Thoracic Perivascular Adipose Tissue Research Articles

β-Adrenergic Stimulation-Induced PVAT Dysfunction in Male Sex: A Role for 11β-Hydroxysteroid Dehydrogenase-1.

Long-term β-adrenoceptor (β-AR) stimulation is a pathological mechanism associated with cardiovascular diseases resulting in endothelial and perivascular adipose tissue (PVAT) dysfunction. In this study, we aimed to identify whether β-adrenergic signaling has a direct effect on PVAT. Thoracic aorta PVAT was obtained from male Wistar rats and cultured ex vivo with the β-AR agonist isoproterenol (Iso; 1 µM) or vehicle for 24 hours. Conditioned culture medium (CCM) from Iso-treated PVAT induced a marked increase in aorta contractile response, induced oxidative stress, and reduced nitric oxide production in PVAT compared to vehicle. In addition, Iso-treated PVAT and PVAT-derived differentiated adipocytes exhibited higher corticosterone release and protein expression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), an enzyme responsible for de novo synthesis of corticosterone. Macrophages exposed to Iso also exhibited increased corticosterone release in response to β-AR stimulation. Incubation of Iso-treated PVAT and PVAT-derived differentiated adipocytes with β3-AR antagonist restored aorta contractile function modulated by Iso-CCM and normalized 11β-HSD1 protein expression. These results show that β3-AR signaling leads to upregulation of 11β-HSD1 in PVAT, thus increasing corticosterone release and contributing to impair the anticontractile function of this tissue.

A cell atlas of thoracic aortic perivascular adipose tissue: a focus on mechanotransducers.

Perivascular adipose tissue (PVAT) is increasingly recognized for its function in mechanotransduction. However, major gaps remain in our understanding of the cells present in PVAT, as well as how different cells contribute to mechanotransduction. We hypothesized that snRNA-seq would reveal the expression of mechanotransducers, and test one (PIEZO1) to illustrate the expression and functional agreement between single-nuclei RNA sequencing (snRNA-seq) and physiological measurements. To contrast two brown tissues, subscapular brown adipose tissue (BAT) was also examined. We used snRNA-seq of the thoracic aorta PVAT (taPVAT) and BAT from male Dahl salt-sensitive (Dahl SS) rats to investigate cell-specific expression mechanotransducers. Localization and function of the mechanostransducer PIEZO1 were further examined using immunohistochemistry (IHC) and RNAscope, as well as pharmacological antagonism. Approximately 30,000 nuclei from taPVAT and BAT each were characterized by snRNA-seq, identifying eight major cell types expected and one unexpected (nuclei with oligodendrocyte marker genes). Cell-specific differential gene expression analysis between taPVAT and BAT identified up to 511 genes (adipocytes) with many (≥20%) being unique to individual cell types. Piezo1 was the most highly, widely expressed mechanotransducer. The presence of PIEZO1 in the PVAT but not the adventitia was confirmed by RNAscope and IHC in male and female rats. Importantly, antagonism of PIEZO1 by GsMTX4 impaired the PVAT's ability to hold tension. Collectively, the cell compositions of taPVAT and BAT are highly similar, and PIEZO1 is likely a mechanotransducer in taPVAT.NEW & NOTEWORTHY This study describes the atlas of cells in the thoracic aorta perivascular adipose tissue (taPVAT) of the Dahl-SS rat, an important hypertension model. We show that mechanotransducers are widely expressed in these cells. Moreover, PIEZO1 expression is shown to be restricted to the taPVAT and is functionally implicated in stress relaxation. These data will serve as the foundation for future studies investigating the role of taPVAT in this model of hypertensive disease.

Nicotine Impairs the Anti-Contractile Function of Perivascular Adipose Tissue by Inhibiting the PPARγ-Adiponectin-AdipoR1 Axis.

Nicotine is an addictive compound found in cigarette smoke that leads to vascular dysfunction and cardiovascular diseases. Perivascular adipose tissue (PVAT) exerts an anti-contractile effect on the underlying vasculature through the production of adipokines, such as adiponectin, which acts on adiponectin receptors 1 (adipoR1) to cause vasorelaxation. Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that regulates adiponectin gene expression and PVAT development. This study aimed to determine the effect of nicotine on the anti-contractile function of PVAT via the PPARγ-adiponectin-adipoR1 axis. Male Sprague Dawley rats were divided into a control group (given normal saline), a nicotine group (given 0.8 mg/kg of nicotine), and a nicotine + PPARγ agonist group (given nicotine and 5 mg/kg of telmisartan). Thoracic aorta PVAT was harvested after 21 days of treatment. The results showed that nicotine reduced the anti-contractile effect of PVAT on the underlying thoracic aorta. Nicotine also decreased the gene and protein expression of PPARγ, adiponectin, and adipoR1 in PVAT. Treatment with telmisartan restored the anti-contractile effect of PVAT and increased the gene and protein expression of PPARγ, adiponectin, and adipoR1 in PVAT. In conclusion, nicotine attenuates the anti-contractile function of PVAT through inhibition of the PPARγ-adiponectin-adipoR1 axis.

