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 2131 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 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.

In primary hyperparathyroidism, an increased risk of developing the cardiovascular disease may exist due to increased activity of the renin-angiotensin-aldosterone system. The aim of this study was to evaluate the relationship between parathyroid hormone and aldosterone in patients with primary hyperparathyroidism. The study included 48 patients with primary hyperparathyroidism and 30 healthy subjects who matched age and gender to the study group. This study was conducted at the Center for Laboratory medicine, Clinical center of Vojvodina, Novi Sad, Serbia. In addition to clinical data and laboratory determination of the concentration of total and ionized calcium, phosphorus, measurements of parathyroid hormone, vitamin D, direct renin, and aldosterone were performed by the method of chemiluminescent technology. Compared to the controls, the study group had statistically significantly higher values of aldosterone ( p = 0.028 ), total calcium ( p = 0.01 ), ionized calcium ( p = 0.003 ) and parathyroid hormone ( P ≤ 0.001 ) Serum aldosterone and parathyroid hormone levels were correlated positively in patients with primary hyperparathyroidism ( r = 0.509 , p < 0.05 ). A statistically significant positive correlation between renin and parathyroid hormone ( r = 0.688 , p < 0.05 ) and renin and calcium ( r = 0.673 , p < 0.05 ) was determined in hyperparathyroid patients. In multivariate regression analysis, the strongest predictive variable of aldosterone secretion was parathyroid hormone ( p = 0.011 ). An independent relationship between parathyroid hormone and aldosterone in patients with primary hyperparathyroidism and the correlation between renin and parathyroid hormone as well as with calcium indicate not only the direct but also the indirect associations between parathyroid hormone and aldosterone in primary hyperparathyroidism. These findings may represent another possible model of renin-angiotensin-aldosterone-induced organ damage.

Backgrounds and Objectives: Obstructive sleep apnea (OSA) is associated with increased morbidity and mortality. OSA is an independent risk factor for many different conditions, especially cardiovascular diseases. The purpose of this study was to ascertain the comorbidity profile of non-obese patients with newly diagnosed OSA and evaluate the risk for cardiovascular disease and mortality. The present study also aimed to establish predictors for OSA severity. Materials and Methods: This study included 138 newly diagnosed patients who underwent polysomnographic analysis. The 10-year risk for cardiovascular disease was assessed using a newly validated prediction model: Systematic Coronary Risk Evaluation (SCORE-2). In addition, the Charlson Comorbidity Index (CCI) was assessed as a widely-used example of a mortality comorbidity index. Results: The study population included 138 patients: 86 males and 52 females. Patients were stratified, according to AHI (apnea/hypopnea index), into four groups: 33 patients had mild OSA (5 ≤ AHI < 15), 33 patients had moderate OSA (15 ≤ AHI < 30), 31 patients had severe OSA (AHI ≥ 30), and 41 individuals had AHI < 5, which were a part of the control group. SCORE-2 increased in line with OSA severity and was higher in OSA groups compared to the control group (H = 29.913; DF = 3; p < 0.001). Charlson Index was significantly higher in OSA patients compared to controls (p = 0.001), with a higher prevalence of total comorbidities in the OSA group of patients. Furthermore, CCI 10-year survival score was significantly lower in the OSA group, suggesting a shorter survival of those patients with a more severe form of OSA. We also examined the prediction model for OSA severity. Conclusions: Determining the comorbidity profile and estimation of the 10-year risk score of OSA patients could be used to classify these patients into various mortality risk categories and, according to that, provide them with adequate treatment.

