Objective:Adipocytes express renin-angiotensin system (RAS) components, including angiotensinogen (Agt), the precursor to angiotensin II (AngII), and the angiotensin type 1a receptor (AT1aR). The RAS contributes to atherosclerosis, and AngII infusion causes abdominal aortic aneurysm (AAA) formation. We studied effects of adipocyte Agt or AT1aR deficiency on diet-induced atherosclerosis and AngII-induced AAAs in male low-density lipoprotein receptor (Ldlr)-deficient mice. Methods:For atherosclerosis, adipocyte Agt- or AT1aR-deficient Ldlr-deficient mice and littermate controls were fed a Western diet for 3 months. For AAAs, adipocyte Agt- or AT1aR-deficient Ldlr -/- mice and littermate controls were fed a Western diet and infused with AngII (1000 ng/kg/min) for 28 days. Atherosclerosis was quantified en face in the aortic arch by the percent of intimal surface area covered by an atherosclerotic lesion. Serum (cholesterol, triglyceride) and plasma renin activity were quantified at study end point. AAAs were quantified in vivo by ultrasound quantification of abdominal aortic lumen diameters in anesthetized mice or at study end point by quantifying maximal external abdominal aortic diameter and AAA incidence (percent). Systolic blood pressure was quantified in AngII-infused mice by tail cuff plethysmography. Adipocyte size was quantified in tissue sections of white adipose tissue. Male Ldlr -/- mice were fed a standard diet or a Western diet (1 or 3 months) and Agt or AT1aR messenger RNA (mRNA) abundance quantified in periaortic fat (PAF) by reverse transcriptase polymerase chain reaction. Results:There was no effect of adipocyte Agt deficiency on body weight, serum cholesterol concentrations, or atherosclerotic lesions of Western diet-fed Ldlr -/- mice. Adipocyte Agt deficiency had no effect on body weight, serum cholesterol concentrations, abdominal aortic lumen diameter, AAA incidence, or atherosclerosis of AngII-infused Ldlr -/- mice. There was no effect of adipocyte AT1aR deficiency on body weight, serum cholesterol concentrations, or atherosclerotic lesions of Western diet-fed Ldlr -/- mice. Control, but not adipocyte AT1aR-deficient mice lost weight during AngII infusion. The size of adipocytes in white adipose tissue was increased in adipocyte AT1aR-deficient mice with no significant influences on abdominal aortic lumen diameter, AAA incidence, or atherosclerosis of AngII-infused mice. In mice fed a Western diet for 1 or 3 months, Agt mRNA abundance in abdominal PAF increased over time in both diet groups, with modest diet-induced decreases in thoracic PAF Agt mRNA abundance. There was an effect of diet duration on AT1aR mRNA abundance in thoracic PAF, and an interaction between diet and time on abdominal PAF AT1aR mRNA abundance. Conclusions:Adipocyte Agt or AT1aR deficiency had minimal effects on atherosclerosis or AngII-induced AAAs. However, adipocyte AT1aR-deficient mice exhibited increased adipocyte size. Western diet-induced regulation of Agt or AT1aR mRNA abundance in PAF may have contributed to these findings. Clinical Relevance:These studies address the role of components of the renin-angiotensin system (RAS), namely, angiotensinogen and angiotensin type 1a receptors, within adipocytes on experimental disease models of atherosclerosis and abdominal aortic aneurysms. Adipocytes express these RAS components and dysfunctional adipose tissue in humans has been linked both to atherosclerosis and abdominal aortic aneurysm severity. Our findings do not support a major role for these adipocyte RAS components in either disease model. Thus, although drugs targeting the RAS may be beneficial in the treatment of these disorders, they are likely not more effective in the obese population experiencing either of these disorders.

Cigarette smoking is a risk factor for abdominal aortic aneurysms (AAAs), with studies suggesting a higher smoking-related AAA risk in women than men. We examined nicotine's effects on angiotensin II (AngII)-induced AAAs in male and female low-density lipoprotein receptor-deficient (Ldlr-/-) mice. Moreover, we defined effects of gonadectomy (GDX) of both sexes on nicotine-induced regulation of AAAs. Male and female Ldlr-/- mice (8-12 weeks of age) were infused with AngII with or without nicotine. Mice under- went sham or GDX surgeries prior to infusions of AngII and nicotine. In males, one or both testes were removed. AAA incidence, size, severity, and serum indices of nicotine metabolism were quantified. Effects of testosterone or estrogen on abdominal aortic smooth muscle cells (SMCs) were assessed. Nicotine increased aortic rupture in males, with modest effects in females. GDX reduced AAA incidence in male mice but had modest effects in females. Serum ratios of trans-3-hydroxycotinine to cotinine, an index of nicotine metabolism, were higher in females and increased by GDX in both sexes. Co-infusion of nicotine with AngII increased matrix metalloproteinase 2 (MMP2) mRNA in abdominal aortas of males, but not females. Similarly, testosterone increased MMP2 mRNA in male, but not female abdominal aortic SMCs. Testosterone reduced markers of a contractile SMC phenotype in SMCs from males, with no effects of estrogen in females. In conclusion, nicotine augments AngII-induced AAAs to a greater extent in males, with sex differences related to influences of sex hormones on nicotine metabolism, aortic MMP2 expression, and markers of a contractile SMC phenotype.

Serotonin (5-HT) has been implicated in cerebral aneurysm rupture, but it is unclear whether 5-HT plays a role in aortic aneurysm development and rupture, despite well known contractile effects of 5-HT through aortic 5-HT receptors. Abdominal aortic aneurysms (AAAs) induced by angiotensin II (AngII) infusion to mice exhibit periaortic inflammation and are prone to rupture. Periaortic fat (PAF), a potential source of 5-HT through tryptophan hydroxylase 1 (Tph1), has been implicated in AAA development. We quantified mRNA abundance of 5-HT receptors (Htr1b, Htr2a, Htr2b, Htr3a, and Htr7) and Tph1 in thoracic and abdominal aortas and surrounding PAF. Compared with other 5-HT receptors, we detected high levels of serotonin 3 receptor type a (Htr3a) mRNA in the abdominal aortas and abdominal PAF. Tph1 mRNA and 5-HT immunostaining were detected in aortas and PAF, with 5-HT levels higher in abdominal than thoracic PAF, and higher in epididymal white than interscapular brown fat. AngII infusion facilitated evoked [3H]5-HT release from thoracic PAF and modestly reduced 5-HT levels in thoracic PAF and brown fat. Based on a high level of Htr3a mRNA in abdominal aortas and PAF, we investigated the development of AngII-induced AAAs when serotonin 3 receptors were pharmacologically antagonized with tropisetron. Tropisetron abrogated abdominal aortic lumen diameters, aneurysm (distal thoracic aneurysm and AAA) incidence, maximal AAA diameters, and aortic weights of AngII-infused male mice. These findings indicate a novel role for serotonin 3 receptor in AAA development, with a potential clinically relevant contribution for PAF as a local source of 5-HT. SIGNIFICANCE STATEMENT: Aortic aneurysms are life-threatening vascular disorders with no effective therapeutics. This study identified antagonism of the serotonin 3 receptor as a potential therapeutic target to reduce the formation and severity of experimentally-induced aneurysms in the thoracic and abdominal aorta. Additionally, periaortic fat was identified as a potential site for serotonin production in the development of aortic aneurysms.

