Absence Of Perivascular Adipose Tissue Research Articles

Abstract 4125587: Vasomotor function and its regulation by perivascular adipose tissue in metabolic syndrome vary depending on severity and combination of metabolic parameters

Metabolic syndrome (MetS) is a cluster of risk factors for cardiovascular disease. Because perivascular adipose tissue (PVAT) regulates arterial tone by releasing vasorelaxing/vasocontracting factors, the effects of PVAT on vasomotor function have been heterogeneously reported. Various PVAT functions can result from the severity or combination of metabolic parameters in MetS. Therefore, we compared the effects of mesenteric arterial PVAT on the nitric oxide-dependent vasorelaxation response among the three metabolic syndrome (MetS) model strains. We compared male OLETF, Zucker fatty (ZF), and SHRSPZF (SPZF) rats aged 20 weeks to lean controls, LETO, ZF +/+ , and Wistar-Kyoto (WKY) rats. To determine the effects of PVAT on vasodilators, we used organ bath techniques to assay nitroprusside-induced relaxation of isolated mesenteric arterial ring preparations with intact or removed PVAT. The waist circumference to length ratio in OLETF and ZF rats was higher than that in SPZF rats, and the systolic blood pressure was higher in SPZF rats than in the other groups. In the serum, there was no significant difference in triglyceride levels. Still, glucose and thiobarbituric acid reactive substance levels were higher in OLETF and SPZF rats than in ZF rats. Relaxation in ZF rats did not change compared to that in ZF +/+ rats and the presence of PVAT. In contrast, relaxation in OLETF rats was lower than in LETO rats, and there was no difference between the presence and absence of PVAT. However, relaxation in SPZF rats was lower than in WKY rats, and relaxation increased to the same level as in WKY rats in the presence of PVAT. This study unveils novel insights, suggesting that high serum glucose and oxidative stress cause arterial dysfunction, whereas high blood pressure induces PVAT functional changes. These findings underscore the complex interplay between metabolic disorders and vascular health, potentially paving the way for more targeted interventions in patients with MetS.

Targeting BRD4-HK2 reverses the meta-inflammatory shift in perivascular adipose tissue and rescues cardiometabolic vascular dysfunction

Abstract Background/Introduction Reducing vascular inflammation is crucial to halt and reverse cardiometabolic damage. BRD4, an epigenetic pan-inflammatory activator whose inhibition has shown promising results in cancer, may play an important role in this context. However, its role in cardiometabolic disease remains unclear. Purpose To investigate BRD4-related transcriptional programmes and therapeutic modulation in translational models of cardiometabolic disease. Methods We dissected small arteries (100-300 μM) from visceral fat biopsies in healthy volunteers (n=16) and patients with obesity and hypertension (n=16), a highly prevalent cardiometabolic phenotype. We assessed the ex vivo effects of BRD4 inhibition on vascular function by pressure myography in the presence or absence of perivascular adipose tissue (PVAT), at baseline and after incubation with the BRD4 inhibitor RVX-208 or with anti-inflammatory/anti-metabolic drugs. We used a cardiometabolic mouse model (high-fat diet+L-NAME supplementation) that mirrored our human phenotype and orally administered RVX-208 (150 mg/kg) or vehicle for 5 days (n=6 per group) to confirm the in vivo effect of chronic BRD4 inhibition. Finally, we investigated the molecular pathways involved in the perivascular cross-talk in an in vitro model of human adipocytes (hADSCs) and endothelial cells (HAECs) using gene overexpression/silencing strategies. We assessed ROS and nitric oxide levels (confocal microscopy), protein and gene expressions (Western blot; qPCR), transcriptional (custom qPCR array; chromatin immunoprecipitation (ChIP)) and metabolic changes (metabolomics, lipidomics, mitochondrial swelling). Results Vascular inflammation, remodeling and function were altered in cardiometabolic patients/mice. RVX-208 attenuated ex vivo vascular dysfunction to a greater extent than anti-IL-1β, anti-IL-6 receptor and anti-TNF-α. In vessels with intact PVAT we observed a stronger effect, due to the restoration of healthy PVAT phenotype (Figure 1). In PVAT, Hexokinase-2 (Hk2) - a glycolytic enzyme implicated in mitochondrial dysfunction and inflammation - was the top downregulated gene by RVX-208 treatment; ChIP confirmed increased binding of BRD4 to the Hk2 promoter. In line, metabolomics and lipidomics assays revealed a PVAT glycolytic shift with increased fatty acid storage in disease states, which was reversed by BRD4 inhibition. The in vitro study showed that: i) healthy adipocytes overexpressing HK2 shift to an inflammatory phenotype; ii) their secretome induces an inflammatory phenotype in HAECs; iii) ex vivo PVAT-selective inhibition of HK2 ameliorates vascular dysfunction (Figure 2). Conclusions We identified BRD4-HK2 interaction at the PVAT level as a novel mediator of cardiometabolic damage. Its targeting rescues vascular dysfunction by reversing the PVAT meta-inflammatory shift. Epigenetic modulators of meta-inflammation may represent a promising strategy in patients with obesity and hypertension.

