvet and Quignard-Boulangé, 2011) together with increased macrophage infiltration (Weisberg et al., 2003). Adipose tissue macrophages in obesity tend to acquire macrophage (M1) polarization with increased expression of proinflammatory cytokines (Lumeng et al., 2007). Under such circumstances, adipose inflammation in obesity contributes to insulin resistance, glucose intolerance, and cardiovascular dysfunction (Jiao and Xu, 2008 Elkhatib et al., 2019). Interestingly, ACE2 deficiency enhanced M1 polarization of adipose tissue macrophages, increased adipose tissue inflammation, exacerbated the metabolic dysfunction, and worsened the associated cardiovascular function in diet-induced obesity (Thatcher et al., 2012 Patel et al., 2016a). Indeed, recent evidence implicates obesity as a risk factor for severe ARDS in patients with COVID-19 (Simonnet et al., 2020). Whereas no change in adipose tissue ACE2 expression levels was observed in patients with obesity compared with lean individuals (Pinheiro et al., 2017), the increased disease severity could be attributed to the overall inflammatory state that results from an obesity-triggered systemic RAS imbalance (Engeli et al., 2005).

Moreover, functional and clinical evidence supports the role of vascular inflammation induced by metabolic diseases in vascular impairment and CVD (Assar et al., 2016 Ormazabal et al., 2018). Patients with type 2 diabetes mellitus have reduced levels of ACE2 expression in various organs, which correlates with inflammatory changes especially in the kidney (Bindom and Lazartigues, 2009). Along the same lines, high levels of VEGF were observed that were correlated with high fasting blood glucose and glycosylated hemoglobin (HbA1c) levels (Zhang et al., 2018). Increases in VEGF reflected the severity of endothelial dysfunction in patients with diabetes and may lead to microvascular complications. In addition, higher levels of VEGF are associated with diabetes-induced central and peripheral neuropathy (Atif et al., 2017 Jerić et al., 2017 Tang et al., 2018). Collectively, vascular inflammation in patients with CVD and higher expression of VEGF in patients with diabetes could be additional factors to enhance viral entry and viral spread, which consequently increase COVID-19 severity in those patients. It is noteworthy that increased usage of VEGF inhibitors could induce cardiovascular and nephrological consequences as such, extreme care should be taken in treatment with such patients (Kikuchi et al., 2019). Moreover, it is noteworthy that patients who previously recovered from SARS-CoV-1 infection reported high blood pressure as well as altered metabolism 12 years later (Wu et al., 2017). Knowing the similarities between SARS-CoV-1 and -2, a similar profile could also occur in patients with COVID-19.

ACE2 is also implicated in the autoimmune disease of rheumatoid arthritis. Mouse models of antigen-induced arthritis portrayed elevated levels of neutrophils in the knee joints, and treatments with Ang1–7 or its analog, AVE0991, were able to reduce neutrophil migration in joints and periarticular tissue (da Silveira et al., 2010). Likewise, the elevated levels of proinflammatory cytokines TNF-α, chemokine (C-X-C motif) ligand, and IL-1β were reduced by Ang1–7 treatment together with hypernociception, the pain index of arthritis (da Silveira et al., 2010). Interestingly, this effect was not mediated by hypotensive or vasodilatory actions. Thus, Ang1–7 is able to mediate these effects by inhibiting local production of cytokines and, importantly, by hindering leukocyte-endothelium adhesion and rolling on the microvasculature of the knee joint. Importantly, Ang1–7 receptors have been detected on microvessels as well as leukocytes (Nie et al., 2009). These findings suggest that loss of ACE2 activity in patients with arthritis may inflame symptoms and cytokine production. Significantly, a recent meta-analysis showed some indication that autoimmune diseases might be associated with an increased risk of COVID-19 severity and mortality (Liu et al., 2020b) on the other hand, emerging reports indicate that SARS-CoV-2 infection precedes the development of various autoimmune disorders (Galeotti and Bayry, 2020). In either case, the role of ACE2 expression or activity alterations has not been examined.

Renal Disease

ACE2 in Renal Disease.