Background We have previously reported that male mice exposed to maternal separation and early weaning (MSEW), a model of early life stress, show sympathetic activation and increased blood pressure in response to a chronic high‐fat diet. The goal of this study was to investigate the contribution of the renin–angiotensin–aldosterone system to the mechanism by which MSEW increases blood pressure and vasomotor sympathetic tone in obese male mice. Methods and Results Mice were exposed to MSEW during postnatal life. Undisturbed litters served as controls. At weaning, both control and MSEW offspring were placed on a low‐fat diet or a high‐fat diet for 20 weeks. Angiotensin peptides in serum were similar in control and MSEW mice regardless of the diet. However, a high‐fat diet induced a similar increase in angiotensinogen levels in serum, renal cortex, liver, and fat in both control and MSEW mice. No evidence of renin–angiotensin system activation was found in adipose tissue and renal cortex. After chronic treatment with enalapril (2.5 mg/kg per day, drinking water, 7 days), an angiotensin‐converting enzyme inhibitor that does not cross the blood–brain barrier, induced a similar reduction in blood pressure in both groups, while the vasomotor sympathetic tone remained increased in obese MSEW mice. In addition, acute boluses of angiotensin II (1, 10, 50 μg/kg s.c.) exerted a similar pressor response in MSEW and control mice before and after enalapril treatment. Conclusions Overall, elevated blood pressure and vasomotor sympathetic tone remained exacerbated in MSEW mice compared with controls after the peripheral inhibition of angiotensin‐converting enzyme, suggesting a mechanism independent of angiotensin II.

The University of Kentucky has required that all researchers and research-eligible individuals complete RCR training every 2 years to ensure there is at least a baseline of RCR training throughout the wider research community. The overall goal is to create a research climate that fosters RCR across the institution for approximately 14,400 researchers and research eligible faculty, staff, and trainees engaged in research or creative work. A systematic data strategy was developed and implemented to identify individuals required to complete the RCR training and included real-time data tracking using data analytic tools and sophisticated data models. The RCR training consists of the completion of online asynchronous modules and an in-person RCR training event where case studies are discussed to apply the learning gained from the online modules and prepare participants to practice ethical decision-making in future real-world problem-solving. A train-the-trainer approach was implemented to conduct the in-person RCR training of all researchers and research eligible individuals. A library of interactive case scenarios was developed for the mandatory in-person RCR training applicable to various disciplines across the institution. A survey of participants on the RCR training provided empirical data from approximately 12% of participants. Over 72% of respondents indicated the training positively impacted their behavior. An initiative of this magnitude and reach is the broadest of its kind in the United States (US) and the article outlines the procedures and experiences in implementing the project.

Background Novel and comprehensive approaches are needed to address shortcomings in the diversity and inclusiveness of the scientific workforce. In response to this need and informed by multiple programs and data sources, we created the Research Scholars Program (RSP). The RSP is a yearlong program for early-career faculty with an overall objective to overcome barriers to the academic success, retention, progression, and promotion of groups underrepresented in biomedical and behavioral research. The goal of the RSP is to increase research confidence and productivity, build a supportive research community, and reduce isolation by providing personal and group research enrichment to junior faculty through professional development, mentorship, and networking. Methods We adapted evidence-based approaches for our institutional context and vetted the RSP across our campus. The resulting RSP consists of three main elements: (1) five levels of Mosaic Mentorship; (2) group and tailored professional development programming; and (3) scientific and social networking. To determine the potential of the RSP to improve research confidence critical to success, we used a modified shortened version of the Clinical Research Appraisal Inventory (CRAI-12) to assess participants’ confidence in performing a variety of research tasks before and after program participation. We collected information about retention, promotion, and grants submitted and awarded. Additionally, we conducted semi-structured exit interviews with each scholar after program participation to identify programmatic strengths and areas for improvement. Data for Cohorts 1 and 2 ( N = 12) were analyzed. Results Our assessment finds, with one exception, increasing confidence in participants’ research skills across all items, ranging from 0.4 (4.7%) to 2.6 (40.6%). In their exit interviews, the Research Scholars (RS) described their improved productivity and increased sense of belonging and support from others. Research Scholars noted numerous components of the RSP as strengths, including the Mosaic Mentorship model, professional development programming, and opportunities for both informal and formal interactions. Respondents identified time pressure, a lack of feedback, and unclear expectations of the various mentorship roles as areas in which the program can improve. Conclusion Preliminary findings indicate that the RSP is successful in building the research confidence of underrepresented and disadvantaged early-career faculty. While this report focuses on the development and protocol of the RSP, additional cohorts and data will provide the evidence base to support dissemination as a national model of research professional development. Such programming is critical to ensure sustainable support structures, institutional networks, infrastructure, and resources that will improve discovery and equity through inclusive excellence.

Background: When aortic cells are under stress, such as increased hemodynamic pressure, they adapt to the environment by modifying their functions, allowing the aorta to maintain its strength. To understand the regulation of this adaptive response, we examined transcriptomic and epigenomic programs in aortic smooth muscle cells (SMCs) during the adaptive response to AngII (angiotensin II) infusion and determined its importance in protecting against aortic aneurysm and dissection (AAD). Methods: We performed single-cell RNA sequencing and single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) analyses in a mouse model of sporadic AAD induced by AngII infusion. We also examined the direct effects of YAP (yes-associated protein) on the SMC adaptive response in vitro. The role of YAP in AAD development was further evaluated in AngII-infused mice with SMC-specific Yap deletion. Results: In wild-type mice, AngII infusion increased medial thickness in the thoracic aorta. Single-cell RNA sequencing analysis revealed an adaptive response in thoracic SMCs characterized by upregulated genes with roles in wound healing, elastin and collagen production, proliferation, migration, cytoskeleton organization, cell-matrix focal adhesion, and PI3K-PKB/Akt (phosphoinositide-3-kinase–protein kinase B/Akt) and TGF-β (transforming growth factor beta) signaling. ScATAC-seq analysis showed increased chromatin accessibility at regulatory regions of adaptive genes and revealed the mechanical sensor YAP/transcriptional enhanced associate domains as a top candidate transcription complex driving the expression of these genes (eg, Lox, Col5a2, Tgfb2 ). In cultured human aortic SMCs, cyclic stretch activated YAP, which directly bound to adaptive gene regulatory regions (eg, Lox ) and increased their transcript abundance. SMC-specific Yap deletion in mice compromised this adaptive response in SMCs, leading to an increased AAD incidence. Conclusions: Aortic stress triggers the systemic epigenetic induction of an adaptive response (eg, wound healing, proliferation, matrix organization) in thoracic aortic SMCs that depends on functional biomechanical signal transduction (eg, YAP signaling). Our study highlights the importance of the adaptive response in maintaining aortic homeostasis and preventing AAD in mice.