Transcriptional regulation of hexokinase 2 by BRD4 drives perivascular adipose tissue meta-inflammation in cardiometabolic disease

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Holcim Stiftung SwissLife Stiftung Background/Introduction BRD4 is an epigenetic reader that modulates inflammatory transcriptional programmes implicated in cancer. The therapeutic potential of BRD4 modulation in cardiometabolic disorders remains elusive. Purpose We investigate BRD4-related transcriptional programmes and therapeutic modulation in mouse and human models of cardiometabolic disease. Methods Small arteries (100-300 μM) from healthy subjects (n=16) and patients with obesity and hypertension (n=16) were dissected from visceral fat biopsies and mounted on a pressurised myograph to assess ex-vivo vascular function. Vasorelaxation to acetylcholine and acetylcholine+L-NAME was evaluated at baseline and after incubation with the BRD4 inhibitor RVX-208 and with selective anti-inflammatory and anti-metabolic drugs. Vasorelaxation was assessed in the presence or in the absence of perivascular adipose tissue (PVAT). An experimental mouse model of cardiometabolic disease (high-fat diet+L-NAME supplementation) was orally administered RVX-208 (150 mg/kg) or vehicle for 10 days (n=6 each group) to test in vivo effect of chronic BRD4 inhibition on endothelial and PVAT functions. Vascular and PVAT levels of cytosolic and mitochondrial ROS and nitric oxide were assessed by confocal microscopy; protein and gene expression were measured by Western blot and qPCR. Transcriptional changes upon BRD4 inhibition were investigated by a custom PCR array and confirmed by ChIP, qPCR and Western blot and further characterised by metabolomics, lipidomics and mitochondrial swelling assays. Results Endothelial-dependent vasorelaxation and tissue levels of TNF-α, IL-1β and IL-6 were altered in the vessel wall and PVAT from cardiometabolic patients and mice. RVX-208 substantially attenuated ex-vivo vascular dysfunction. Distinct ex-vivo modulation of well-established inflammatory pathways showed that the effect observed with BRD4 inhibition was stronger than with anti-IL-1β, anti-IL-6 receptor and anti-TNF-α. The effect was more pronounced in vessels with intact PVAT, suggesting a restoration of the PVAT anti-contractile phenotype. (Figure 1). Gene expression profiling in PVAT from cardiometabolic mice unveiled hexokinase-2 (HK2) - a glycolytic enzyme implicated in mitochondrial dysfunction and inflammation - as the top downregulated gene by RVX-208 treatment. Increased binding of BRD4 to HK2 promoter in PVAT samples from cardiometabolic mice was confirmed by ChIP assays. Metabolomics assays further validated the findings, showing a PVAT glycolytic shift in condition of disease. Finally, ex-vivo selective inhibition of HK2 restored vascular dysfunction (Figure 2). Conclusions Targeting the deleterious BRD4-HK2 interplay restores cardiometabolic-related vascular dysfunction reversing the PVAT meta-inflammatory shift. Epigenetic modulators of meta-inflammatory pathways might represent a promising strategy to prevent and vascular dysfunction, key signature of cardiometabolic disease.Figure 1Figure 2

TRANSCRIPTIONAL REGULATION OF HEXOKINASE-2 BY BRD4 DRIVES PERIVASCULAR ADIPOSE TISSUE META-INFLAMMATION IN CARDIOMETABOLIC DISEASE

Objective: To investigate BRD4-related transcriptional programmes and therapeutic modulation in mouse and human models of cardiometabolic disease. Design and method: Small arteries (0.1-0.3 mm) from healthy subjects (n=16) and patients with obesity and hypertension (n=16) were dissected from visceral fat biopsies and mounted on a pressurised myograph to assess the ex-vivo effects of BRD4 inhibition on vascular function. Vasorelaxation to acetylcholine and acetylcholine+L-NAME was evaluated, in the presence or in the absence of perivascular adipose tissue (PVAT), at baseline and after incubation with the BRD4 inhibitor RVX-208 and with selective anti-inflammatory and anti-metabolic drugs. A cardiometabolic mouse (high-fat diet+L-NAME supplementation) was orally administered RVX-208 (150 mg/kg) to test in vivo effect of chronic BRD4 inhibition. ROS and nitric oxide were assessed by confocal microscopy; protein and gene expression were measured by Western blot and qPCR. Transcriptional changes upon BRD4 inhibition were investigated by a custom qPCR array, confirmed by ChIP, qPCR and Western blot and further characterised by metabolomics, lipidomics and mitochondrial swelling assays. Results: Endothelial-dependent vasorelaxation and tissue levels of TNF-alpha, IL-1beta, and IL-6 were altered in the vessel wall and PVAT from cardiometabolic patients and mice. RVX-208 substantially attenuated ex-vivo vascular dysfunction, with an impact greater when compared to anti-IL-1beta, anti-IL-6 receptor and anti-TNF-alpha. The effect was more pronounced in vessels with intact PVAT, suggesting a restoration of the PVAT anti-contractile phenotype. Gene expression profiling in PVAT from cardiometabolic mice unveiled hexokinase-2 (HK2) - a glycolytic enzyme implicated in mitochondrial dysfunction and inflammation - as the top downregulated gene by RVX-208 treatment. Increased binding of BRD4 to HK2 promoter in PVAT samples from cardiometabolic mice was confirmed by ChIP assays. Metabolomics assays further validate the findings, showing a PVAT glycolytic shift in condition of disease. Finally, ex-vivo selective inhibition of HK2 restored vascular dysfunction. Conclusions: Targeting the deleterious BRD4-HK2 interplay restores cardiometabolic-related vascular dysfunction reversing the PVAT meta-inflammatory shift. Epigenetic modulators of meta-inflammatory pathways might represent a promising strategy to prevent and reverse vascular dysfunction, key signature of cardiometabolic disease.