Abstract 121: A Cell Atlas Of The Dahl SS Thoracic Aortic Perivascular Adipose Tissue: Delving Into Single-Cell Data

Nearly every blood vessel is surrounded by a layer of fat aptly named perivascular adipose tissue (PVAT). PVAT is an important regulator of vascular tone known largely for its anti-contractile secretions. Recently, however, new PVAT functions have been discovered. These findings include mechanosensing and immune cell regulating capabilities. Further research would be facilitated by the existence of a map of the cells that make up PVAT and showing how they differ from the cells in other types of adipose tissue. Here, we build this map using single-nucleus RNA-sequencing (snRNA-seq) to construct a thoracic aorta PVAT (tPVAT) cell atlas. We then test our hypothesis that cell populations, cell-type proportions, and gene expression patterns differ between tPVAT and brown adipose tissue (BAT). To accomplish this, snRNA-seq was performed on two samples of both tPVAT and BAT that were collected from male Dahl SS rats fed a normal diet. The resulting sequencing files were quality controlled and processed using standard snRNA-seq pipelines. A total of 30,352 and 29,813 nuclei were profiled in tPVAT and BAT respectively. Both tissues were primarily represented by adipocytes, smooth muscle cells, and endothelial cells with a few differences in proportions (Fig. 1). Notably, there is a population of white adipocytes representing 0.3% (89/30352) of nuclei in tPVAT that are absent in BAT. We also find many genes that are differentially expressed in each cell type between the two tissues. This cell atlas provides valuable information to understand the cells that participate in recognized and newly discovered mechanisms of PVAT.

Angiotensin II type 2 receptor as a novel activator of brown adipose tissue in obesity.

The angiotensin II type 2 receptor (AT2R) exerts vasorelaxant, anti-inflammatory, and antioxidant properties. In obesity, its activation counterbalances the adverse cardiovascular effects of angiotensin II mediated by the AT1R. Preliminary results indicate that it also promotes brown adipocyte differentiation in vitro. Our hypothesis is that AT2R activation could increase BAT mass and activity in obesity. Five-week-old male C57BL/6J mice were fed a standard or a high-fat (HF) diet for 6 weeks. Half of the animals were treated with compound 21 (C21), a selective AT2R agonist, (1 mg/kg/day) in the drinking water. Electron transport chain (ETC), oxidative phosphorylation, and UCP1 proteins were measured in the interscapular BAT (iBAT) and thoracic perivascular adipose tissue (tPVAT) as well as inflammatory and oxidative parameters. Differentiation and oxygen consumption rate (OCR) in the presence of C21 was tested in brown preadipocytes. In vitro, C21-differentiated brown adipocytes showed an AT2R-dependent increase of differentiation markers (Ucp1, Cidea, Pparg) and increased basal and H+ leak-linked OCR. In vivo, HF-C21 mice showed increased iBAT mass compared to HF animals. Both their iBAT and tPVAT showed higher protein levels of the ETC protein complexes and UCP1, together with a reduction of inflammatory and oxidative markers. The activation of the AT2R increases BAT mass, mitochondrial activity, and reduces markers of tissue inflammation and oxidative stress in obesity. Therefore, insulin reduction and better vascular responses are achieved. Thus, the activation of the protective arm of the renin-angiotensin system arises as a promising tool in the treatment of obesity.

High intensity exercise improves the metabolic syndrome in an aged murine model, but it abrogates the vascular function improvement induced by moderate exercise