AngII (angiotensin II) infusion in mice has been used to provide mechanistic insight into human abdominal aortic aneurysms for over 2 decades. This is a technically facile animal model that recapitulates multiple facets of the human disease. Although numerous publications have reported abdominal aortic aneurysms with AngII infusion in mice, there remain many fundamental unanswered questions such as uniformity of describing the pathological characteristics and which cell type is stimulated by AngII to promote abdominal aortic aneurysms. Extrapolation of the findings to provide insight into the human disease has been hindered by the preponderance of studies designed to determine the effects on initiation of abdominal aortic aneurysms, rather than a more clinically relevant scenario of determining efficacy on the established disease. The purpose of this review is to enhance understanding of AngII-induced abdominal aortic pathologies in mice, thereby providing greater insight into the human disease.

Introduction: Ascending thoracic aortic aneurysm (ATAA) progression to dissection (ATAD) is associated with a high risk of mortality. Since the specific molecular and cellular changes leading to dissection are poorly understood, we compared transcriptome profiles of aortic smooth muscle cells (SMCs) between ATAD, ATAA, and controls. Methods: We performed single-cell RNA sequencing (scRNA-seq) of ascending aorta tissues from patients with acute ATAD (dissected and non-dissected areas were collected separately, 3 women, 5 men) and ATAA without dissection (3 women, 4 men), and from control subjects without aortic diseases (transplant donors, 3 women, 5 men). SMCs clusters were analyzed and immunofluorescence staining was performed to confirm expression of key genes. Mice exposed to β-aminopropionitrile monofumarate (BAPN) starting at P21 were used to investigate the changes of mitochondria-related genes during disease progression in undissected mice. Results: Compared with controls, SMCs in ATAA exhibited an adaptive response characterized by upregulation of a subset of focal adhesion and contractile genes and a pro-fibroblast phenotype. These expression changes were decreased in SMCs in ATAD, which instead exhibited significant increases in genes for inflammatory response, glycolysis, senescence and cell death. Additionally, mitochondrial genes including tricarboxylic acid (TCA) cycle genes (e.g. SDHB , FH , and MDH2 ) and mitochondria genome genes (e.g. MT-ND4 , MT-CO2 , and MT-CYB ), exhibited increased expression in ATAAs, and downregulated in ATADs. Immunofluorescence staining of corresponding proteins confirmed some key findings. Consistent with human data, scRNA-seq showed increased mitochondria-related genes in SMCs in BAPN mouse model at P35 when aneurysms form, which were decreased in the proximal aortic tissues after the onset of dissection deaths in surviving mice at P42. Conclusions: Our data suggest a dynamic change from mitochondrial compensation to failure with progression from normal to aortic aneurysms to dissections, which ultimately increases cell death pathways.

Prostate apoptosis response-4 (Par-4) is a tumor suppressor that induces apoptosis in cancer cells. However, the physiological function of Par-4 remains unknown. Here we show that conventional Par-4 knockout (Par-4-/-) mice and adipocyte-specific Par-4 knockout (AKO) mice, but not hepatocyte-specific Par-4 knockout mice, are obese with standard chow diet. Par-4-/- and AKO mice exhibit increased absorption and storage of fat in adipocytes. Mechanistically, Par-4 loss is associated with mdm2 downregulation and activation of p53. We identified complement factor c3 as a p53-regulated gene linked to fat storage in adipocytes. Par-4 re-expression in adipocytes or c3 deletion reversed the obese mouse phenotype. Moreover, obese human subjects showed lower expression of Par-4 relative to lean subjects, and in longitudinal studies, low baseline Par-4 levels denoted an increased risk of developing obesity later in life. These findings indicate that Par-4 suppresses p53 and its target c3 to regulate obesity.

BACKGROUND:Obesity increases the risk for human abdominal aortic aneurysms (AAAs) and enhances Ang II (angiotensin II)-induced AAA formation in C57BL/6J mice. Obesity is also associated with increases in perivascular fat that expresses proinflammatory markers including SAA (serum amyloid A). We previously reported that deficiency of SAA significantly reduces Ang II-induced inflammation and AAA in hyperlipidemic apoE-deficient mice. In this study. we investigated whether adipose tissue-derived SAA plays a role in Ang II-induced AAA in obese C57BL/6J mice. METHODS:The development of AAA was compared between male C57BL/6J mice (wild type), C57BL/6J mice lacking SAA1.1, SAA2.1, and SAA3 (TKO); and TKO mice harboring a doxycycline-inducible, adipocyte-specific SAA1.1 transgene (TKO-Tgfat; SAA expressed only in fat). All mice were fed an obesogenic diet and doxycycline to induce SAA transgene expression and infused with Ang II to induce AAA. RESULTS:In response to Ang II infusion, SAA expression was significantly increased in perivascular fat of obese C57BL/6J mice. Maximal luminal diameters of the abdominal aorta were determined by ultrasound before and after Ang II infusion, which indicated a significant increase in aortic luminal diameters in wild type and TKO-TGfat mice but not in TKO mice. Adipocyte-specific SAA expression was associated with MMP (matrix metalloproteinase) activity and macrophage infiltration in abdominal aortas of Ang II-infused obese mice. CONCLUSIONS:We demonstrate for the first time that SAA deficiency protects obese C57BL/6J mice from Ang II-induced AAA. SAA expression only in adipocytes is sufficient to cause AAA in obese mice infused with Ang II.

Objective: Turner syndrome women (monosomy X) have high risk of aortopathies consistent with a role for sex chromosomes in disease development. We demonstrated that sex chromosomes influence regional development of Ang II (angiotensin II)-induced aortopathies in mice. In this study, we determined if the number of X chromosomes regulates regional development of Ang II-induced aortopathies. Approach and Results: We used females with varying numbers of X chromosomes (XX female mice [XXF] or XO female mice [XOF]) on an C57BL/6J (ascending aortopathies) or low-density lipoprotein receptor deficient (Ldlr(-/-)) background (descending and abdominal aortopathies) compared with XY males (XYM). To induce aortopathies, mice were infused with Ang II. XOF (C57BL/6J) exhibited larger percent increases in ascending aortic lumen diameters than Ang II-infused XXF or XYM. Ang II-infused XOF (Ldlr(-/-)) exhibited similar incidences of thoracic (XOF, 50%; XYM, 71%) and abdominal aortopathies (XOF, 83%; XYM, 71%) as XYM, which were greater than XXF (XXF, 0%). Abdominal aortic lumen diameters and maximal external diameters were similar between XOF and XYM but greater than XXF, and these effects persisted with extended Ang II infusions. Larger aortic lumen diameters, abdominal aortopathy incidence (XXF, 20%; XOF, 75%), and maximal aneurysm diameters (XXF, 1.020.17; XOF, 1.96 +/- 0.32 mm; P=0.027) persisted in ovariectomized Ang II-infused XOF mice. Data from RNA-seq demonstrated that X chromosome genes that escape X-inactivation (histone lysine demethylases Kdm5c and Kdm6a) exhibited lower mRNA abundance in aortas of XOF than XXF (P=0.033 and 0.024, respectively). Conversely, DNA methylation was higher in aortas of XOF than XXF (P=0.038). Conclusions: The absence of a second X chromosome promotes diffuse Ang II-induced aortopathies in females.