Transcriptional regulation of Hexokinase-2 by BRD4 drives perivascular adipose tissue meta-inflammation in cardiometabolic disease

Aim: To investigate BRD4-related transcriptional programmes in mouse and human models of cardiometabolic disease. Methods: Small arteries (0.1-0.3 mm) dissected from visceral fat biopsies from healthy subjects (n=16) and patients with obesity and hypertension (n=16) were mounted on a pressurized myograph to assess the acute ex-vivo effects of BRD4 inhibition on vascular function. Vasorelaxation to acetylcholine and acetylcholine+L-NAME was evaluated, in the presence or in the absence of perivascular adipose tissue (PVAT), at baseline and after incubation with the BRD4 inhibitor RVX-208 and with selective anti-inflammatory and anti-metabolic drugs. A cardiometabolic mouse (high-fat diet+L-NAME supplementation) was orally administered RVX-208 (150 mg/kg) to test in vivo effect of chronic BRD4 inhibition. ROS and nitric oxide were assessed by confocal microscopy; protein and gene expression by Western blot and qPCR. Transcriptional changes upon BRD4 inhibition were investigated by a custom PCR array, confirmed by ChIP, and characterised by metabolomics, lipidomics and mitochondrial swelling. Results: Endothelial-dependent vasorelaxation and vascular and perivascular TNF-alpha, IL-1beta, IL-6 were altered in cardiometabolic patients and mice. RVX-208 substantially attenuated ex-vivo vascular dysfunction, with an impact greater than anti-IL-1beta, anti-IL-6 receptor and anti-TNF-alpha. The effect was more pronounced in vessels with intact PVAT, suggesting a restoration of the PVAT anti-contractile phenotype. Gene expression profiling in PVAT unveiled hexokinase-2 (HK2) - a glycolytic enzyme implicated in mitochondrial dysfunction and inflammation - as the top downregulated gene by RVX-208 treatment. Increased binding of BRD4 to HK2 promoter in PVAT samples from cardiometabolic mice was confirmed by ChIP assays. Metabolomics assays further validated the findings by demonstrating a glycolytic shift in PVAT under disease conditions. Finally, ex vivo selective inhibition of HK2 rescued vascular dysfunction. Conclusion: Targeting the deleterious BRD4-HK2 interplay restores cardiometabolic vascular dysfunction via reversal of the PVAT meta-inflammatory shift, highlighting a novel potential target to fight cardiometabolic pandemics.

Therapeutic modulation of epigenetic readers rescues obesity-induced vascular dysfunction: role of endothelium and perivascular adipose tissue

Abstract Background/Introduction Bromodomain and extraterminal proteins (BET), particularly BRD4, are epigenetic readers modulating transcriptional programs implicated in inflammation, cancer and renal disease. Therapeutic potential of BRD4 modulation in cardiometabolic disorders remains elusive. Purpose We investigate BRD4-related transcriptional programs and therapeutic modulation in mouse and human models of cardiometabolic disease. Methods Small arteries (100-300 μM) from healthy subjects (n=13) and patients with obesity (n=13) were dissected from visceral fat biopsies collected during elective laparoscopy and mounted on a pressurised myograph to assess the ex-vivo effects of BRD4 inhibition on vascular function. Vasorelaxation to acetylcholine and acetylcholine+L-NAME were evaluated at baseline and after incubation with the BRD4 inhibitor RVX-208. Anti-IL-1β, anti-IL-6 receptor and anti-TNF-α were also employed to assess the relative contribution of distinct BRD4-related pathways. Vasorelaxation was performed in the presence or in the absence of perivascular adipose tissue (PVAT). An experimental mouse model of cardiometabolic disease (high-fat diet+L-NAME supplementation) was orally administered RVX-208 (150 mg/kg) or vehicle for 10 days (n=6 each group) to test in vivo effect of chronic BRD4 inhibition on endothelial and PVAT functions. Vascular and PVAT levels of cytosolic and mitochondrial ROS and nitric oxide were assessed by confocal microscopy; protein and gene expression was measured by Western blot and qPCR. Transcriptional changes upon BRD4 inhibition were investigated by a custom PCR array. Results In patients with obesity, endothelial-dependent vasorelaxation and vascular nitric oxide availability were impaired. ROS levels, as well as TNF-α, IL-1β and IL-6, were increased in the vessel wall and PVAT. In these subjects, RVX-208 substantially attenuated ex-vivo vascular dysfunction. The effect was more pronounced in vessels with intact PVAT, suggesting a restoration of the PVAT anti-contractile phenotype (Figure 1). Distinct ex-vivo modulation of well-established inflammatory pathways showed that the effect observed with BRD4 inhibition was stronger than with anti-IL-1β, anti-IL-6 receptor and anti-TNF-α. In vivo, chronic treatment with RVX-208 restored endothelial vasorelaxation by rescuing PVAT dysfunction. Gene expression profiling in PVAT from cardiometabolic mice unveiled hexokinase-2 (HK2) - a glycolytic enzyme implicated in mitochondrial dysfunction and inflammation - as the top downregulated gene by RVX-208 treatment (Figure 2). Conclusions Therapeutic modulation of BRD4 restores cardiometabolic-related vascular dysfunction and PVAT anti-contractile properties. These effects are explained by a suppression of PVAT inflammation (IL-1β, IL-6 and TNF-α), likely driven by HK2 activation. BET inhibitors might represent promising epi-drugs for the prevention and treatment of vascular dysfunction, a hallmark of cardiometabolic disease.

