Role of angiotensin II in SARS-CoV-2 pathophysiology in a hamster model of COVID-19

OBJECTIVES/GOALS: Hamsters develop COVID-19 similarly to people because the SARS-CoV-2 spike protein binds with high affinity to hamster ACE2 resulting in host cell entry and replication. Our goal was to establish a hamster model that mirrors the lung and brain pathophysiology observed in COVID-19. METHODS/STUDY POPULATION: Hamsters infected with SARS CoV-2 are sacrificed on day 1 and day 6 postinfection. Lung histopathology scoring model was implemented for assessment all pathological relevant changes in the lungs of infected animals on tissue sections stained with hematoxylin and eosin. To quantify the extent and severity of lung pathology, two scoring systems were used: the first evaluated all relevant changes in the lungs of the infected animals and the second evaluated only the pathology associated with the pulmonary vasculature. Percentage of airway affected, airway severity, bronchiolar epithelial hyperplasia, alveoli affected, alveolar severity, type II pneumocyte hyperplasia and vessels affected were analyzed. Total airway score plus total lung alveolar score give lung histopathology score. RESULTS/ANTICIPATED RESULTS: Compared to the control hamster, the hamsters day 1 postinfection, exhibited a higher total airway score [9.00 ± 1.35 vs. 0.25 ± 0.1;p DISCUSSION/SIGNIFICANCE: Establishing this outstanding small animal model of COVID-19 will facilitate studies investigating diagnostics, prognosis and response to treatment in COVID-19 disease. These studies will provide insights that will complement on-going clinical trials on angiotensin type 1 receptor (AT1R) blockers (ARBs) in COVID-19.

A novel mouse model of COVID-19

OBJECTIVES/GOALS: Rodents are the most widely used experimental animals to study disease mechanisms due to their availability and cost-effectiveness. An international drive to investigate the pathophysiology of COVID-19 is inhibited by the resistance of rats and mice to SARS-CoV-2 infection. Our goal was to establish an appropriate small animal model. METHODS/STUDY POPULATION: To recreate the cytokine storm that is associated with COVID-19, we injected angiotensin converting enzyme 2 knockout (ACE2KO) mice (C57BI/6 strain) with lipopolysaccharide (LPS) intraperitoneally and measured the expression of multiple cytokines as a function of time and LPS dose. We then chose a minimum dose (500ug/kg) and time (3h) when multiple cytokines were elevated to measure lung injury scores using a point-counting technique on tissue sections stained with hematoxylin and eosin. The data are expressed as mean percentage of grid points lying within the peribronchial and superficial area in up to 20 fields. Percentage of peribronchial and superficial intrapulmonary hemorrhage, congestion, neutrophil infiltration and area of alveolar space were all assessed. RESULTS/ANTICIPATED RESULTS: Compared to the wildtype group (WT-G), the LPS-injected ACE2KO mice (LPS-G) exhibited a higher percentage of peribronchial intrapulmonary hemorrhage [(%): LPS-G, 10.56 ± 2.06 vs. WT-G, 5.59 ± 0.53;p DISCUSSION/SIGNIFICANCE: Establishing this novel mouse model of COVID-19 will facilitate studies investigating tissue-specific mechanisms of pathogenesis in this disease. This model can also be used to discover novel therapeutic targets and the design of clinical trials focusing on diagnostics, treatments and outcomes in COVID-19.

Dietary Empagliflozin Regulates Angiotensin Converting Enzyme 2 (ACE2) and Transmembrane Serine Protease 2 (TMPRSS2) in Kidney Cortex of Mice