ACE2 is abundantly expressed on the vascular endothelium supplying the kidneys and on the tubular and glomerular epithelium (Lely et al., 2004). Mice lacking ACE2 develop late-onset nephrotic glomerulosclerosis (Oudit et al., 2006) and more profound diabetic renal injury (Wong et al., 2007), where ACE2 activity was shown to be protective by reducing local AngII levels (Batlle et al., 2012). Indeed, in a related study of diabetic mice, RAS imbalance involving ACE2 downregulation was found to be involved in renal damage mediated via elevated reactive oxygen species (Chang et al., 2011). Importantly, the renoprotective effects of ACE2 are not only restricted to chronic disorders. While renal ischemia and reperfusion trigger acute kidney injury via activation of inflammatory cascades involving increased migration of leukocytes and release of cytokines, treatment with the Ang1–7 analog AVE0991 reduced renal injury and inhibited leukocyte migration in mice post–ischemia reperfusion (Barroso et al., 2012). These findings broadly categorize the ACE2 axis once again as anti-inflammatory. However, the role of ACE2/Ang1–7 appears to be rather complex and should be approached with caution, as other studies have conversely demonstrated that this pathway may contribute to certain forms of kidney injury in diabetes, including fibrogenesis and renal hypertrophy (Tikellis et al., 2008), possibly affected by differences in disease stage, locally activated signaling pathways, and dosages of the activating or inhibiting agents used (Zimmerman and Burns, 2012).

ACE2 and Renal Symptoms in Patients with SARS-CoV-2.

Acute kidney injury was identified in up to 25% of patients admitted with COVID-19 (Fanelli et al., 2020), representing a higher mortality risk in these patients (Wilson and Calfee, 2020). A similar profile was seen in two other studies in which patients showing progressive increases in blood urea nitrogen and creatinine had higher mortality rates (Chen et al., 2020b Wang et al., 2020a). The mechanism behind this renal injury could be multifactorial. Although ischemic injury and inflammatory cytokines are likely to contribute to renal damage, direct SARS-CoV-2 entry through ACE2 to the renal cells could still be a major factor. SARS-CoV-2 entry in a human kidney cell line required priming by both TMPRSS2 and the endosomal cysteine proteases cathepsin B and L (Hoffmann et al., 2020). However, viral entry in ACE2-expressing HEK293 cells was reported to be mediated through endocytosis in which other proteins, such as PIKfyve, (Phosphoinositide kinase, FYVE-type zinc finger containing phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase type III) two pore segment channel 2, and cathepsin L, were found essential for virus entry (Ou et al., 2020). Significantly, a recent study showed cellular damage and direct ultrastructural evidence of viral infection in proximal tubular epithelial cells in postmortem examination of a patient with COVID-19 and acute kidney injury (Farkash et al., 2020). Yet other studies argue against a causal relationship between COVID-19 infection and acute kidney injury (Wang et al., 2020b). Indeed, the same factors that contribute to SARS-CoV-2 infection severity and involve alteration of ACE2 activity/expression, including age, obesity, and diabetes, contribute to increased risk of acute renal dysfunction (Fanelli et al., 2020), making the establishment of causal relationships and the examination of a role for ACE2 difficult in this context.

The Reproductive System

ACE2 in Reproductive Diseases.

Although ACE1, AngII, AT1R, AT2R, ACE2, Ang1–7, and Ang1–7 receptors are all involved in the reproductive physiology in both males and females, ACE2 was found to play an important role in the testis, with expression specifically enriched in Leydig cells and cells in the seminiferous tubules (Donoghue et al., 2000 Pan et al., 2013). In males, ACE1/AngII and ACE2/Ang1–7 balance drives optimal male fertility, including steroidogenesis, epididymal contractility, and sperm cell function. Males with impaired spermatogenesis do not have detected levels of Ang1–7 or Ang1–7 receptors in seminiferous tubules compared with healthy males (Reis et al., 2010). Also, lower levels of ACE2, Ang1–7, and Ang1–7 receptors in the testis are correlated to severe impairment in spermatogenesis and lower testicular weight (Reis et al., 2010). ACE2 is specifically implicated in spermatogenesis because of its presence in Leydig cells, which produce sex hormones. Moreover, ACE2 is being increasingly considered as a therapeutic mechanism to improve male fertility (Pan et al., 2013). In addition, the expression of ACE2, Ang1–7, and Ang1–7 receptors in human ovaries is directly affected by gonadotropin, suggesting an important role in ovarian physiology, follicular development, and ovulation (Pereira et al., 2009 Reis et al., 2011). Collectively, disruption in ACE2 in male and female reproductive cells may contribute to reproductive impairments and polycystic ovarian syndrome, respectively. Furthermore, its presence within the testes makes them vulnerable to possible damage by SARS-CoV-2.