Lipoprotein lipase activation is a novel vascular anti-contractile mechanism that is lost in aging

Increasing incidence of cardiovascular events with age can be attributed to the natural deterioration in organ function, and manifests through a range of vascular phenotypes that include, endothelial dysfunction, hypercontractility, stiffening and remodeling, inflammation, and oxidative stress. Given the aging global population, the identification and translation of novel mechanisms and therapeutics will be essential to increase longevity and prevent premature death and disability. Physiologically, perivascular adipose tissue (PVAT) is known to impact vasculature by exerting an anti-contractile effect mediated by the biosynthesis and liberation of a myriad of anti-contractile molecules (e.g., adipokines). However, this anti-contractile function declines with age and in related diseases. Therefore, our study aimed to restore the anti-contractile effect of PVAT in arteries from aged animals. Specifically, we predicted that increasing lipoprotein lipase (LPL) activity would reinstate the anti-contractile effect of PVAT on aged arteries. To assess this hypothesis, mesenteric resistance arteries (MRA), with (+) and without (-) PVAT, were isolated from male Fisher 344 rats aged 3 (3M) or 16 months (16M) and mounted on a wire myograph. Phenylephrine (PE) concentration-response curves were measured in the presence or absence of LPL activator, NO-1886 (Ibrolipim, 10uM). As expected, PVAT exerted significant anti-contractile effect in MRA, regardless of age [PE 100uM (%), 3M (-PVAT): 112.4±13.3 vs. 3M (+PVAT): 27.6±28.5 {Figure 1.a.} and 16M (-PVAT): 115.4±23.1 vs. 16M (+PVAT): 35.0±34.1 {Figure 1.b.}]. Contrary to our hypothesis, NO-1886 had no effect on the anti-contractile effect of PVAT in 3M or 16M rats [PE 100uM (%), 3M (+PVAT +vehicle): 30.0±34.3 vs. 3M (+PVAT +NO-1886): 47.9±29.8 {Figure 1.c.} and 16M (+PVAT +vehicle): 48.6±42.3 vs. 16M (+PVAT +NO-1886): 60.0±36.9 {Figure 1.d.}]. Surprisingly, we observed an effect of NO-1886 in MRA in the absence of PVAT. Specifically, NO-1886 significantly decreased phenylephrine-induced contraction in 3M rats, but not 16M rats [PE 100uM (%), 3M (-PVAT +vehicle): 112.4±13.3 vs. 3M (-PVAT +NO-1886): 37.8±22.2 {Figure 1.e.} and 16M (-PVAT +vehicle): 115.4±23.1 vs. 16M (-PVAT +NO-1886): 103.1±23.1 {Figure 1.f.}]. Overall, this suggests that LPL has an important physiological role in controlling vascular contraction and this novel anti-contractile mechanism of LPL diminishes with biological age. Funding: NHLBI (R00HL151889), NIGMS (1P20GM103641 - Pilot Project), NIA (K01AG061263), UofSC SOM (Startup funds) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PS-BPB09-4: ARTERIAL SITES AND SEX DIFFERENCES IN ENHANCING VASORELAXATION RESPONSE BY PERIVASCU-LAR ADIPOSE TISSUE IN METABOLIC SYNDROME RATS

Objective: Regulation of arterial tone by perivascular adipose tissue (PVAT) differs with sex. We have demonstrated that PVAT compensates vascular tone when vascular dysfunc-tion occurs in mesenteric arteries of male SHRSP. Z-Leprfa/IzmDmcr (SPZF) rats, but such compensation function disappears in the late stage of metabolic syndrome (MetS). In con-trast, the favorable effects of PVAT remain in female SPZF even after the effects disappear in age-matched males. Design and method: Therefore, in this study, we investigated whether the sex differences in PVAT response are observed in another arterial sites in SPZF and in another MetS model, SHR/NDmcr-cp (CP) rats as well. Renal arteries were isolated from male and female SPZF rats and CP rats at 23 weeks of age. Ring preparations with and without PVAT were made. Vasodilation and mRNA transcript levels in PVAT were examined using organ bath methods and quantitative real-time PCR, respectively. Results, and Conclusions: In renal arteries without PVAT of SPZF and CP rats, acetylcholine-induced relaxations were smaller in female than in male. The presence of PVAT increased the relaxations in CP rats only. In contrast, sodium nitroprusside-induced relaxations, nevertheless presence or ab-sence of PVAT, were smaller in female than in male in SPZF rats, but sex differences were not observed in CP rats. Apelin mRNA levels in female CP rats were the highest among groups. The enhancements of acetylcholine-induced relaxations by PVAT were positively correlated with apelin mRNA levels in PVAT. These results demonstrated that sex difference in enhancing vasorelaxation response by PVAT in renal arteries differs to that in mesenteric arteries in SPZF rats at the same age and to that in another MetS model strain, CP rats, at the same age and arterial sites. This discrepancy observed in female rats between two MetS strains may be associated with the difference in arterial dysfunction mechanism; namely, impairment of only endothelial nitric oxide production in CP rats, and deterioration of re-sponse to nitric oxide in smooth muscle in SPZF rats. Furthermore, apelin levels of PVAT may be involved in the appearance of modulation of PVAT on arterial tone.