Abstract Introduction Compelling evidence supports that regular moderate exercise has a positive impact on cardiovascular disease (CVD) incidence and prognosis [1]. Nevertheless, emerging clinician and experimental data in healthy individuals suggests that long-term, high-intensity endurance exercise might increase vascular stiffness and enhance coronary calcification [2,3]. In patients with CV risk factors, it has been hypothesized that the beneficial effects of strenuous exercise on risk factors could outweigh its deleterious effects, but this remains unproven [4]. Purpose To analyze how increasing doses of exercise impinge on the vascular system in a murine model of aged rats with metabolic syndrome and kidney disease. Methods Seven-week-old male Zucker obese rats, a metabolic syndrome model, were subjected to left nephrectomy. At 26 weeks of age, rats were assigned to a sedentary (SED), moderate intensity (MOD, 15 cm/s, 40 min/day), or high intensity (INT, 25 cm/s, 60 min/day) group, and trained 5 days/week for 10 weeks. An echocardiogram and a glucose tolerance test were performed 24 hours after the last training bout. At sacrifice, vascular reactivity was assayed in the descending thoracic aorta, and EC50 calculated for every experiment. Expression of oxidant and antioxidant mRNA markers was analyzed in thoracic perivascular adipose tissue (tPVAT). Cytokines levels (TNFα, IL10, IL6, Selectin, ICAM1 and Adiponectin) were analyzed in plasma samples. Results Rats in the INT group had a significantly lower body weight (10.99±0.35 g/mm vs 12.23±0.58 g/mm) and a higher heart weight (32.44±0.72 mg/mm vs 31.28±0.78 mg/mm), indexed by tibia length, than the SED group. Both MOD and INT exercise improved glucose tolerance compared with SED (Fig 1a). On echocardiography, INT rats presented with LV septum hypertrophy and dilation, mildly reduced ejection fraction and fractional shortening compared with SED. Aortic diameter was also increased in the INT group (3.62±0.11 mm vs 3.25±0.13 mm). In vascular reactivity assays, the MOD group showed improved endothelial function, indicated by a larger carbachol-induced relaxation than SED and INT (Fig. 1b). However, MOD and INT were no longer different after adding the antioxidant Tempol (Fig1c). No differences were found in tunica media fibrosis amongst the groups (19.12±0.45%; 26.97±2.52%; 25.98±2.14%). In tPVAT, the mRNA expression of prooxidants (NOX2, p47) was higher, and antioxidant markers (SOD2, GCH1) was lower in the INT group than MOD and SED (Fig. 2). TNFα levels were higher in plasma of INT rats than SED. Conclusion In a rodent model, both intense and moderate exercise improved the CV risk profile, but only moderate training improved vascular function. Oxidative stress may, at least partially, account for the lack of benefits of intensive exercise. Our results suggest that the deleterious effects of strenuous physical activity could persist even in individuals with CV risk factor. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Fondo de Investigaciόn Sanitaria (FIS), Instituto de Salud Carlos III

PIEZO1 mechanoreceptor activation reduces adipogenesis in perivascular adipose tissue preadipocytes.

During hypertension, vascular remodeling allows the blood vessel to withstand mechanical forces induced by high blood pressure (BP). This process is well characterized in the media and intima layers of the vessel but not in the perivascular adipose tissue (PVAT). In PVAT, there is evidence for fibrosis development during hypertension; however, PVAT remodeling is poorly understood. In non-PVAT depots, mechanical forces can affect adipogenesis and lipogenic stages in preadipocytes. In tissues exposed to high magnitudes of pressure like bone, the activation of the mechanosensor PIEZO1 induces differentiation of progenitor cells towards osteogenic lineages. PVAT’s anatomical location continuously exposes it to forces generated by blood flow that could affect adipogenesis in normotensive and hypertensive states. In this study, we hypothesize that activation of PIEZO1 reduces adipogenesis in PVAT preadipocytes. The hypothesis was tested using pharmacological and mechanical activation of PIEZO1. Thoracic aorta PVAT (APVAT) was collected from 10-wk old male SD rats (n=15) to harvest preadipocytes that were differentiated to adipocytes in the presence of the PIEZO1 agonist Yoda1 (10 µM). Mechanical stretch was applied with the FlexCell System at 12% elongation, half-sine at 1 Hz simultaneously during the 4 d of adipogenesis (MS+, mechanical force applied; MS-, no mechanical force used). Yoda1 reduced adipogenesis by 33% compared with CON and, as expected, increased cytoplasmic Ca2+ flux. MS+ reduced adipogenesis efficiency compared with MS-. When Piezo1 expression was blocked with siRNA [siPiezo1; NC=non-coding siRNA], the anti-adipogenic effect of Yoda1 was reversed in siPiezo1 cells but not in NC; in contrast, siPiezo1 did not alter the inhibitory effect of MS+ on adipogenesis. These data demonstrate that PIEZO1 activation in PVAT reduces adipogenesis and lipogenesis and provides initial evidence for an adaptive response to excessive mechanical forces in PVAT during hypertension.

Thoracic perivascular adipose tissue inhibits VSMC apoptosis and aortic aneurysm formation in mice via the secretome of browning adipocytes.