Background: Severe food restriction (sFR) due to various psychological, environmental, and economical reasons can have adverse consequences to cardiovascular functioning and health. Less understood are the long-term risks for developing cardiovascular disease after the sFR period has ended. The renin-angiotensin system (RAS) is responsible for regulating blood pressure and we have found that the RAS is chronically upregulated months after the sFR is over and body weight (BW) recovered due to refeeding (sFR-Refed). AIM: Determine the role of the kidney in the chronic up-regulation of the RAS in sFR-Refed rats. Methods: We investigated the long-term consequences of sFR after refeeding on kidney structure and function in sFR-Refed rats. Female Fischer rats (3-months-old) were maintained on normal chow (Ctrl) ad libitum or a 60% caloric restricted diet for 2 weeks. Thereafter, all rats received regular chow ad libitum for 3 months. Kidney function was analyzed by precision ultrasound. ACE expression (qPCR) and activity (fluorescent assay) were measured and renal pathology was assessed by H&E staining. Results: After 2 weeks of sFR, rats lost 15% of their initial BW [DFinal/Initial): Ctrl, 1.50±0.80 vs sFR, -15.4±1.1; p<0.001; n=8]. After 3 months of refeeding, there was no detectable difference in BW, blood pressure and heart rate between Ctrl and sFR-Refed groups. However, the renal artery blood flow was reduced by 13% [(mm/s): Ctrl, 255 ± 12 vs sFR-Refed, 199 ± 7.8; p<0.02; n=4-8 Glomeruli size was reduced [(mm): Ctrl, 399 ± 6.2 vs sFR-Refed, 383 ± 3.8; p<0.05; n=9] and renal AT1R mRNA expression was increased by 1.3-fold [(fold of Ctrl): Ctrl, 1.00 ± 0.060 vs sFR-Refed, 1.29 ± 0.040; p<0.005; n=8]. Conclusion: In summary, AT1Rs in the renal cortex are up-regulated under conditions in which kidney structure and function are impaired months after the sFR period has ended and BW is restored to normal levels. These findings suggest increased renal AT1R activity contributes to the long-term renal dysfunction observed in sFR-Refed rats. Further research is needed to understand how women who are subjected either voluntarily (e.g., crash diets) or involuntarily (e.g., very low food security) to periods of inadequate caloric intake could be at increased risk for developing renal disease later in life. AHA: 940246 (AS); IH 1R01HL119380 (KS) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

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.

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.

Objective: Due to their affordability and availability, rodents are the most often employed experimental animals to explore disease mechanisms. Rats and mice's resistance to SARS-CoV-2 infection is impeding a worldwide effort to study the pathogenesis of COVID-19. Our aim was to develop a suitable small animal model of COVID-19 disease to investigate mechanisms of brain and lung inflammation. Methods: Lipopolysaccharide (LPS) was administered intraperitoneally to wildtype (WT) and angiotensin converting enzyme 2 knockout (ACE2KO) mice of the C57BI/6 strain in order to mimic the cytokine storm that results from COVID-19. Since the SARS‐CoV‐2 utilizes human ACE2 as the receptor for entry with subsequent downregulation of ACE2, these mice deficient in ACE2 gene may have a similar mechanism observed in COVID‐19 patients. We selected a minimal dose (500ug/kg) and time period (3h) when several cytokines were elevated to determine the severity of lung injury using a point-counting method on tissue sections stained with hematoxylin and eosin. The data are expressed as a mean percentage of grid points located in the superficial and peribronchial area in up to 20 fields. Congestion, neutrophil infiltration, percentage of peribronchial and superficial intrapulmonary hemorrhage and alveolar space area were all assessed. Results: To see the effect of LPS, WT mice (WT-G) were injected with a minimal dose of LPS (WT LPS-G). We observed a much larger percentage of peribronchial intrapulmonary hemorrhage in WT LPS-G compared to WT-G [(%): WT LPS-G, 12.3±1.8 vs. WT-G, 6.67±1.3; p<0.05; n=5]. Significantly higher percentage of the peribronchial congestion was seen in the LPS group [(%): WT LPS-G, 1.78±0.46 vs. WT-G, 0.770±0.18; p<0.05; n=5]. We implemented this lung histology scoring criteria in ACE2KO mice to see effect of LPS in mice deficient in ACE2 gene. The LPS-injected ACE2KO mice (ACE2KO LPS-G) exhibited a higher percentage of peribronchial intrapulmonary hemorrhage compared to WT-G [(%): ACE2KO LPS-G, 10.6±2.1 vs. WT-G, 6.67±1.3; p<0.05; n=5], peribronchial neutrophil infiltration [(%): ACE2KO LPS-G, 2.50±0.68 vs. WT-G, 0.640±0.54; p<0.05; n=5] and superficial neutrophil infiltration [(%): LPS-G, 2.14±0.59 vs. WT-G, 0.720±0.56; p<0.05; n=5]. These lung pathologies led to 1.6-fold higher lung histopathology scores in the ACE2KO LPS-G compared to WT-G for peribronchial intrapulmonary hemorrhage, 3.9-fold higher scores for peribronchial neutrophil infiltration and 3.0-fold higher scores for superficial neutrophil infiltration. On-going studies are investigating injury in brain since COVID-19 exacerbates brain pathologies. Conclusion: Research on the tissue-specific etiology of the disease will greatly benefit from the creation of this distinctive COVID-19 animal model. This model will facilitate studies investigating diagnostics, prognosis and response to treatments in COVID-19 disease. TL1TR001431 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.