Contribution of RAS, ROS and COX-1-derived prostanoids to the contractile profile of perivascular adipose tissue in cafeteria diet-induced obesity

AimsObesity can lead to the loss of the anticontractile properties of perivascular adipose tissue (PVAT). Given that cafeteria (CAF) diet reflects the variety of highly calorie and easily accessible foods in Western societies, contributing to obesity and metabolic disorders, we sought to investigate the impact of CAF diet on PVAT vasoactive profile and the involvement of renin-angiotensin system, oxidative stress, and cyclooxygenase pathway. Main methodsMale Balb/c mice received standard or CAF diet for 4 weeks. Oral glucose tolerance and insulin sensitivity tests were performed, and fasting serum glucose, cholesterol and triglyceride parameters were determined. Vascular reactivity, fluorescence and immunofluorescence analyzes were carried out in intact thoracic aorta in the presence or absence of PVAT. Key findingsCAF diet was effective in inducing obesity and metabolic disorders, as demonstrated by increased body weight gain and adiposity index, hyperlipidemia, hyperglycemia, glucose intolerance and insulin insensitivity. Importantly, CAF diet led to a significant decrease in aortic contractility which was restored in the presence of PVAT, exhibiting therefore a contractile profile. The contractile effect of PVAT was associated with the activation of AT1 receptor, reactive oxygen species, cyclooxygenase-1, thromboxane A2 and prostaglandin E2 receptors. SignificanceThese findings suggest that the contractile profile of PVAT involving the renin-angiotensin system activation, reactive oxygen species and cyclooxygenase-1 metabolites may be a protective compensatory adaptive response during early stage of CAF diet-induced obesity as an attempt to restore the impaired vascular contraction observed in the absence of PVAT, contributing to the maintenance of vascular tone.

Neutrophil Extracellular Traps Contribute to Perivascular Adipose Tissue Dysfunction in High‐Fat Diet Obese Mice

BackgroundObesity triggers functional changes in the perivascular adipose tissue (PVAT), favoring the release of vasoconstrictor factors and the activation of contractile mechanisms in vascular smooth muscle cells. In the PVAT of obese individuals, there is infiltration of immune cells, including neutrophils. A neutrophil defense mechanism is the formation of neutrophil extracellular traps (NETs), a cytotoxic framework that allows maintenance and expansion of inflammatory processes. We hypothesize that PVAT dysfunction in obese mice is due to increased neutrophil infiltration, NETs formation and inflammation.MethodsSix weeks‐old male Balb/C mice received control (CD) or high fat diet (HFD) for 18 weeks. Vascular function was assessed in endothelium‐intact mesenteric arteries in the presence or absence of PVAT. Mice were treated with DNAse type 1 (10 mg/kg for 14 days, every other day). Molecular mechanisms were investigated. Experiments were approved by the Ethics Committee on Animal Research of the Ribeirao Preto Medical School, University of Sao Paulo (protocol nº 209/2016)ResultsValues are expressed relatively to the contraction triggered by 120 mM KCl [Emax (% KCl)]. In Balb/C mice, the PVAT decreased the contractile response in control arteries [Control PVAT (‐): 123.2±8.1 n=6; Control PVAT (+): 64.4±4.0 n=6]. However, HFD promoted partial loss of PVAT anti‐contractile effect [HFD PVAT (‐): 127.9±6.9 n=6; HFD PVAT (+): 98.3±7.4 n=6]. In PVAT of obese mice there was a greater amount of neutrophils, compared to PVAT from control mice [Control: 8.0% n=5; HFD: 18.9% n=5]. In PVAT of obese mice there was greater formation of NETs, and production of interleukin 6 (IL‐6), compared to PVAT from control mice [NETs in pg/ml – Control: 30.5±2.7 n=6; HFD: 71.4±3.2 n=6; IL‐6 in pg/100 mg of PVAT – Control: 45.6±1.9 n=6; HFD: 98.4±3.1 n=6]. DNAse treatment reduced the formation of NETs and IL‐6 in the PVAT of obese mice [NETs in pg/ml – HFD+DNAse: 18.3±1.2 n=6; IL‐6 in pg/100 mg of PVAT – HFD: 48.1±3.4 n=6]. DNAse treatment restored PVAT anticontractile function in obese mice [HFD+DNAse PVAT (+): 71.3±7.4 n=6]. Adipose tissue of obese humans exhibited greater formation of NETs and production of IL‐6 compared to adipose tissue of eutrophic humans [NETs in pg/ml – Eutrophic: 20.5±3.7 n=7; Obese: 79.2±1.3 n=7; IL‐6 in pg/100 mg of adipose tissue – Eutrophic: 9.7±2.9 n=7; Obese: 27.7±2.6 n=7].ConclusionThese results indicate that, in obesity, there is a greater neutrophils infiltration in the PVAT and, consequently, greater formation of NETs and inflammation, events that contribute to PVAT dysfunction.