Combined neprilysin (NEP) inhibition (sacubitril) and angiotensin type 1 receptor (AT1R) antagonism (valsartan) is used in the treatment of congestive heart failure and is gaining interest for other angiotensin II (AngII)-related cardiovascular diseases. In addition to heart failure, AngII promotes hypertension, atherosclerosis, and abdominal aortic aneurysms (AAAs). Similarly, NEP substrates or products have broad effects on the cardiovascular system. In this study, we examined NEP inhibition (with sacubitril) and AT1R antagonism (with valsartan) alone or in combination on AngII-induced hypertension, atherosclerosis, or AAAs in male low-density lipoprotein receptor-deficient mice. Preliminary studies assessed drug delivery via osmotic minipumps for simultaneous release of sacubitril and/or valsartan with AngII over 28 days. Mice were infused with AngII (1000 ng/kg per minute) in the absence (vehicle) or presence of sacubitril (1, 6, or 9 mg/kg per day), valsartan (0.3, 0.5, 1, 6, or 20 mg/kg per day), or the combination thereof (1 and 0.3, or 9 or 0.5 mg/kg per day of sacubitril and valsartan, respectively). Plasma AngII and renin concentrations increased 4-fold at higher valsartan doses, indicative of removal of AngII negative feedback on renin. Sacubitril doubled plasma AngII concentrations at lower doses (1 mg/kg per day). Valsartan dose-dependently decreased systolic blood pressure, aortic atherosclerosis, and AAAs of AngII-infused mice, whereas sacubitril had no effect on atherosclerosis or AAAs but reduced blood pressure of AngII-infused mice. Combination therapy with sacubitril and valsartan did not provide additive benefits. These results suggest limited effects of combination therapy with NEP inhibition and AT1R antagonism against AngII-induced hypertension, atherosclerosis, or AAAs. SIGNIFICANCE STATEMENT: The combination of valsartan (angiotensin type 1 receptor antagonist) and sacubitril (neprilysin inhibitor) did not provide benefit above valsartan alone on AngII-induced hypertension, atherosclerosis, or abdominal aortic aneurysms in low-density lipoprotein receptor-deficient male mice. These results do not support this drug combination in therapy of these AngII-induced cardiovascular diseases.

Angiotensin converting enzyme 2 (ACE2) is an enzyme that limits activity of the renin-angiotensin system (RAS) and also serves as a receptor for the SARS-CoV-2 Spike (S) protein. Binding of S protein to ACE2 causes internalization which activates local RAS. ACE2 is on the X chromosome and its expression is regulated by sex hormones. In this study, we defined ACE2 mRNA abundance and examined effects of S protein on ACE2 activity and/or angiotensin II (AngII) levels in pivotal tissues (lung, adipose) from male and female mice. In lung, ACE2 mRNA abundance was reduced following gonadectomy (GDX) of male and female mice and was higher in XX than XY mice of the Four Core Genotypes (FCG). Reductions in lung ACE2 mRNA abundance by GDX occurred in XX, but not XY FCG female mice. Lung mRNA abundance of ADAM17 and TMPRSS2, enzymes that shed cell surface ACE2 and facilitate viral cell entry, was reduced by GDX in male but not female mice. For comparison, adipose ACE2 mRNA abundance was higher in female than male mice and higher in XX than XY FCG mice. Adipose ADAM17 mRNA abundance was increased by GDX of male and female mice. S protein reduced ACE2 activity in alveolar type II epithelial cells and 3T3-L1 adipocytes. Administration of S protein to male and female mice increased lung AngII levels and decreased adipose ACE2 activity in male but not female mice. These results demonstrate that sex differences in ACE2 expression levels may impact local RAS following S protein exposures.

Male C57BL/6J mice exposed to maternal separation and early weaning (MSEW), a mouse model of early life stress, display increased blood pressure (BP) and sympathetic activation compared to obese controls when fed a high fat diet (HF). Moreover, HF-fed MSEW males display exacerbated BP responses to the acute stimulation of the adipose afferent reflex (AAR) in epididymal white adipose tissue (eWAT). The aim of this study was to investigate the contribution of endogenous factors that could stimulate fat sensory neurons. MSEW and control (C) mice (n=8/group) were placed on a LF or HF (10% and 60% Kcal from fat, respectively) for 16 weeks. Then, serum obtained by decapitation and adipose tissue samples were collected to measure mRNA and protein expression of 15 factors and receptors known to activate sensory neurons. No differences were found across measurements on LF. Plasma AGT and AngII were decreased in HF-fed MSEW compared to C (AGT: 760±48 vs. 1267±161 ng/ml, p<0.05; AngII; 413±57 vs. 1082±340 pmol/l, p<0.07, Attoquant) and no differences were found in leptin (103±6 vs. 104±4 ng/ml, p<0.87). In eWAT, MSEW and C showed similar AGT (2.1±0.4 vs. 1.9±0.3 ng/ml per g tissue), AngII (1.7±0.2 vs. 2.3±0.5 pg AngII/mg tissue), ACE 1 activity (21.5±1.2 vs. 20.0±0.9 RFU/min/μg protein, p<0.33) and leptin (102.8±6.1 vs. 104.5±6.8 ng/mg of tissue, p<0.87). However, HF-fed MSEW showed increased eWAT mRNA expression of tryptophan hydroxylase 1 (Tph1), the rate limiting enzyme in serotonin (5-HT) synthesis (10.2±2.9 vs. 1.6±0.3 2 -ΔΔct , p<0.03). SERT-Tph1-MAO signaling pathway protein expression was activated, and fat serotonin concentration was also increased in eWAT from obese MSEW mice compared to C (16.58±1.5 vs. 8.5±2.1 ug/mg of tissue, p<0.01). Acute stimulation of eWAT with serotonin (10-6 M, 4 sites, 2 ul/site) tend to increase pressor response in MSEW mice (p<0.066, n=2-3). Unlike in female MSEW mice, our study demonstrates that MSEW does not increase circulating and tissue AGT, Ang II and leptin in male mice. Taken together, these data suggest that increased local serotonin could be endogenously sensitizing the sensory neurons in obese MSEW mice contributing to chronic AAR stimulation, directly via TRPV1 channels, or indirectly, via acid-sensing ion channels.