Abdominal aortic aneurysm (AAA) is a dangerous vascular disease without any effective drug therapies so far. Emerging evidence suggests the phenotypic differences in perivascular adipose tissue (PVAT) between regions of the aorta are implicated in the development of atherosclerosis evidenced by the abdominal aorta more vulnerable to atherosclerosis than the thoracic aorta in large animals and humans. The prevalence of thoracic aortic aneurysms (TAA) is much less than that of abdominal aortic aneurysms (AAA). In this study we investigated the effect of thoracic PVAT (T-PVAT) transplantation on aortic aneurysm formation and the impact of T-PVAT on vascular smooth muscle cells. Calcium phosphate-induced mouse AAA model was established. T-PVAT (20 mg)was implanted around the abdominal aorta of recipient mice after removal of endogenous abdominal PVAT (A-PVAT) and calcium phosphate treatment. Mice were sacrificed two weeks after the surgery and the maximum external diameter of infrarenal aorta was measured. We found that T-PVAT displayed a more BAT-like phenotype than A-PVAT; transplantation of T-PVAT significantly attenuated calcium phosphate-induced abdominal aortic dilation and elastic degradation as compared to sham control or A-PVAT transplantation. In addition, T-PVAT transplantation largely preserved smooth muscle cell content in the abdominal aortic wall. Co-culture of T-PVAT with vascular smooth muscle cells (VSMCs) significantly inhibited H2O2- or TNFα plus cycloheximide-induced VSMC apoptosis. RNA sequencing analysis showed that T-PVAT was enriched by browning adipocytes and anti-apoptotic secretory proteins. We further verified that the secretome of mature adipocytes isolated from T-PVAT significantly inhibited H2O2- or TNFα plus cycloheximide-induced VSMC apoptosis. Using proteomic and bioinformatic analyses we identified cartilage oligomeric matrix protein (COMP) as a secreted protein significantly increased in T-PVAT. Recombinant COMP protein significantly inhibited VSMC apoptosis. We conclude that T-PVAT exerts anti-apoptosis effect on VSMCs and attenuates AAA formation, which is possibly attributed to the secretome of browning adipocytes.

Comparative Proteomic Analysis of tPVAT during Ang II Infusion.

Perivascular adipose tissue (PVAT) homeostasis plays an important role in maintaining vascular function, and PVAT dysfunction may induce several pathophysiological situations. In this study, we investigated the effect and mechanism of the local angiotensin II (Ang II) on PVAT. High-throughput comparative proteomic analysis, based on TMT labeling combined with LC-MS/MS, were performed on an in vivo Ang II infusion mice model to obtain a comprehensive view of the protein ensembles associated with thoracic PVAT (tPVAT) dysfunction induced by Ang II. In total, 5037 proteins were confidently identified, of which 4984 proteins were quantified. Compared with the saline group, 145 proteins were upregulated and 146 proteins were downregulated during Ang II-induced tPVAT pathogenesis. Bioinformatics analyses revealed that the most enriched GO terms were annotated as gene silencing, monosaccharide binding, and extracellular matrix. In addition, some novel proteins, potentially associated with Ang II infusion, were identified, such as acyl-CoA carboxylase α, very long-chain acyl-CoA synthetase (ACSVL), uncoupling protein 1 (UCP1), perilipin, RAS protein-specific guanine nucleotide-releasing factor 2 (RasGRF2), and hypoxia inducible factor 1α (HIF-1α). Ang II could directly participate in the regulation of lipid metabolism, transportation, and adipocyte differentiation by affecting UCP1 and perilipin. Importantly, the key KEGG pathways were involved in fatty acid biosynthesis, FABP3-PPARα/γ, RasGRF2-ERK-HIF-1α, RasGRF2-PKC-HIF-1α, and STAT3-HIF-1α axis. The present study provided the most comprehensive proteome profile of mice tPVAT and some novel insights into Ang II-mediated tPVAT dysfunction and will be helpful for understanding the possible relationship between local RAS activation and PVAT dysfunction.

A Differential Pattern of Batokine Expression in Perivascular Adipose Tissue Depots From Mice.

Depending on its anatomical placement, perivascular adipose tissue (PVAT) has been found to possess features more (e.g., aortic thoracic) or less (e.g., aortic abdominal) similar to brown/beige adipose tissue in mice, whereas PVAT surrounding the mesenteric arteries and the caudal part of abdominal aorta is similar to white fat. PVAT is thought to influence vascular function through the effects of adipose-secreted molecules on vessels. Brown adipose tissue was recently shown to play differential secretory role via secretion of the so-called batokines but the involvement of differential batokine production in PVAT brown/beige plasticity was unclear. The current study characterizes for the first time the expression of batokines at aortic thoracic PVAT (tPVAT) and aortic abdominal PVAT (aPVAT) in comparison with typical brown and white adipose depots, in basal and thermogenically activated conditions. We found that both PVAT depots increased their expression of genes encoding the batokines bone morphogenetic protein-8b (BMP8B), fibroblast growth factor-21 (FGF21), and kininogen-2 (KNG2) in response to cold, indicating that, under cold-induced thermogenic activation, both thoracic aorta and abdominal aorta would experience intense local exposure to these PVAT-secreted batokines. In contrast, the gene expression levels of growth/differentiation factor-15 and vascular endothelial growth factor-A were induced only in tPVAT. Under short-term high-fat diet-induced thermogenic activation, the thoracic aorta would be specifically exposed to a local increase in PVAT-originating BMP8B, FGF21, and KNG2. Our data support the notion that acquisition of a brown/beige phenotype in PVAT is associated with upregulation of batokines, mainly BMP8B, FGF21, and KNG2, that can differentially target the vascular system.