ACE2 and Reproductive Impairment in Patients with SARS-CoV-2.

SARS-CoV–induced viral testicular tissue damage has been reported. SARS-CoV leads to germ destruction, loss of spermatozoon in seminiferous tubules, and leukocyte infiltration (Xu et al., 2006). The possibility of viral orchitis after SARS-CoV-2 infection potentially leading to testicular damage and infertility remains high (Cardona Maya et al., 2020). As such, it is recommended that proper care is given to male patients with COVID-19 to guard against possible orchitis, and follow-up in males recovering from COVID-19 is equally important. Furthermore, exacerbation of polycystic ovarian syndrome symptoms in females may also need proper follow-up.

The Digestive System

ACE2 and the Digestive System.

Although ACE2 is a key component of the RAS system, it has an independent role in the gut. ACE2 is necessary for the expression of transporters of neutral amino acids in the small intestine (Hashimoto et al., 2012 Perlot and Penninger, 2013). Furthermore, ACE2 has been shown to confer protection against colitis. This was also directly correlated with a reduced uptake of tryptophan, one of the main functions of ACE2 in the gut. A reduction in tryptophan results in a reduction in the mammalian target of rapamycin pathway in the small intestine. This impairs the expression of antimicrobial peptides, leading to an altered intestinal microbiome and thus an increased susceptibility to colitis (Perlot and Penninger, 2013). Hence, ACE2 has a key role in maintaining a protective healthy biome in the gut, and its function involves a mammalian target of rapamycin pathway.

ACE2 and Digestive Impairment in Patients with SARS-CoV-2.

Reports from Wuhan highlighted the enteric involvement of COVID-19: 2%–10% of patients with COVID-19 initially presented to hospitals with gastrointestinal symptoms, such as diarrhea, abdominal pain, and vomiting (Chen et al., 2020b Yang et al., 2020). Patients expressed symptoms 1 to 2 days prior to the development of respiratory symptoms and fever. Moreover, patients admitted to intensive care units were more likely to report abdominal pain and anorexia (Wang et al., 2020a). The intestine represents one of the biggest immune system compartments and is highly vascularized and innervated. Previous studies reported that the gut impacts the viral immune and neuronal systems and also affects pulmonary function (Mowat and Agace, 2014 Shenoy et al., 2014). In fact, the progression of intestinal infection, inflammation, and subsequent live virus emergence in the lungs suggests the development of sequential respiratory infection. Intestinal cells are highly permissive to coronaviruses regardless of the stage of cell differentiation, suggesting a high sensitivity of the intestinal epithelial cells to these viruses (Cinatl et al., 2004 Zhou et al., 2017). Furthermore, ACE2 is expressed in the oral cavity, particularly in epithelial cells of the tongue. Also, lymphocytes in the oral cavity express ACE2 receptors in a similar density as those in the lungs (Xu et al., 2020). The stomach and the esophagus have not yet displayed infected epithelial cells. SARS-CoV-2 infection could have been initiated by eating raw food from the Wuhan market, the center of coronavirus outbreak. Yet, ACE2 being located in the basolateral membrane of the oral mucosa makes the viral uptake via this route less likely to be efficient. However, its presence on the apical surface of the enterocytes and the detection of the virus in stool samples provides a possible explanation and highlights the possibility of fecal-to-oral transmission, as stated by previous studies (Yeo et al., 2020).

The Bile Duct

ACE2 and Biliary Impairment in Patients with SARS-CoV-2.