HomeHypertensionVol. 77, No. 2Connecting Generations of Scientists in the Council on Hypertension Through Harriet Dustan Free AccessReview ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessReview ArticlePDF/EPUBConnecting Generations of Scientists in the Council on Hypertension Through Harriet Dustan Stephanie W. Watts, Barbara T. Alexander, Chris Baylis, Nancy J. Brown, Lisa A. Cassis, Kate M. Denton, Bina Joe, Lilach O. Lerman, Suzanne Oparil, Jane F. Reckelhoff, Kathryn Sandberg, Rhian M. Touyz Stephanie W. WattsStephanie W. Watts Correspondence to: Stephanie W. Watts, Department of Pharmacology and Toxicology, Michigan State University, 1355 Bogue St, Rm B445, E Lansing, MI 48824. Email E-mail Address: [email protected] From the Department of Pharmacology and Toxicology, Michigan State University, East Lansing (S.W.W.) , Barbara T. AlexanderBarbara T. Alexander Department of Physiology (B.A.), University of Mississippi, Jackson , Chris BaylisChris Baylis Department of Physiology and Functional Genomics, University of Florida, Gainesville (C.B.) , Nancy J. BrownNancy J. Brown https://orcid.org/0000-0001-7109-3142 School of Medicine, Yale University, New Haven, CT (N.B.) , Lisa A. CassisLisa A. Cassis https://orcid.org/0000-0002-0962-0578 Department of Pharmacology and Nutritional Sciences, University of Kentucky, Lexington (L.C.) , Kate M. DentonKate M. Denton Cardiovascular Disease Program, Monash University, Melbourne, Australia (K.D.) , Bina JoeBina Joe Department of Physiology and Pharmacology, University of Toledo, OH (B.J.) , Lilach O. LermanLilach O. Lerman https://orcid.org/0000-0002-3271-3887 Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN (L.L.) , Suzanne OparilSuzanne Oparil Division of Cardiovascular Disease, University of Alabama, Birmingham (S.O.) , Jane F. ReckelhoffJane F. Reckelhoff https://orcid.org/0000-0002-6606-4133 Department of Cell and Molecular Biology (J.R.), University of Mississippi, Jackson , Kathryn SandbergKathryn Sandberg Department of Medicine, Georgetown University, Washington, DC (K.S.) , and Rhian M. TouyzRhian M. Touyz Institute of Cardiovascular and Medical Science, University of Glasgow, Scotland (R.M.T.). Originally published4 Jan 2021https://doi.org/10.1161/HYPERTENSIONAHA.120.16623Hypertension. 2021;77:296–307This year has made me think about time and how we connect with one another. Anyone in any part of this world reading this knows that this has been the strangest and most difficult year that we will (hopefully) ever live. How do you keep connected when you can't actually be with one another? Talk directly with someone? Argue science face to face? Online platforms have been magnificent for allowing us to touch one another. The reason they work, limited though they may be, is because there is a foundation of connectedness that already exists.The Council on Hypertension, my scientific home since 1992, has this very strong foundation. This was evidence by the successful 74th conference of the Council on Hypertension, held virtually in September 2020. My immense thanks to our leaders—Dr Curt Sigmund, Dr Karen Griffin, all council leaders, as well as the one and only Susan Kunish of the American Heart Association (AHA)—for being able to make this happen. The organizational work necessary for executing this meeting is something of which I am in awe.This was not the same as the fall meeting we are used to: poster competition for our trainees on the first night of the meeting accompanied by a little wine, food, and friends; oral presentations that challenge what you think and help you learn while you sit before the scientist speaking; poster sessions where you can talk deeply about science with others; the Excellence in Hypertension dinner where we honor those that tread new paths in hypertension research. I would be remiss in not mentioning the Trainee Advocacy Mixer, to which all attendees are invited. I've danced with so many of my colleagues at this event, and I am better for it because we share together a human experience.Though this year's meeting was not what we had hoped for, excellent things still happened. Engaging in the different sessions of this meeting, delving into the poster presentations, and talking with you, my colleagues, online put a smile on my face every day. We could talk about science because we knew this family of scientists—this council—was committed to research, and we could push one another in questions, in asking the so whats of one's research. This foundation is at least one reason why this could work (as well as the awesome organization!).I was privileged to give what is the 13th (!) Harriet Dustan Lecture at this 2020 virtual meeting. Dr Harriet Pearson Dustan, MD (Figure 1), was the first Editor-in-Chief of this journal, published by the AHA. She was also the second woman president of the AHA, as well as the first woman on the Board of Governors of the American Board of Internal Medicine. Dr Ed Frohlich, also a past Editor-in-Chief of this journal, wrote a beautiful memorial for Dr Dustan when she passed away in 1999.1 He describes how important she was to the genesis of this council, arriving at the Research Division of the Cleveland Clinic to work the Dr Irvine H. Page and Dr Arthur C. Corcoran (these two distinguished scientists also have Council awards named after them!). Her work broke ground on some of the mechanisms and antihypertensive medications we now take for granted: the actions of chlorothiazide, the role of sodium and obesity in hypertension to name just a few. Her work and her presence, I would argue, is also part of the foundation of this council.Download figureDownload PowerPointFigure 1. Dr Harriet Pearson Dustan. Photograph provided by S.O.It is thus that I wished to connect those that are a distance in time from Dr Dustan—our current trainees—to those that have been honored with the Dustan Award since its inception. At my request, the Trainee Advocacy Committee (TAC) of the Council on Hypertension asked its group of trainees to pose a series of questions revolving around this profession of hypertension research. What did they really want to learn from this esteemed group of women?These scientists—the Dustan Awardees—were almost unanimous in answering questions posed by the TAC. The Executive Committee of the TAC—Drs Hana Itani, Brandi Wynne, and Guto Montezano—helped me winnow the many questions posed by trainees down to eight. These eight were chosen because they reflected what the trainees as a whole were asking, as well as timely concerns that would be of interest to many.Here, I share both the questions posed by the TAC and the Dustan Awardee responses with you. Before doing this, though, please let me introduce to you the Dustan Awardees (Figures 2 and 3). These women have an unparalleled expertise in hypertension research, a wisdom that is hard fought and well-earned, and a graciousness in sharing for which I am immensely thankful. In the references, I provide a weblink to each of their professional pages where available.2–13 In Figures 1 and 2, the year of their receipt of the Dustan Award is accompanied by their name, current title(s), and photograph. Beneath each photo is either (1) the year they joined the Council on Hypertension (formerly known as the Council of High Blood Pressure Research) or (2) the year they became a Fellow of AHA. Typically, becoming a Fellow requires years of research and engagement in this Council, so most of these scientists have been involved with this Council for far before the date they became a Fellow of AHA.Download figureDownload PowerPointFigure 2. Dustan Awardees of the Council on Hypertension from 2008 to 2013. The far left lists the year the award was received, awardee current title, photograph, and name. Beneath each photograph is the year the awardee became either (1) a member of the council or (2) a Fellow of the American Heart Association (FAHA).Download figureDownload PowerPointFigure 3. Dustan Awardees of the Council on Hypertension from 2014 to 2019. The far left lists the year the award was received, awardee current title, photograph, and name. Beneath each photograph is the year the awardee became either (1) a member of the council or (2) a Fellow of the American Heart Association (FAHA).I've not attributed any one response to a particular awardee, nor a question posed (at the diamond ♦) by a particular trainee. However, some answers may reveal the responder. A bullet (•) indicates the response of one Dustan Awardee. For some questions, Dustan Awardees may not have provided a response; this was completely their choice.What these esteemed scientists share with you is nothing short of remarkable. They've put their immense brain power, as well as their hearts, into the words you next read. Please. Find a big cup of coffee, a comfortable place to sit, and just read.Questions From the TAC, Responded to by Dustan Awardees♦ How Do You Tackle the Overwhelming Concern of Staying Funded?Hard work and perseverance. However, to be honest, it is not easy. A career in the field of academic science is very challenging. To maintain funding is one of the hardest and most stressful components of this career choice. You must have a passion and a great love for this career to make it worthwhile. However, I personally think it is the best profession and career path in the world. Let's be honest, we get paid to do what we love: ask questions, seek answers, mentor, and teach.As a young investigator, I was hampered by working in relatively underfunded areas (pregnancy, aging, and sexual dimorphism), so I tried to compensate by writing grants to multiple agencies. While the National Institutes of Health (NIH) has always been the major support of my research, I have also been supported by AHA, Baxter Extramural Grant Program, the March of Dimes, and several pharmaceutical companies. I also applied to as many intramural sources as were available. As a mature investigator, I branched out into NO and chronic kidney disease research, and while these were better funded areas, the competition was very high. Basically, I resigned myself to developing/writing grant applications almost continually and always striving for overlapping funds. Now that I am retired, a great weight has left me.Fortunately, I have been continually funded by the NIH for close to 2 decades. The key, in my opinion, is to recognize that meritorious science always strikes a chord with peers and funding agencies. To remain meritorious in the face of adversities takes consistency in approach. The business of science is not for the frail at heart but for those who realize that they are tough enough to remain creative for the long haul.I remember that there are other options in life. If this doesn't work out, then something else will turn up. It is not the end of the world. Give it your best shot, but be ready to move on if you need too.I have also looked at other people and thought if they can do it, so can I! Is that arrogant? Perhaps you need a little (arrogance) to succeed.One word: perseverance. I have never been one to get a grant on the first submission; this happens rarely for me. Many times, my ideas are too way out there, so the good thing is that I don't struggle with innovation or significance. Mostly, I struggle with the word mechanism. What I consider mechanism is not necessarily the same as many reviewers, so I constantly have to push myself to delve deeper into areas that are not that interesting to me or outside my realm of comfort to address criticisms around mechanism. I persevere through all this, try my best to rise to the challenge of responding to reviewers. Not a genius by any means, just a stubborn woman who doesn't give up.To stay funded, you have to publish. If you don't publish using funds you were lucky enough to be provided, you will have trouble staying funded for very long.Find a research area that is hot, but not too competitive, and that suits your talents. Get started early and decide on a field of interest. Apply for funding early and often. Practice your writing skills and identify diverse funding sources. Make it a priority to network and advocate for biomedical research.The secret of remaining funded is flexibility, innovation, and collaboration. While focusing on a central research theme, you may need to continuously reinvent your research approaches and platforms and adjust them to cutting-edge scientific developments and unmet needs. You need to build on and keep developing your strengths, so that you can leverage them to address pressing questions in innovative ways. Last, but not least, you need to remain abreast of new developments not only in your own but also in other fields, so you can bridge multidisciplinary areas and build synergies.Funding is fundamental to research and advancement of knowledge. As we know, research is expensive, but importantly, ignorance is more expensive. Hence, ensuring a research portfolio that is sustainable with long-term financial support is critical—a difficult ambition especially during challenging funding times.From my personal experience of 25 years as a researcher, and from gaining insights through my involvement as a member of numerous grant panels, I share the following tips:Submit the highest quality grant application, with robust pilot data. If you feel the grant is not in its best form, delay submission. If you are aware of the limitations and weaknesses, address them before submission because the reviewers will definitely pick these up and your grant will be downgraded. You can't hide what you know as weaknesses.It is very useful to get expert opinion about your grant before it is submitted. Get your colleagues, collaborators, mentors, etc, to critically review your grant before submission. Ask for constructive criticism and honest opinions. It is always better to get your grant rejected by your colleagues than by the grant panel.Ensure you respect the guidelines and objectives of the granting agencies. Grants that are poorly prepared, that do not fulfill the requirements of the specific agency, and that may not align with the mission of the funding agency will not be well received.Ensure you submit your grant to the appropriate funding agency.To maintain continual funding, it is advisable to have at least 2 grants that are staggered. This is not easy, but it is a safety net if one grant is not funded.I submit many grant applications 6–10 every year, not only as Principal Investigator but also as Coinvestigator.In the past 4 years, I have written 2 R01s (original and resubmissions for both), a program project, a Support of Competitive research on sex differences, and a Centers of Biomedical Research Excellence grant, which is a training grant for junior faculty (original and resubmission in phase 1 and just resubmitted in phase 2). Of all of these, only 1 of the R01s was funded and the phase 1 Centers of Biomedical Research Excellence. I'm awaiting study section on the other R01 and the phase 2 Centers of Biomedical Research Excellence.The bottom line is you can't get discouraged if the grants aren't funded with the first submission or even not discussed (and on multiple submissions)! I am very persistant, and I keep resubmitting the grants, making them better each time, until I'm successful. I also believe it is very important to get involved with your professional society and present your work at meetings where people who will review your grants will see it (hopefully, we will get back to that model soon!). I actually enjoy writing research grants because it gives me an opportunity to catch up on the literature and come up with new ideas.It is very important to ask compelling questions. It is also important to tell a compelling story to grant reviewers. It is really important to seek input from those around you to test the importance of the question and your communication. I have found that I know a young investigator will succeed when answering the question becomes a greater driver than getting the grant.