Chronic stress induced perivascular adipose tissue impairment of aortic function and the therapeutic effect of exercise.

What is the central question of this study? Thoracic perivascular adipose tissue (tPVAT) is known to, in part, regulate aortic function: what are the effects of unpredictable chronic mild stress (UCMS) on the tPVAT regulation of aortic function and what is the role of exercise training in alleviating the potential negative actions of UCMS on tPVAT? What is the main finding and its importance? UCMS causes tPVAT to disrupt endothelium-dependent dilatation, increases inflammatory cytokine production and diminishes tPVAT-adiponectin. Exercise training proved efficacious in preventing tPVAT-mediated disruption of aortic function. The data support a tPVAT mechanism through which chronic stress negatively impacts vascular health, which adds to our knowledge of how psychological disorders might increase the risk of cardiovascular disease. Chronic stress is a major risk for cardiovascular disease. Perivascular adipose tissue (PVAT) has been shown to regulate vascular function; however, the impact of chronic stress and the comorbidity of metabolic syndrome (MetS) on thoracic (t)PVAT is unknown. Additionally, aerobic exercise training (AET) is known to combat the pathology of MetS and chronic stress, but the role of tPVAT in these actions is also unknown. Therefore, the purpose of this study was to examine the effects of unpredictable chronic mild stress (UCMS) on the tPVAT regulation of aortic function and the preventative effect of AET. Lean (LZR) and obese (OZR) Zucker rats (16-17weeks old) were exposed to 8weeks of UCMS with and without treadmill exercise (AET). In LZR, UCMS impaired aortic endothelium-dependent dilatation (EDD) (assessed ex vivo by wire myography) and aortic stiffness (assessed by elastic modulus) with no change in OZR subject to UCMS. However, both LZR and OZR UCMS tPVAT impaired EDD compared to respective controls. LZR and OZR subject to UCMS had higher oxidative stress production, diminished adiponectin and impaired aortic nitric oxide levels. Divergently, UCMS induced greater inflammatory cytokine production in LZR UCMS tPVAT, but not in OZR UCMS tPVAT. AET prevented the tPVAT impairment of aortic relaxation with UCMS in LZR and OZR. Additionally, AET reduced aortic stiffness in both LZR and OZR. These beneficial effects on tPVAT regulation of the aorta are likely due to AET preservation of adiponectin, reduced oxidative stress and inflammation, and enhanced nitric oxide. UCMS impaired tPVAT-regulated aortic function in LZR, and augmented MetS-induced EDD in OZR. Conversely, AET in combination with UCMS largely preserved aortic function and the tPVAT environment, in both groups.

Liver circadian clock disruption alters perivascular adipose tissue gene expression and aortic function in mice.

The liver plays a central role that influences cardiovascular disease outcomes through regulation of glucose and lipid metabolism. It is recognized that the local liver molecular clock regulates some liver-derived metabolites. However, it is unknown whether the liver clock may impact cardiovascular function. Perivascular adipose tissue (PVAT) is a specialized type of adipose tissue surrounding blood vessels. Importantly, cross talk between the endothelium and PVAT via vasoactive factors is critical for vascular function. Therefore, we designed studies to test the hypothesis that cardiovascular function, including PVAT function, is impaired in mice with liver-specific circadian clock disruption. Bmal1 is a core circadian clock gene, thus studies were undertaken in male hepatocyte-specific Bmal1 knockout (HBK) mice and littermate controls (i.e., flox mice). HBK mice showed significantly elevated plasma levels of β-hydroxybutyrate, nonesterified fatty acids/free fatty acids, triglycerides, and insulin-like growth factor 1 compared with flox mice. Thoracic aorta PVAT in HBK mice had increased mRNA expression of several key regulatory and metabolic genes, Ppargc1a, Pparg, Adipoq, Lpl, and Ucp1, suggesting altered PVAT energy metabolism and thermogenesis. Sensitivity to acetylcholine-induced vasorelaxation was significantly decreased in the aortae of HBK mice with PVAT attached compared with aortae of HBK mice with PVAT removed, however, aortic vasorelaxation in flox mice showed no differences with or without attached PVAT. HBK mice had a significantly lower systolic blood pressure during the inactive period of the day. These new findings establish a novel role of the liver circadian clock in regulating PVAT metabolic gene expression and PVAT-mediated aortic vascular function.