High-Carbohydrate Diet Enhanced the Anticontractile Effect of Perivascular Adipose Tissue Through Activation of Renin-Angiotensin System

The perivascular adipose tissue (PVAT) is an active endocrine organ responsible for release several substances that influence on vascular tone. Increasing evidence suggest that hyperactivation of the local renin-angiotensin system (RAS) in the PVAT plays a pivotal role in the pathogenesis of cardiometabolic diseases. However, the local RAS contribution to the PVAT control of vascular tone during obesity is still not clear. Since the consumption of a high-carbohydrate diet (HC diet) contributes to obesity inducing a rapid and sustained increase in adiposity, so that the functional activity of PVAT could be modulated, we aimed to evaluate the effect of HC diet on the PVAT control of vascular tone and verify the involvement of RAS in this effect. For that, male Balb/c mice were fed standard or HC diet for 4 weeks. Vascular reactivity, histology, fluorescence, and immunofluorescence analysis were performed in intact thoracic aorta in the presence or absence of PVAT. The results showed that HC diet caused an increase in visceral adiposity and also in the PVAT area. Phenylephrine-induced vasoconstriction was significantly reduced in the HC group only in the presence of PVAT. The anticontractile effect of PVAT induced by HC diet was lost when aortic rings were previously incubated with angiotensin-converting enzyme inhibitor, Mas, and AT2 receptors antagonists, PI3K, nNOS, and iNOS inhibitors, hydrogen peroxide (H2O2) decomposing enzyme or non-selective potassium channels blocker. Immunofluorescence assays showed that both Mas and AT2 receptors as well as nNOS and iNOS isoforms were markedly expressed in the PVAT of the HC group. Furthermore, the PVAT from HC group also exhibited higher nitric oxide (NO) and hydrogen peroxide bioavailability. Taken together, these findings suggest that the anticontractile effect of PVAT induced by HC diet involves the signaling cascade triggered by the renin-angiotensin system through the activation of Mas and AT2 receptors, PI3K, nNOS, and iNOS, leading to increased production of nitric oxide and hydrogen peroxide, and subsequently opening of potassium channels. The contribution of PVAT during HC diet-induced obesity could be a compensatory adaptive characteristic in order to preserve the vascular function.

Anticontractile Effect of Perivascular Adipose Tissue But Not of Endothelium Is Enhanced by Hydrogen Sulfide Stimulation in Hypertensive Pregnant Rat Aortae.

Perivascular adipose tissue (PVAT) modulates the vascular tone. Hydrogen sulfide (H2S) is synthetized by cystathionine gamma-lyase (CSE) in brown PVAT. Modulation of vascular contractility by H2S is, in part, adenosine triphosphate (ATP)-sensitive potassium channels dependent. However, the role of PVAT-derived H2S in hypertensive pregnancy (HTN-Preg) is unclear. Therefore, we aimed to examine the involvement of H2S in the anticontractile effect of PVAT in aortae from normotensive and hypertensive pregnant rats. To this end, phenylephrine-induced contractions in the presence and absence of PVAT and endothelium in aortae from normotensive pregnant (Norm-Preg) and HTN-Preg rats were investigated. Maternal blood pressure, fetal-placental parameters, angiogenesis-related biomarkers, and H2S levels were also assessed. We found that circulating H2S is elevated in hypertensive pregnancy associated with angiogenic imbalance, fetal and placental growth restrictions, which revealed that there is H2S pathway activation. Moreover, under stimulated H2S formation PVAT, but not endothelium, reduced phenylephrine-induced contractions in aortae from HTN-Preg rats. Also, H2S synthesis inhibitor abolished anticontractile effects of PVAT and endothelium. Furthermore, anticontractile effect of PVAT, but not of endothelium, was eliminated by ATP-sensitive potassium channels blocker. In accordance, increases in H2S levels in PVAT and placenta, but not in aortae without PVAT, were also observed. In conclusion, anticontractile effect of PVAT is lost, at least in part, in HTN-Preg aortae and PVAT effect is ATP-sensitive potassium channels dependent in normotensive and hypertensive pregnant rat aortae. PVAT but not endothelium is responsive to the H2S stimulation in hypertensive pregnant rat aortae, implying a key role for PVAT-derived H2S under endothelial dysfunction.

Abstract P061: Identification Of Piezo1 Channels In Perivascular Adipose Tissue (pvat) And Their Potential Role In Vascular Function