♦ What Is Your Opinion on Where Studies in Sex Differences in Hypertension Stand? Has This Progressed in the Way You Hope? If Not, What Needs to Be Done?In terms of the Council on Hypertension and inclusion of research related to sex differences and women's health, we have certainly come a long way since I first joined the AHA in 1998. Jane Reckelhoff, Kathryn Sandberg, Christine Maric, Sandra Davidge, Kate Denton, and Virginia Miller have had a very strong and positive impact on my career and my interest to pursue sex as a biological variable (SABV) in my research questions. Collectively, these women have also made a huge contribution to our understanding of how sex matters in cardiovascular and renal disease. They have also been major players in heightening awareness for the importance of inclusion of sex differences in cardiovascular, renal, and hypertension research. Yet, this role is not just limited to women. Joey Granger has led the charge to increase awareness in the field of women's health research within the Council on Hypertension and beyond by his pursuit to study the pregnancy-specific disease preeclampsia. During his service in leadership roles within our Council, he has strived to make women's health an important component of programming at our annual meeting. Although the importance of SABV may not be the first thought someone has when approaching a new direction, or asking a research-based question, I think that the field of sex differences has advanced. With more and more women joining the ranks of academic and biomedical science, I think the movement to consider sex will continue to grow. The advocates listed above have made a great impact in the field of sex differences and women's health, and clearly, the momentum to move this area of research forward has had a strong impact on NIH. These efforts will continue to facilitate the importance of sex differences research. However, I think it is very important for those of us that work in this field to continue to serve as advocates and encourage others to think about SABV as a standard part of their research questions and experimental design.When I published my first paper on sexual dimorphism and the kidney,14 the most substantial comment I received from one reviewer was "why are you studying females?" This annoyed me, so I rewrote the Introduction and sent a grumpy response to the Editor. The article was accepted, and things have improved enormously since then! The increasing recognition that sex is a critical determinant of disease progression and optimization of treatment has advanced hypertension research and therapies. The requirement by NIH to study both sexes or to justify the use of only one has been a critical turning point for studies on sex differences. Nevertheless, not all investigators are on board; I reviewed a grant about a year ago where the principal investigator explained that he was only working on males because females were too complicated. True of course, but not an acceptable response! While it is clear that more attention is now paid to female physiology/disease, we need to continue advocating for full equality.Great progress has been made in this area in the last 10 years. I still think females are underrepresented in preclinical studies. It's too easy to say it increases the number of groups required—too hard, too expensive.I study sex differences in general in the area of obesity-hypertension and more recently largely in the area of vascular diseases. With the NIH mandate that investigators include both sexes, this will help move this field forward to some extent. However, most investigators do not have significant expertise, or even interest, in understanding the basis of sex differences they may find in their mediator of interest using this approach (ie, inclusion of both sexes). Thus, I think this has helped us, but what it may have hurt is investigators who focus on sex differences as their main area of interest, as now everyone does this, albeit not to much avail or mechanistic insight.Much progress has been done over the past decade in integrating studies in sex differences in hypertension. These include requirements from the NIH to ensure identification of sex-based differences in each study and publication of many papers in that area.There is no doubt that the research community is addressing sex differences in hypertension much better now than in the past.A PubMed search indicated that in the 1950s only 1 to 2 papers were published per year; in the 1980s, this increased to about 60 per year while in 1990, there were roughly 200 papers/year. In 2019, 814 papers on sex differences in hypertension were published.The positive trajectory in published papers indicates the growing recognition of the importance of sex differences in hypertension. I feel we are addressing this more in experimental studies than in clinical studies. Inclusion of women in more clinical studies and trials needs to be emphasized. We have not yet done enough, and as a community, we need to continue to lobby on the importance of addressing sex differences in hypertension. It is really encouraging that funding agencies are stressing sex and gender as factors that need to be included in research.Progress in investigating sex differences in hypertension has been disappointing for a variety of reasons. First, there is a commonly held belief that sex differences in blood pressure and responses to treatment are unimportant and not deserving of further study. Further, the belief of the public and health care community that estrogen is harmful for older persons and should not be studied further has created a major impediment to further research in the area. Another particularly vexing challenge is the controversy regarding the relative benefits and risks of blood pressure lowering in pregnant women. To address these gaps in knowledge about sex differences in the pathogenesis of hypertension and its vascular complications, more support is needed to examine these issues in both animal models and humans. These issues have been discussed in depth in a recent report of the National Heart, Lung, and Blood Institute Working Group on Hypertension.15Awareness of the importance of considering the biological impact of sex is growing within the scientific community. This is apparent when one measures as a function of year, the number of abstracts presented at major scientific meetings that focus on hypertension in the female or on sex differences in hypertension; however, there are still too many publications that do not disaggregate clinical findings by sex, and the majority of basic science research on animal models of hypertension remains focused on the male. Funding agencies should increase the number of requests for applications on female models of hypertension or on sex differences in hypertension. Publishers should require disaggregation of clinical data by sex.Certainly, the fact that SABV is now part of the NIH applications is a plus for expanding sex differences research in general. However, at NIH, there is no follow through on whether any studies are actually done on males and females once the grants are funded. This needs to be changed. There are encouraging data based on the increase in the number of papers that include both sexes or even include females only now though. This suggests that the SABV mandate is being taken seriously by some investigators whom I'm sure are pleasantly surprised that they are finding sex differences in their studies.For hypertension research, the newest guidelines for clinical care (2017) include women as an other group category. This is ironic since women make up >50% of the population, and most women over the age of 70 are hypertensive. Preeclampsia was not even mentioned. We know that the mechanisms of hypertension and the responses to treatment regimens are different in men and women, but the clinical treatment guidelines have failed to recognize this. Thus, we investigators need to raise awareness about the necessity of more research into the mechanisms of hypertension in women at all ages, so that in future clinical guidelines, treatment specific for women is included or at least discussed.The Dustan Award recognizes female investigators. Some women investigators are interested in sex differences in hypertension. There are many important questions to be addressed in the field of hypertension.♦ How Do You Differentiate Yourself From Your Mentor if You Decide to Stay in the Same Institution?I received my PhD, completed my postdoctoral training, and then transitioned to a tenure-track faculty position, all within the same institution, although not all within the same department. You may be surprised to find that Jen Sullivan and Justin Grobe followed a similar pathway. What do we all have in common? I think we all have a passion for what we do coupled to a very strong desire to succeed. Dr John Hall—the Chair of the Department of Physiology at the University of Mississippi Medical Center—always states that luck is a part of success in science. Clearly, I have been very lucky. I lucked out to find myself in a great department with a fabulous postdoctoral mentor, Dr Joey Granger. I lucked out to stay in the same department for my academic career. Yet, my ability to succeed and develop my own independent area of research also involved a lot of hard work and perseverance. My mentor, Dr Joey Granger, not only encouraged me to pursue my own area of independent research as a senior postdoctoral fellow in his lab but he also provided the resources to help me initiate this journey. Another bit of luck also involved my chosen area of research interest, study of how adverse events in early life program increased cardiovascular risk in later life. This area of research was very novel 20 years ago when I embarked on my journey toward independence. With encouragement from Jane Reckelhoff, I included male and female offspring in my very first study, and the study of sex differences has remained a very strong focus within my research. Through a love of giving back to my professional societies by providing service, I greatly expanded my network of connections. Looking back, I think this was also an important part of my career development as these opportunities greatly expanded my ability to meet others. Thus, is it possible stay in one place, become independent from your mentor, and thrive? Yes! However, be prepared to work hard and develop your own area of research. Also, do not forget to reach out to others.You need to have a frank discussion with your mentor on how to do this—if the two of you can agree on areas where you can continue to collaborate and other distinct areas where you can work independently, your path to academic promotion will be clear. If not, it might be worth moving to another institution or shifting fields of interest.In my opinion, the successful development of an academic career is enhanced by experiencing different environments and working with collaborators, some of whom will be specific to certain career stages. Even if a junior researcher can establish a truly independent research career in the same institution as her primary mentor, third parties (eg, grant reviewers), may question that independence.I did this by bringing in new, yet complimentary technology that did not exist in my mentor's laboratory. Along with new technology, I could bring a new established collaborator as a co-principal investigator on my first grant application. As a result, including the novel technological adaptation, the ability of a new investigator to network was a skill that was appreciated by the review panel.An important question, particularly relevant for Australia where the places you can move to are limited. There are only about 40 universities in the whole country. To do this successful, you must have the support of your mentor. If you have this you need to identity a related branch of research that you can clearly make your own, it has to be distinct and in a direction they have agreed not to follow in their own work.This is tough one. I have had many trainees stay on at my institution. The path we have used is to find them an area that is their own, to agree not to have coauthored publications until they achieve tenure and promotion, and then to collaborate thereafter if mutually agreeable. I also ask them to become truly independent, meaning I don't edit all their papers or grants if I am not a coauthor or co-principal investigator. While this may seem cruel to some, it is a true path to independence. These approaches work best if your previous trainee does not become faculty in your same department or college.When you do experiments, you inevitably find unexpected results that lead you in a new direction. A generous mentor will enable you to follow those results where they take you. The mentor must then become a sponsor who introduces you to others. It is important for you to network as well.It is preferable for the mentee to identify a research area continuous with their previous research during training, to ensure accumulating sufficient experience and prolonged track record to be competitive for funding during grant applications. It might be close to the mentor's area but not preferably directly competing. It is helpful for mentees whose mentor diverges away from a research area selected by the trainee or directs them to an area that the mentor does not wish to pursue.At the same time, as long as the mentee needs infrastructure/financial support from the mentor, it is important to have the mentor engaged and interested in this new direction.

Angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor and an enzyme of the renin-angiotensin system (RAS), is on the X chromosome and stimulated by estrogen. Male sex is a risk factor for SARS-CoV-2 severity. Previous investigators demonstrated that the SARs-CoV-2 Spike (S) protein decreases tissue ACE2 by protein internalization or shedding. This study defined sex differences in tissue ACE2 expression and their impact on SARS-CoV-2 S protein regulation of ACE2 activity and AngII levels. Male and female intact or gonadectomized (GDX) low density lipoprotein receptor deficient ( Ldlr -/- ) mice, and Four Core Genotype (FCG) male (XY or XX) or female (XX or XY) mice were fed a Western diet for 4 months. In lung, ACE2 mRNA abundance was similar in male and female mice and reduced by GDX (Male XY intact: 1.04 ± 0.15; Female XX intact: 1.13 ± 0.13; Male XY GDX: 0.11 ± 0.03; Female XX GDX: 0.18 ± 0.04 ΔΔCt; P<0.05). Lungs from XX mice had higher ACE2 mRNA abundance than XY mice regardless of gonadal sex (P<0.05), and GDX reduced ACE2 mRNA abundance in lungs of XX, but not XY females (XX Female GDX: 0.18 ± 0.04; XY Female GDX: 0.38 ± 0.09; P<0.05). In adipose, XX females had higher ACE2 mRNA abundance than XY males (XX female: 5.4 ± 0.7; XY male: 1.0 ± 0.1; P<0.05), regardless of gonadal sex (XY females: 3.3 ± 0.7; XX males: 1.5 ± 0.3; P<0.05). Male XY and female XX Ldlr -/- mice were administered vehicle or SARS-CoV-2 S protein (2 nmol/kg, ip, 3 doses) with tissue harvest six hours later. In lung, AngII levels were increased by S protein in male, but not female mice (Male, vehicle: 12.3 ± 2.3; Male, S protein: 33.6 ± 7.1; Female, vehicle: 16.1 ± 2.0; Female, S protein: 20.2 ± 1.3 pg/μg protein; P<0.05). In adipose, ACE2 activity was reduced by S protein in male, but not female mice (Male, vehicle: 63.6 ± 13.9; Male, S protein: 26.1 ± 1.9; Female, vehicle: 32.5 ± 1.9; Female, S protein: 25.1 ± 1.3 RFU/hr/mg tissue; P<0.05). SARS-CoV-2 S protein (35 nM) decreased ACE2 activity in type II lung alveolar cells (Vehicle: 2.0 x 10 4 ; S protein: 1.2 x 10 4 RFU/10 6 cells) and 3T3-L1 adipocytes (Vehicle: 2.1 x 10 4 ± 0.3 x 10 4 ; S protein: 1.1 x 10 4 ± 0.8 x 10 3 RFU/10 5 cells; P<0.05). Biologic sex regulation of ACE2 may protect females from SARS-CoV-2 S protein-mediated ACE2 reductions and activation of the local RAS.