Defining the lineage of thermogenic perivascular adipose tissue.

Brown adipose tissue can expend large amounts of energy, and therefore increasing its size or activity is a promising therapeutic approach to combat metabolic disease. In humans, major deposits of brown fat cells are found intimately associated with large blood vessels, corresponding to perivascular adipose tissue (PVAT). However, the cellular origins of PVAT are poorly understood. Here, we determine the identity of perivascular adipocyte progenitors in mice and humans. In mice, thoracic PVAT develops from a fibroblastic lineage, consisting of progenitor cells (Pdgfra+; Ly6a+; Pparg-) and preadipocytes (Pdgfra+; Ly6a+; Pparg+), which share transcriptional similarity with analogous cell types in white adipose tissue. Interestingly, the aortic adventitia of adult animals contains a population of adipogenic smooth muscle cells (SMCs) (Myh11+; Pdgfra-; Pparg+) that contribute to perivascular adipocyte formation. Similarly, human PVAT contains presumptive fibroblastic and SMC-like adipocyte progenitors, as revealed by single nucleus RNAseq. Taken together, these studies define distinct populations of progenitor cells for thermogenic PVAT, providing a foundation for developing strategies to augment brown fat activity.

Mineralocorticoid Receptor in Myeloid Cells Mediates Angiotensin II-Induced Vascular Dysfunction in Female Mice

Enhanced mineralocorticoid receptor (MR) signaling is critical to the development of endothelial dysfunction and arterial stiffening. However, there is a lack of knowledge about the role of MR-induced adipose tissue inflammation in the genesis of vascular dysfunction in women. In this study, we hypothesize that MR activation in myeloid cells contributes to angiotensin II (Ang II)-induced aortic stiffening and endothelial dysfunction in females via increased pro-inflammatory (M1) macrophage polarization. Female mice lacking MR in myeloid cells (MyMRKO) were infused with Ang II (500 ng/kg/min) for 4 weeks. This was followed by determinations of aortic stiffness and vasomotor responses, as well as measurements of markers of inflammation and macrophage infiltration/polarization in different adipose tissue compartments. MyMRKO mice were protected against Ang II-induced aortic endothelial stiffening, as assessed via atomic force microscopy in aortic explants, and vasorelaxation dysfunction, as measured by aortic wire myography. In alignment, MyMRKO mice were protected against Ang II-induced macrophage infiltration and M1 polarization in visceral adipose tissue (VAT) and thoracic perivascular adipose tissue (tPVAT). Collectively, this study demonstrates a critical role of MR activation in myeloid cells in the pathogenesis of vascular dysfunction in females associated with pro-inflammatory macrophage polarization in VAT and tPVAT. Our data have potential clinical implications for the prevention and management of cardiovascular disease in women, who are disproportionally at higher risk for poor outcomes.

Perivascular Adipose Tissue Inflammation in Ischemic Heart Disease.

There is growing recognition that adipose tissue-derived proatherogenic mediators contribute to obesity-related cardiovascular disease. We sought to characterize regional differences in perivascular adipose tissue (PVAT) phenotype in relation to atherosclerosis susceptibility. Approach and Results: We examined thoracic PVAT samples in 34 subjects (body mass index 32±6 kg/m2, age 59±11 years) undergoing valvular, aortic, or coronary artery bypass graft surgeries and performed transcriptomic characterization using whole-genome expression profiling and quantitative polymerase chain reaction analyses. We identified a highly inflamed region of PVAT surrounding the human aortic root in close proximity to coronary takeoff and adjoining epicardial fat. In subjects undergoing coronary artery bypass graft, we found 300 genes significantly upregulated (false discovery rate Q<0.1) in paired samples of PVAT surrounding the aortic root compared with nonatherosclerotic left internal mammary artery. Genes encoding proteins mechanistically implicated in atherogenesis were enriched in aortic PVAT consisting of signaling pathways linked to inflammation, WNT (wingless-related integration site) signaling, matrix remodeling, coagulation, and angiogenesis. Overexpression of several proatherogenic transcripts, including IL1β, CCL2 (MCP-1), and IL6, were confirmed by quantitative polymerase chain reaction and significantly bolstered in coronary artery disease subjects. Angiographic coronary artery disease burden quantified by the Gensini score positively correlated with the expression of inflammatory genes in PVAT. Moreover, periaortic adipose inflammation was markedly higher in obese subjects with striking upregulation (≈8-fold) of IL1β expression compared to nonobese individuals. Proatherogenic mediators that originate from dysfunctional PVAT may contribute to vascular disease mechanisms in human vessels. Moreover, PVAT may adopt detrimental properties under obese conditions that play a key role in the pathophysiology of ischemic heart disease. Graphic Abstract: A graphic abstract is available for this article.