The possible involvement of hepatotoxicity in patients with COVID-19 is questionable. Liver injury is found to be more prevalent in severe than in mild cases of COVID-19, and significant increases in liver enzymes and bilirubin were reported in 20%–37% of cases (Chen et al., 2020b Huang et al., 2020). Taking this into account, it is not clear whether liver injury in patients with COVID-19 is mainly due to direct entry of the virus into liver cells or induced by certain drugs after treatment. ACE2 has not been detected on hepatocytes but rather on cholangiocytes of the bile ducts (Hamming et al., 2004). However, the role of ACE2 in liver disease is of great importance, as it participates in the regulation of liver inflammation, tissue remodeling, and liver fibrosis (Rajapaksha et al., 2019). The protective pathway of ACE2 is thought to be mediated through a significant reduction in AngII-induced fibrosis, TGF-β, and NADPH oxidase (Rajapaksha et al., 2019). A study by Chai et al. identified cholangiocyte-specific expression of ACE2, which could have been a possible route of SARS-CoV-2 entry and hence could lead to profound liver toxicity in patients with COVID-19 (X. Chai et al., preprint, DOI: Moreover, ACE2 expression and activity were shown to be induced as a response to chronic liver injury (Osterreicher et al., 2009). As such, this begs the question of whether patients with chronic hepatic inflammatory conditions might be at a higher risk of SARS-CoV-2–induced liver damage. Subsequently proper monitoring of liver function should be undertaken to guard against liver injury in patients with COVID-19.

The Nervous System

ACE2 and the Brain.

ACE2 is also found in the brain (Xu et al., 2011). The highest activity of ACE2 in the central nervous system is detected in the hypothalamus (Xu et al., 2011). AngII has been shown to reduce levels of ACE2 expression in cerebral and medullary astrocytes, although the exact role is still elusive. Conversely, ACE2 overexpression in the paraventricular nucleus has been shown to directly impede AngII-induced hypertension (Sriramula et al., 2011). As such, aside from the local neurogenic effects, even within the brain, ACE2 can impart cardioprotective roles.

ACE2 and Central Nervous System Symptoms in Patients with SARS-CoV-2.

Of the most common atypical symptoms in patients with COVID-19 are the neurologic signs, which were present in 45.5% of patients with severe cases (L. Mao et al., preprint, DOI: In this study, headache (13.1%), dizziness (16.8%), and impaired consciousness (14.8%) were the most common signs of viral neuroinvasion. Other studies also reported the prevalence of neurologic symptoms as well (Baig et al., 2020 Chen et al., 2020b Wang et al., 2020a). Moreover, a new COVID-19 case was reported to have altered mental status that developed into hemorrhagic necrotizing encephalopathy (Poyiadji et al., 2020). Moreover, a sudden loss of smell and/or taste is common in patients infected with SARS-CoV-2, possibly occurring without any other typical symptoms, which highlights central nervous system involvement as a COVID-19 manifestation (Gautier and Ravussin, 2020). Previous studies detected SARS-CoV in the brain. The virus was administered via the intranasal route, where it enters through the olfactory neurons and heavily infects several areas of the brain, including piriform and infralimbic cortices, basal ganglia, and midbrain with first- or second-order connections with the olfactory bulb (Netland et al., 2008). Other sites, including those in the brainstem, were shown to have been possibly infected via the oral route. In this study, SARS-CoV infection induced neuronal death in absence of encephalitis in mice. Moreover, SARS-CoV is thought to invade the peripheral nerve terminals and then get access to the central nervous system via the synapse-connected route (Li et al., 2012, 2013). More studies have documented the neuroinvasion of other coronaviruses, in which it was shown to spread through peripheral nerves into the medullary neurons involved in the peristaltic movement of the digestive system, leading to vomiting (Li et al., 2020c). The transfer between nerves could be through clathrin-mediated endocytosis/exocytosis pathway (Li et al., 2013). As for SARS-CoV-2, the latency period of the virus could be sufficient for neuroinvasion. Besides, severe development of COVID-19 could be linked to neuroinvasion and subsequent dysfunction in the cardiorespiratory center in the brainstem, as reported elsewhere (Netland et al., 2008). On the other hand, in addition to viral entry to neurons via ACE2 receptors, brain ischemia could also take place as a result of downregulation of ACE2 expression (Simões E Silva et al., 2017). As such, care should be taken to guide toward proper treatment and prevention of possible neuroinvasion of SARS-CoV-2–induced cardiorespiratory failure (Li et al., 2020c). Altered consciousness could be as devastating as respiratory distress syndrome.

