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Principal Investigator: Hans Ackerman
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2020
Award: $44,930
Funding agency: National Institute of Allergy and Infectious Diseases
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). Two common medications- ACE inhibitors (ACEi) and angiotensin receptor blockers (ARB)- may potentially increase the expression of ACE2 in 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 164 C57Bl6/J male and female mice from Jackson laboratory. 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 study, a small pilot study was conducted with 10 mice to determine the average daily water consumption by body weight for male and female mice. The main study has 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. As of this report, all 164 mice have been sacrificed and tissue collection has been completed.
Protein abundance is being assessed by Western Blot and ELISA. Assay validation and optimization has occurred using standard laboratory mice. Gene expression will be determined by quantitative polymerase chain reaction (qPCR). Tissue expression will be investigated by use of immunohistochemical staining. Neither measurement of gene expression nor tissue histology has commenced at this point.
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