Persons with type 2 diabetes mellitus (T2D) are at greater risk for poor prognosis, including renal pathology, when infected with Covid‐19 virus. A newer class of medications for T2D is the gliflozin class, including Empagliflozin (EMPA), which inhibits the renal proximal tubule sodium glucose cotransporter, type 2 (SGLT2) resulting in glucosuria and reduction in hyperglycemia. Whether these medications alter susceptibility and responses to Covid‐19, and similar infections, is not known. The angiotensin 2 converting enzyme (ACE2) is the receptor for Covid‐19, while transmembrane serine protease 2 (TMPRSS2) is a proteinase expressed on the surface of epithelial cells and facilitates virus uptake. In general, ACE2 is thought to be renoprotective, as it converts Ang I (Ang 1‐10) to Ang 1‐9 and Ang II (Ang 1‐8) to Ang 1‐7. Ang 1‐8, in particular, is a potent vasoconstrictor and natriferic agent. Our aim was to determine how chronic EMPA affected ACE2 and TMPRSS2 protein levels and ACE2 activity in the kidney cortex of mice. Adult male (M) and female (F) mice of three strains (n = 8/group), i.e., C57Bl6 (C57), Swiss Webster (SW), and Tally Ho/Jng (TH) were fed high‐fat control diet (C, 60% milk‐fat) or EMPA‐containing high‐fat diet (E, 0.01%) for 12 weeks. TH mice are insulin resistant and obese, with a tendency to develop T2D over time. SW are lean and the closest genetically related background strain to TH. EMPA increased glucosuria but not body weight (p = 0.38 for treatment, 3‐way ANOVA);however kidney weight (normalized to body weight) was 2‐22% increased (depending on group) by EMPA (p = 0.002 for treatment). Kidney cortex protein levels of ACE2 and TMPRSS2 were determined by conventional western blotting (to observe band size and antibody specificity) and by dot blotting (to compare all samples on the same blot). Dot blotting revealed significantly higher ACE2 protein in males (p < 0.0001) relative to females and in the TH and SW strains (p < 0.0001), relative to C57. There was a significant sex‐by‐strain interaction in that the TH females were not significantly lower than TH males. Similarly, cortical TMPRSS2 was lower in females (p < 0.0001) and in C57 mice (p < 0.0001), but also reduced by EMPA (p = 0.030). ACE2 activity was determined in 10 μg kidney cortex by calculating the slope (over 2 hours) of the difference in fluorescence generated in the presence of the ACE2 substrate (Mca‐YVADAPK(Dnp)‐OH) with the addition of the ACE2‐specific antagonist (MLN) minus fluorescence with addition of both captopril (ACE antagonist) plus MLN. Slopes were: (RFU/min, mean ± sem): MC57C‐ 19.9 ± 5.5;MC57E‐ 31.8 ± 5.2;MSWC‐ 20.2 ± 1.9;MSWE‐ 25.7 ± 3.6;MTHC‐ 18.8 ± 3.6;MTHE‐ 34.7 ± 11.3;FC57C‐ 4.7 ± 1.2;FC57E‐ 9.7 ± 3.4;FSWC‐ 4.9 ± 1.2;FSWE‐ 4.4 ± 1.8;FTHC‐ 9.0 ± 1.8;and FTHE‐ 16.2 ± 2.3. Three‐way ANOVA revealed significant effects of both sex (p < 0.0001) and treatment (p = 0.0052) in that Empagliflozin and male sex increased the slope (activity). Overall, male sex was the greatest determinant of differences in ACE2 protein and activity and TMPRSS2 protein. EMPA increased ACE2 activity, but reduced TMPRSS2 protein levels. In general, sex differences were attenuated in the TH strain. There is the potential for these relative increases to play a role in increased sensitivity of male, obese, and/or T2D subjects to pathology associated with Covid‐19;however, the beneficial effect of enhanced ACE2 to reduce Ang II may also provide countering protection.

Brain pathophysiology in SARS-CoV-2 disease

OBJECTIVES/GOALS: The SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus-2), which underlies the current COVID-19 pandemic, among other tissues, also targets the central nervous system (CNS). The goal of this study is to investigate mechanisms of neuroinflammation in Lipopolysaccharides (LPS)-treated mouse model and SARS-CoV-2-infected hamsters. METHODS/STUDY POPULATION: In this research I will assay vascular reactivity of cerebral vessels to assess vascular dysfunction within the microcirculation. I will determine expression of proinflammatory cytokines, coagulation factors and AT1 receptors (AT1R) in isolated microvessels from the circle of Willis to assess inflammation, thrombosis and RAS activity in the microvasculature. LPS and SARS-CoV-2, are both associated with coagulopathies and because of that I will measure concentration of PAI-1, von Willebrand Factor, thrombin and D-dimer to assess the thrombotic pathway in the circulation. Histology and immunohistochemistry will assess immune cell type infiltration into the brain parenchyma, microglia activation and severity of neuroinflammation and neural injury. RESULTS/ANTICIPATED RESULTS: We hypothesize that under conditions of reduced ACE2 (e.g., SARS-CoV-2 infection), AT1R activity is upregulated in the microvasculature. In the presence of an inflammatory insult, these AT1Rs promote endothelialitis and immunothrombosis through pro-thrombotic pathways and pro-inflammatory cytokine production leading to endothelial dysfunction in the microvasculature, blood brain barrier (BBB) injury, deficits in cognition and increased anxiety. We will test this hypothesis through 2 aims: Aim 1: Determine the role of the pro-injury arm of the RAS in the pathophysiology of the brain in animal models of neuroinflammation and COVID-19. Aim 1: Determine the role of the protective arm of the RAS in the pathophysiology of the brain in animal models of neuroinflammation and COVID-19. DISCUSSION/SIGNIFICANCE: This study will provide insights that will complement on-going clinical trials on angiotensin type 1 receptor (AT1R) blockers (ARBs) in COVID-19. This research is a necessary first step in understanding mechanisms of brain pathogenesis that can set the groundwork for future studies of more complex models of disease.