Abstract P142: Sex-differences In M1-like Macrophage Counts In Aortic Perivascular Adipose Tissue Precedes High Fat Diet-induced Hypertension In Dahl S Rats

Perivascular adipose tissue (PVAT) may connect adiposity to hypertension because of its functions and proximity to blood vessels. Immune cells in adipose tissue are proposed to couple adiposity to hypertension development in a sex-specific fashion. It is unknown if sex-differences exist in PVAT’s immune community during the onset of adiposity-induced hypertension. Both sexes of the Dahl S rat strain become equally hypertensive (table) when fed a high fat (HFD) diet. We hypothesized that both sexes have similar immune cell composition in PVAT with the development and progression of HFD-induced hypertension. Male and female Dahl S rats were fed a regular (10% calories from fat; CD) diet or a HFD (60%) from weaning. Thoracic aorta PVAT (APVAT) was harvested at 10 (pre-hypertension), 17 (onset) or 24 (chronic) weeks (w) of diet. Macrophages (subtypes), neutrophils, mast, T (subtypes), B, NK cells were measured by flow cytometry. At 10 w, HFD females had 5X the number of M1-like macrophages vs HFD males. At 17 w, CD females had 10X the number of M2-like macrophages vs CD males. At 17 w and 24 w, males had greater number of CD4 (2X) and CD8 (1.5X) memory T cells vs females, independent of the diet (table). In summary, sex-differences in M1-like macrophage counts in APVAT precedes the development of HFD-induced hypertension in Dahl S rats. The progression of hypertension is associated with memory T cells in males and M2-like macrophages in females. This study is foundational to understand the sex-specific roles of these immune cells in regulating vascular tone in HFD-induced hypertension, underscoring the need for novel sex-specific anti-hypertensive immune modulators.

The Influence of Photoperiod on the Action of Exogenous Leptin on Gene Expression of Proinflammatory Cytokines and Their Receptors in the Thoracic Perivascular Adipose Tissue (PVAT) in Ewes.

Leptin resistance is either a condition induced by human obesity or a natural phenomenon associated with seasonality in ruminants. In the cardiovascular system, the leptin resistance state presence is a complex issue. Moreover, the perivascular adipose tissue (PVAT) appears to be crucial as a source of proinflammatory cytokines and as a site of interaction for leptin contributing to endothelium dysfunction and atherosclerosis progression. So the aim of this study was to examine the influence of the photoperiod on the action of exogenous leptin on gene expression of selected proinflammatory cytokines and their receptors in thoracic PVAT of ewe with or without prior lipopolysaccharide (LPS) stimulation. The experiment was conducted on 48 adult, female ewes divided into 4 group (n = 6 in each): control, with LPS intravenous (iv.) injection (400 ng/kg of BW), with leptin iv. injection (20 μg/kg BW), and with LPS and 30-minute-later leptin injection, during short-day (SD) and long-day (LD) seasons. Three hours after LPS/control treatment, animals were euthanized to collect the PVAT adherent to the aorta wall. The leptin injection enhanced IL1B gene expression only in the LD season; however, in both seasons leptin injection intensified LPS-induced increase in IL1B gene expression. IL1R2 gene expression was increased by leptin injection only in the SD season. Neither IL6 nor its receptor and signal transducer gene expressions were influenced by leptin administration. Leptin injection increased TNFA gene expression regardless of photoperiodic conditions. Only in the SD season did leptin treatment increase the gene expression of both TNFα receptors. To conclude, leptin may modulate the inflammatory reaction progress in PVAT. In ewe, the sensitivity of PVAT on leptin action is dependent upon the photoperiodic condition with stronger effects stated in the SD season.

Site-specific impairment of perivascular adipose tissue on advanced atherosclerotic plaques using multimodal nonlinear optical imaging

