Understanding spatial and temporal dynamics of the SARS-CoV-2 receptor, ACE2

National Institute Of Allergy And Infectious Diseases

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Abstract

This project is part of NIAID intramural basic science research response to the COVID-19 pandemic. The SARS-CoV-2 coronavirus (which causes COVID-19) enters cells through binding with angiotensin converting enzyme 2 (ACE2). It is currently unknown whether two common blood pressure medications- ACE inhibitors (ACEi) and angiotensin receptor blockers (ARB)- may potentially increase the expression of ACE2 in tissues commonly impacted during SARS-CoV-2 infection, including the lungs, small intestines, and vasculature, thereby increasing the abundance of the molecular target ACE2 for SARS-CoV-2 to bind. Neither ACE inhibitors (ACEi) nor ARB directly bind to ACE2, but they may modulate ACE2 expression indirectly by changing the circulating levels of angiotensin-II, the major substrate for ACE2. These medications are also commonly used among patients deemed high-risk for complications of COVID-19 due to pre-existing hypertension. Preclinical data in rats shows that monotherapy with ACEi or ARB is sufficient to increase Ace2 in cardiac tissue, while combination therapy does not; the effect on Ace2 expression in other tissues relevant to COVID-19, such as lung alveolar cells and the vasculature, has not been studied. Determining whether these medications increase, decrease, or have no effect on ACE2 expression is of pressing concern, as well as whether combination therapy might be advantageous in higher-risk individuals. Therefore, the goal of this pre-clinical study is to compare the impact of ACEi and angiotensin receptor blocker (ARB) combination therapy versus monotherapy, and discontinuation of these therapies, on the temporal dynamics and tissue distribution of Ace2 in male and female C57Bl/6J mice. The findings of this study will help illuminate how use of these two common drug classes changes expression of the SARS-CoV-2 target receptor Ace2. This project was funded in May 2020 and work began in June 2020. Significant materials included the use of mice over two cohorts- the first cohort of 164 C57Bl6/J male and female mice, and second cohort of 80 male and female C57Bl6/J mice- from Jackson laboratory. The first cohort study was conducted from June through August of 2020 and was performed during the 2020 fiscal year; the second cohort was conducted from November 2020 through January of 2021 and was performed wholly during the 2021 fiscal year. The first cohort was designed with temporal endpoints- mice were collected every three days to assess whether distinct short term changes in Ace2 expression could be correlated with lisinopril treatment, losartan treatment, or both against untreated controls. While day-to-day Ace2 levels were variable, by Day 21 of treatment with lisinopril, a trend emerged of higher Ace2 across tissues in both male and female mice. Therefore, the second cohort was designed to test tissue Ace2 levels in a larger sample size of male and female mice specifically after 21 days of treatment with lisinopril, losartan, or both, as well as after 21 days of treatment withdrawal. This second cohort assigned 5 male and 5 female mice to each treatment group (ACEi, ARB, both, or neither) at two timepoints- after 21 days of treatment, and 21 days after cessation of treatment. For both cohorts, Lisinopril and losartan were sourced from the National Institutes of Health Division of Veterinary Resources (NIH/DVR) Pharmacy. Following FDA guidelines, drinking water formulations were made for each drug based on mouse body weight and average daily water consumption. Standard mouse drinking water and bottles from the vivarium were used to formulate the treated water solutions and deliver the drugs. Prior to starting the first cohort a small pilot study was conducted with 10 mice to determine the average daily water consumption by body weight for male and female mice. This study overall had two aims- 1) investigating whether ACE2 expression changes following ACEi or ARB monotherapy and combination therapy; and 2) measuring whether ACE2 expression changes after discontinuation of either monotherapy or combination therapy. Treatment doses were adjusted weekly based on body weight and average volume of consumption to maintain consistent dosing for all mice. At pre-determined timepoints, mice were euthanized under isoflurane anesthesia. Blood was collected, and mice were perfused with cold phosphate buffered saline to remove blood from tissues. Tissue was collected and stored for future processing. Tissue Ace2 was measured in total protein that was extracted from lung, kidney, brain, and small intestine tissue from each mouse. Tissue extraction of protein, and RNA extraction, were both performed in part with use of a Precellys Evolution Cryo (Bertin Instruments). Tissue abundance was measured by ELISA (Abcam) on a plate reader (PerkinElmer) and normalized to total tissue protein measured by BCA assay. RNA was extracted from flash-frozen pieces of perfused lung, kidney, brain, and small intestine. Ace2 gene expression was measured by droplet digital PCR (Bio-Rad QX200 system). Whole blood in EDTA was spun to isolate plasma. Ace2 protein was measured in plasma by ELISA. Histological profiling of tissue Ace2 in the small intestine was performed by immunohistochemistry (IHC) (NIAID Comparative Medicine Branch). As of this report, all mice from both cohorts have been sacrificed and tissue collection has been completed. Tissue processing to extract protein and RNA from all stored tissue has been completed. IHC staining and analysis of small intestine tissue preserved in formalin has been completed. From the first cohort, this study determined that Ace2 protein levels varied significantly by tissue type, with small intestine having the highest abundance of Ace2. Tissue Ace2 did not differ significantly by sex. Within the small sample size at each time point, a trend towards increased tissue Ace2 following lisinopril treatment was observed, necessitating the second cohort to further explore these observations. The major finding from the second cohort (completed entirely within fiscal year 2021) is that Ace2 abundance increased across tissues in mice after 21 days of treatment with lisinopril compared to control treated mice, but that this increase was prevented by combination treatment with losartan. In losartan-only treated mice, Ace2 increased in the small intestine only. In contrast to protein abundance, Ace2 mRNA was not elevated in small intestine tissue from lisinopril or losartan treated mice compared to control; instead, combination treated mice showed significantly decreased Ace2 mRNA compared to control or monotherapy treated mice. Following 21 days of treatment cessation, no tissue differences in Ace2 remained between any treatment group, indicating that the differences at day 21 were dependent on active treatment and could be reversed by cessation. Lastly, Ace2 protein in plasma was not significantly different between any treatment group at day 21, indicating that plasma Ace2 is not a valid biomarker for detecting changes in tissue Ace2. As of August 2021, all experimental data collection and measurement has been completed for this project. The major findings were originally disseminated at the Experimental Biology 2021 Virtual Conference in April 2021, and are currently being finalized as a manuscript for immediate submission to the journal Vascular Pharmacology for consideration in their SARS-CoV-2 special issue. The publication of this manuscript will conclude the research efforts of all associated investigators on this Research Project.