Background: Thoracic aortic aneurysms associated with Marfan syndrome (MFS) carry a high risk of mortality; however, the molecular and cellular processes leading to aortopathy in this population remain poorly understood. We aimed to use single-cell RNA (scRNA) sequencing to define the non-immune cell populations present within the aortic wall in MFS, hypothesizing that these would differ from those of non-aneurysmal control tissue. Methods: We performed scRNA sequencing of ascending aortic aneurysm tissues from MFS patients (n=3) undergoing aneurysm repair and of age-matched, non-aneurysmal control tissue from cardiac transplant donors and recipients (n=4). The Seurat package in R was used for analysis. Differentially expressed genes were identified using edgeR. Results: Eighteen non-immune cell clusters were identified, with conserved gene expression of the largest of the clusters consistent with smooth muscle cells (SMCs; n=6), fibroblasts (n=3), and endothelial cells (n=3). The SMCs and fibroblasts exhibited graded changes in their expression of contractile and extracellular matrix protein genes, supportive of a phenotypic continuum. Additionally, we identified differences in the proportions of non-immune cells in MFS tissues compared to controls. In control tissues, the most common non-immune cells expressed markers of contractile SMC maturity including CNN1 , MYH11 , and SMTN . In contrast, the largest clusters in MFS tissue were most closely related to SMCs on correlation analysis, but displayed increased expression of cyclin genes as well as immune, endothelial, and fibroblast genes indicative of de-differentiated, proliferative SMCs. Additionally, expression of genes associated with SMC phenotypic maturity, including MYH11 and MYOCD , were significantly downregulated in several of the MFS SMC clusters. Conclusion: Our data demonstrate a phenotypic continuum between fibroblasts and SMCs, with aortas from patients with MFS exhibiting an increased proportion of de-differentiated, proliferative SMCs compared to controls. Additionally, markers of SMC maturity were downregulated in SMCs in MFS compared to controls. This may be due to disruption of signaling pathways that promote differentiation.

Objective: Angiotensin II (AngII) actions at angiotensin type 1 receptors (AT1R) contribute to the development of atherosclerosis and abdominal aortic aneurysms (AAAs), two vascular diseases exhibi...