Perivascular adipose tissue (PVAT), as a mechanical support, has been reported to systemically regulate vascular physiology by secreting adipokines and cytokines. How PVAT spatially and locally changes as atherosclerosis progresses is not known, however. We aimed to reveal the molecular changes in PVAT in advanced atherosclerosis based on multimodal nonlinear optical (MNLO) imaging. First, using an atherogenic apolipoprotein E knockout mouse model, we precisely assessed the browning level of thoracic PVAT via a correlative analysis between the size and number of lipid droplets (LDs) of label-free MNLO images. We also biochemically demonstrated the increased level of brown fat markers in the PVAT of atherosclerosis. In the initial stage of atherosclerosis, the PVAT showed a highly activated brown fat feature due to the increased energy expenditure; however, in the advanced stage, only the PVAT in the regions of the atherosclerotic plaques, not that in the nonplaque regions, showed site-specific changes. We found that p-smad2/3 and TGF-β signaling enhanced the increase in collagen to penetrate the PVAT and the agglomeration of LDs only at the sites of atherosclerotic plaques. Moreover, atherosclerotic thoracic PVAT (tPVAT) was an increased inflammatory response. Taken together, our findings show that PVAT changes differentially from the initial stages to advanced stages of atherosclerosis and undergoes spatial impairment focused on atherosclerotic plaques. Our study may provide insight into the local control of PVAT as a therapeutic target.

SIRT3-MEDIATED MACROPHAGE PYRUVATE DEHYDROGENASE ACTIVITY IMPROVES PERIVASCULAR ADIPOSE TISSUE REMODELING IN HYPERTENSIVE MICE

Objective: Infiltrated macrophages are a dominant population in the perivascular adipose tissue (PVAT) microenvironment and actively promote PVAT remodeling in hypertensive mice. However, the molecular mechanism underlying the role of macrophages in initiating metabolic inflammation remains uncertain. Here, we investigate whether Situin-3(SIRT3) is determinant in macrophage activation. Design and method: 10-week-old male WT, SIRT3 KO, and NLRP3 KO mice were infused with Angiotension II (Ang II, 1000ng/kg/min) or normal saline (NS) for 7 days. The morphological and functional changes of thoracic aorta and PVAT were determined by histological analysis and immunofluorescence staining. In vitro studies were performed on bone marrow-derived macrophages (BMDMs) and brown adipocytes isolated from WT or SIRT3 KO mice. Results: We report that Ang II accelerates PVAT inflammation and fibrosis in SIRT3-deficient mice. This effect is associated with attenuating browning markers and lipid droplet-associated protein expression. Immunofluorescence analysis confirmed that there is an increase in NLRP3 inflammasome and secretion of interleukin-1β (IL-1β). In vitro studies indicate that BMDMs from SIRT3 KO mice induce interleukin-IL-1β production by shifting metabolic phenotype from oxidative phosphorylation towards glycolysis. Mechanically, SIRT3 deacetylates and activates pyruvate dehydrogenase E1 alpha (PDHE1a) at lysine 83, loss of SIRT3 lead to decreased PDH activity, and accumulation of pyruvate and lactate metabolites. The PDHK inhibitor dichloroacetate (DCA) suppresses both pro-IL-1β maturation and caspase-1 activation in macrophages in response to Ang II. The blockade of IL-1β from macrophage into brown adipocytes restores expression of brown adipocyte specific markers. Coincidentally, NLRP3 KO mice exhibited a reduction in infiltrated macrophages in PVAT, while partially rescuing brown adipocytes mitochondrial function and collagen accumulation. Conclusions: Taken together, these findings reveal that SIRT3-mediated activity of PDHE1a inhibits macrophage activation. Pharmacological targeting of glycolysis metabolism may therefore represent an effective approach.

Vascular Endothelial Growth Factor-A Deficiency in Perivascular Adipose Tissue Impairs Macrovascular Function.

Objective: Thoracic perivascular adipose tissue (PVAT) has been shown to release factors that influence the functioning of neighboring vascular tissue. Cardiovascular complications of obesity are on the rise; therefore, this study set out to determine if adipose-specific ablation of vascular endothelial growth factor-A (VEGF-A) plays a role in the maintenance of aortic structure and function.Methods: Adipose-specific VEGF-A-deficient mice were previously generated. Fabp4cre(+). VEGFflox/flox and Fabp4cre(−). VEGFflox/flox mice were maintained on chow diet. PVAT gene expression was measured with real-time quantitative PCR. Aortic vasomotor response was assessed with isometric tension measurements. Collagen deposition was analyzed histologically in the vascular media and compared using ratiometric pigment density.Results: PVAT-specific adiponectin expression was decreased in Fabp4cre(+). VEGFflox/flox mice. Isometric tension measurements revealed a dose-dependent dysfunction in response to acetylcholine within the distal aortic segment of Fabp4cre(+). VEGFflox/flox. Fabp4cre(+). VEGFflox/flox mice exhibited increased aortic deposition of collagen within the thoracic adventitial and medial spaces.Conclusion: These data demonstrate that decreased expression of VEGF-A within the surrounding adipose tissue microenvironment of the thoracic aorta has a detrimental effect on aortic integrity and vascular function. Modulation of angiogenic pathways within PVAT may offer an important avenue toward the treatment of adipose tissue dysfunction in obesity and its vascular complications.