ACE inhibitors and angiotensin receptor blockers may increase the risk of severe COVID-19, paper suggests

James Diaz, MD, MHA, MPH & TM, Dr PH, Professor and Head of Environmental Health Sciences at LSU Health New Orleans School of Public Health, has proposed a possible explanation for the severe lung complications being seen in some people diagnosed with COVID-19. The manuscript was published by Oxford University Press online in the Journal of Travel Medicine.

The SARS beta coronaviruses, SARS-CoV, which caused the SARS (Severe Acute Respiratory Syndrome) outbreak in 2003 and the new SARS-CoV-2, which causes COVID-19, bind to angiotensin converting enzyme 2 (ACE2) receptors in the lower respiratory tracts of infected patients to gain entry into the lungs. Viral pneumonia and potentially fatal respiratory failure may result in susceptible persons after 10-14 days.

"Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) are highly recommended medications for patients with cardiovascular diseases including heart attacks, high blood pressure, diabetes and chronic kidney disease to name a few," notes Dr. Diaz. "Many of those who develop these diseases are older adults. They are prescribed these medications and take them every day."

Research in experimental models has shown an increase in the number of ACE2 receptors in the cardiopulmonary circulation after intravenous infusions of ACE inhibitors.

"Since patients treated with ACEIs and ARBS will have increased numbers of ACE2 receptors in their lungs for coronavirus S proteins to bind to, they may be at increased risk of severe disease outcomes due to SARS-CoV-2infections," explains Diaz.

Diaz writes, this hypothesis is supported by a recent descriptive analysis of 1,099 patients with laboratory-confirmed COVID-19 infections treated in China during the reporting period, December 11, 2019, to January 29, 2020. This study reported more severe disease outcomes in patients with hypertension, coronary artery disease, diabetes and chronic renal disease. All patients with the diagnoses noted met the recommended indications for treatment with ACEIs or ARBs. Diaz says that two mechanisms may protect children from COVID-19 infections -- cross-protective antibodies from multiple upper respiratory tract infections caused by the common cold-causing alpha coronaviruses, and fewer ACE2 receptors in their lower respiratory tracts to attract the binding S proteins of the beta coronaviruses.

He recommends future case-control studies in patients with COVID-19 infections to further confirm chronic therapy with ACEIs or ARBs may raise the risk for severe outcomes.

In the meantime he cautions, "Patients treated with ACEIs and ARBs for cardiovascular diseases should not stop taking their medicine, but should avoid crowds, mass events, ocean cruises, prolonged air travel, and all persons with respiratory illnesses during the current COVID-19 outbreak in order to reduce their risks of infection."

Immunotargeting of catalase to the pulmonary endothelium alleviates oxidative stress and reduces acute lung transplantation injury

Vascular immunotargeting may facilitate the rapid and specific delivery of therapeutic agents to endothelial cells. We investigated whether targeting of an antioxidant enzyme, catalase, to the pulmonary endothelium alleviates oxidative stress in an in vivo model of lung transplantation. Intravenously injected enzymes, conjugated with an antibody to platelet-endothelial cell adhesion molecule-1, accumulate in the pulmonary vasculature and retain their activity during prolonged cold storage and transplantation. Immunotargeting of catalase to donor rats augments the antioxidant capacity of the pulmonary endothelium, reduces oxidative stress, ameliorates ischemia-reperfusion injury, prolongs the acceptable cold ischemia period of lung grafts, and improves the function of transplanted lung grafts. These findings validate the therapeutic potential of vascular immunotargeting as a drug delivery strategy to reduce endothelial injury. Potential applications of this strategy include improving the outcome of clinical lung transplantation and treating a wide variety of endothelial disorders.