The vasculature constantly experiences distension/pressure exerted by the blood and responds accordingly to maintain homeostasis. Perivascular adipose tissue (PVAT) is gaining support as a formal blood vessel layer and also experiences these changes. We hypothesized that activation of the mechanotransducer Piezo1 directly increases vascular contraction in a way that might be modified by PVAT. The presence of Piezo1 was investigated at the mRNA level via PCR; protein via immunohistochemistry; and contractility via isolated tissue bath. Rat superior and mesenteric arteries, thoracic aortae, human mesenteric vessels and their PVATs were studied. Piezo1 mRNA (beta2 microglobulin calibrator) was expressed in the aortic vessel (2 -ΔC T =0.011); aortic PVAT (2 -ΔC T =0.0172); mesenteric vessel (2 -ΔC T =.00302), and mesenteric PVAT (2 -ΔC T =0.0219). Both adipocytes (2 -ΔC T =0.0249) and stromal vascular fraction (2 -ΔC T =0.0159) of mesenteric PVAT expressed Piezo1 mRNA. Piezo1 mRNA expression was greater in magnitude (one-way ANOVA) than that of the mechanotransducers Piezo2, TRPV4, TMEM16, and Panx1. Piezo1 protein was present in rat aortic PVAT, rat mesenteric (mes) artery, vein, and PVAT, as well as in human artery, vein, and PVAT. The Piezo1 agonists Yoda and Jedi (1 nM - 10 μM) did not stimulate rat aortic contraction [max <10% phenylephrine (PE) 10 μM contraction] or relaxation independent from vehicle in tissues + or - PVAT (relaxation as % of half maximal PE contraction was: Veh-PVAT=45.3±7.0; Yoda-PVAT=46.7±25.6; Jedi-PVAT= 40.4±10.3; Veh+PVAT= 71.8±19.7; Jedi+PVAT=39.1±13.2; Yoda +PVAT=21.6±10.9). Slightly K+ depolarizing the aorta did not unmask contraction to Yoda. Finally, the Piezo1 antagonist Dooku [10 μM] did not shift the PE curve (-log EC50 values [M]: Veh-PVAT= 7.96±0.12; Dooku-PVAT=7.26±0.22, Veh+PVAT=7.29±0.08; Dooku+PVAT=6.96±0.07). Surprisingly, Dooku [10 μM] directly caused aortic contraction in the absence of PVAT (Dooku 27.2±11.7 vs vehicle 13.5±11.2 %PE contraction), but not in the presence of PVAT vs vehicle (Dooku 2.9±1.9 vs Vehicle 7.3±5.2% PE contraction). Thus, Piezo1 is present and functional in the isolated aorta, important knowledge given that this molecule may serve as a translator of vascular pressure.

Perivascular adipose tissue phenotype and sepsis vascular dysfunction: Differential contribution of NO, ROS and beta 3-adrenergic receptor

AimsVascular dysfunction plays a key role in sepsis but the role of perivascular adipose tissue (PVAT) in this condition is relatively unknown. Main methodsSepsis was induced by cecal ligation and puncture (CLP). The responses of the aorta and superior mesenteric artery to norepinephrine in the presence or absence of PVAT were evaluated. Fluorescent probes measured the production of nitric oxide (NO) and reactive oxygen species (ROS). NO synthases (NOS) and β3-adrenoceptor expression were detected by immunofluorescence and S-nitrosylation by the biotin switch assay. Key findingsAorta and superior mesenteric arteries from septic animals with intact PVAT showed a worsened response to the vasoconstrictor compared to vessels without PVAT. PVAT from the aorta (APVAT) produced NO and ROS whereas PVAT from the superior mesenteric artery (MPVAT) produced only ROS. Septic APVAT exhibited a higher density of NOS-1 and NOS-3. S-nitrosylation was found in APVAT. Donor (PVAT obtained from normal or septic rats):Host (normal vessel without PVAT) experiments showed that L-NAME, ODQ and β3-adrenergic receptor antagonist blocked the septic APVAT anti-contractile effect. None of these compounds affected MPVAT; tempol, but not apocynin, blocked its anti-contractile effect. SignificancePVAT contributes to the anti-contractile effect in the aorta and mesenteric artery of septic rats through different pathways. β3-Adrenergic receptor and NO appear to be key mediators of this effect in APVAT, but not in MPVAT where ROS seem to be a relevant mediator. Therefore, PVAT is a relevant player of sepsis vascular dysfunction.

Postpartum Maternal Vascular Function in a Rat Model of Gestational Obstructive Sleep Apnea