Male bias in ACE2 basic science research: missed opportunity for discovery in the time of COVID-19.

Throughout the world, including the United States, men have worse outcomes from COVID-19 than women. SARS-CoV-2, the causative virus of the COVID-19 pandemic, uses angiotensin-converting enzyme 2 (ACE2) to gain cellular entry. ACE2 is a member of the renin-angiotensin system (RAS) and plays an important role in counteracting the harmful effects mediated by the angiotensin type 1 receptor. Therefore, we conducted Ovid MEDLINE and Embase database searches of basic science studies investigating the impact of the biological variable of sex on ACE2 expression and regulation from 2000, the year ACE2 was discovered, through December 31, 2020. Out of 2,131 publications, we identified 853 original research articles on ACE2 conducted in primary cells, tissues, and/or whole mammals excluding humans. The majority (68.7%) of these studies that cited the sex of the animal were conducted in males, while 11.2% were conducted solely in females; 9.26% compared ACE2 between the sexes, while 10.8% did not report the sex of the animals used. General findings are that sex differences are tissue-specific and when present, are dependent upon gonadal state. Renal, cardiac, and adipose ACE2 is increased in both sexes under experimental conditions that model co-morbidities associated with worse COVID-19 outcomes including hypertension, obesity, and renal and cardiovascular diseases; however, ACE2 protein was generally higher in the males. Studies in Ace2 knockout mice indicate ACE2 plays a greater role in protecting the female from developing hypertension than the male. Studying the biological variable of sex in ACE2 research provides an opportunity for discovery in conditions involving RAS dysfunction and will shed light on sex differences in COVID-19 severity.

Sex-Specific Modulation of Blood Pressure and the Renin-Angiotensin System by ACE (Angiotensin-Converting Enzyme) 2.

We showed ACE (angiotensin-converting enzyme) 2 is higher in the kidney of male compared with female mice. To further investigate this sex difference, we examined the role of ACE2 in Ang-[1-8] (angiotensin [1-8])-induced hypertension and regulation of the renin-angiotensin system in the kidney of WT (wild type) and Ace2 KO (knockout) mice. Mean arterial pressure rose faster in WT male than WT female mice after Ang-[1-8] infusion. This sex difference was attenuated in ACE2 KO mice. Ang-[1-8] infusion reduced glomerular AT1R (angiotensin type 1 receptor) binding in WT female mice by 30%, and deletion of Ace2 abolished this effect. In contrast, Ang-[1-8] infusion increased glomerular AT1R binding in WT male mice by 1.2-fold, and this effect of Ang-[1-8] persisted in Ace2 KO male mice (1.3-fold). ACE2 also had an effect on renal protein expression of the neutral endopeptidase NEP (neprilysin), the enzyme that catabolizes Ang-[1-10] (angiotensin [1-10]), the precursor of Ang-[1-8]. Ang-[1-8] infusion downregulated NEP protein expression by 20% in WT male, whereas there was a slight increase in NEP expression in WT female mice. Deletion of Ace2 resulted in lowered NEP expression after Ang-[1-8] infusion in both sexes. These findings suggest sex-specific ACE2 regulation of the renin-angiotensin system contributes to female protection from Ang-[1-8]-induced hypertension. These findings have ramifications for the current coronavirus disease 2019 (COVID-19) pandemic, especially in hypertension since ACE2 is the SARS-CoV-2 receptor and hypertension is a major risk factor for poor outcomes.

Biological sex impacts COVID-19 outcomes.

The current novel coronavirus disease 2019 (COVID-19) pandemic is revealing profound differences between men and women in disease outcomes worldwide. In the United States, there has been inconsistent reporting and analyses of male-female differences in COVID-19 cases, hospitalizations, and deaths. We seek to raise awareness about the male-biased severe outcomes from COVID-19, highlighting the mechanistic differences including in the expression and activity of angiotensin-converting enzyme 2 (ACE2) as well as in antiviral immunity. We also highlight how sex differences in comorbidities, which can be associated with both age and race, impact male-biased outcomes from COVID-19.

Immediate impact of the COVID-19 pandemic on CTSA TL1 and KL2 training and career development.

Clinical and Translational Science Award (CTSA) TL1 trainees and KL2 scholars were surveyed to determine the immediate impact of the COVID-19 pandemic on training and career development. The most negative impact was lack of access to research facilities, clinics, and human subjects, plus for KL2 scholars lack of access to team members and need for homeschooling. TL1 trainees reported having more time to think and write. Common strategies to maintain research productivity involved time management, virtual connections with colleagues, and shifting to research activities not requiring laboratory/clinic settings. Strategies for mitigating the impact of the COVID-19 pandemic on training and career development are described.