IntroductionDe novo obstructive sleep apnea (OSA) in pregnancy, is associated with adverse gestational outcomes such as preeclampsia, gestational diabetes, and fetoplacental hypoxia. In addition, exposure to chronic intermittent hypoxia (CIH), a model of OSA, induces endothelial dysfunction and enhanced vasoconstriction in pregnant mice. It is currently unknown whether gestational OSA has a long‐term effect on maternal vascular function. We hypothesized that exposure to gestational CIH during late pregnancy will result in postpartum maternal vascular dysfunction.MethodsTimed pregnant female Long‐Evans rats were randomly assigned to two experimental groups: Normoxia (n=6) and CIH (n=6). The CIH group was exposed to five days (gestational days: 15–20) of intermittent hypoxia [6 min cycles of 3 min hypoxia (10% O2) and 3 min normoxia (21% O2)]. Gestational age at delivery was recorded and neonate’s crown‐rump length, abdominal girth, and body weights were measured within 12–16 hours from birth. At weaning (postnatal day 28), dams were euthanized and third‐order mesenteric arteries, and the main uterine artery, and uterine perivascular adipose tissue (PVAT) were excised. To assess endothelium‐dependent relaxation, we performed concentration‐response curves to acetylcholine (ACh: 10−9 – 3×10−5 M) using wire myography. In uterine arteries, we measured responses to ACh in the presence and absence of PVAT (0.1 g) based on our previous findings that uterine PVAT has vasoactive effects on these arteries.ResultsGestational CIH had no effect on the duration of gestation (Normoxia: 22.3 ± 0.21 days vs. CIH: 22.8 ± 1.16 days, p=0.24), litter size (Normoxia: 12.2 ± 1.64 vs. CIH: 10.3 ± 1.40, p=0.34), and neonatal weight at birth (Normoxia: 6.4 ± 0.24 g vs. CIH: 6.7 ± 0.19 g, p=0.16), while it increased neonatal crown‐rump length (Normoxia: 3.98 ± 0.07 cm vs. CIH: 4.19 ± 0.07 cm, p=0.05) and abdominal girth (Normoxia: 4.72 ± 0.10 cm vs. 4.94 ± 0.03 cm, p=0.07). At weaning, dams exposed to gestational CIH were lighter compared to dams exposed to normoxia (Normoxia: 319 ± 6.5 g vs. CIH: 298 ± 8.7 g, p=0.08). Exposure to gestational CIH had no effect on ACh‐induced relaxation in mesenteric resistance arteries (pEC50, Normoxia: 7.80 ± 0.03 vs. CIH: 7.89 ± 0.06, p=0.19). In the absence of PVAT, uterine arteries from dams exposed to gestational CIH had exaggerated responses to ACh compared to dams exposed to normoxia [(−)PVAT/pEC50, Normoxia: 6.77 ± 0.08 vs. CIH: 7.13 ± 0.09, p<0.01], while PVAT normalized this difference [(+)PVAT/pEC50, Normoxia: 6.67 ± 0.08 vs. CIH: 6.70 ± 0.07, p=0.99]. The effects of PVAT were due to its anti‐dilatory influences on uterine arteries from CIH‐treated rats [CIH (−PVAT) vs. CIH (+PVAT), p = 0.003], whereas it had no effect on arteries from control rats [Normoxia (−PVAT) vs. Normoxia (+PVAT), p = 0.77].ConclusionExposure to CIH during gestation resulted in neonatal macrosomia, reduced maternal weight at weaning, and exaggerated postpartum uterine vascular smooth muscle relaxation responses, while it did not affect small resistance artery reactivity. Gestational OSA may impair maternal vascular recovery after birth.Support or Funding InformationUNTHSC Basic Seed Grant

β3 -Adrenoceptor stimulation of perivascular adipocytes leads to increased fat cell-derived NO and vascular relaxation in small arteries.

Background and PurposeIn response to noradrenaline, healthy perivascular adipose tissue (PVAT) exerts an anticontractile effect on adjacent small arterial tissue. Organ bath solution transfer experiments have demonstrated the release of PVAT‐derived relaxing factors that mediate this function. The present studies were designed to investigate the mechanism responsible for the noradrenaline‐induced PVAT anticontractile effect.Experimental Approach In vitro rat small arterial contractile function was assessed using wire myography in the presence and absence of PVAT and the effects of sympathomimetic stimulation on the PVAT environment explored using Western blotting and assays of organ bath buffer.Key ResultsPVAT elicited an anticontractile effect in response to noradrenaline but not phenylephrine stimulation. In arteries surrounded by intact PVAT, the β3‐adrenoceptor agonist, CL‐316243, reduced the vasoconstrictor effect of phenylephrine but not noradrenaline. Kv7 channel inhibition using XE 991 reversed the noradrenaline‐induced anticontractile effect in exogenously applied PVAT studies. Adrenergic stimulation of PVAT with noradrenaline and CL‐316243, but not phenylephrine, was associated with increased adipocyte‐derived NO production, and the contractile response to noradrenaline was augmented following incubation of exogenous PVAT with L‐NMMA. PVAT from eNOS−/− mice had no anticontractile effect. Assays of adipocyte cAMP demonstrated an increase with noradrenaline stimulation implicating Gαs signalling in this process.Conclusions and ImplicationsWe have shown that adipocyte‐located β3‐adrenoceptor stimulation leads to activation of Gαs signalling pathways with increased cAMP and the release of adipocyte‐derived NO. This process is dependent upon Kv7 channel function. We conclude that adipocyte‐derived NO plays a central role in anticontractile activity when rodent PVAT is stimulated by noradrenaline.

Intrauterine exposure to metformin: Evaluation of endothelial and perivascular adipose tissue function in abdominal aorta of adult offspring

The biguanide metformin (MET) has been used during pregnancy for treatment of polycystic ovary syndrome and gestational diabetes. MET crosses the placenta and maternal treatment can expose the progeny to this drug during important phases of body development. Direct vascular protective effects have been described with the treatment of metformin. Nevertheless, it is unclear whether intrauterine exposure to metformin is safe for the vascular system of offspring. Thus, the present study aimed to investigate the intrinsic effects of metformin exposure in utero in the offspring abdominal aorta reactivity, in the presence and absence of perivascular adipose tissue (PVAT) and endothelium. For this, Wistar rats were treated with metformin 293 mg/kg/day (MET) or water (CTR) by gavage during the gestational period. The abdominal aorta reactivity to phenylephrine, acetylcholine, and sodium nitroprusside was evaluated in male adult offspring. It was observed that abdominal aorta relaxation was similar between MET and CTR groups in the presence or absence of PVAT. In addition, the contraction to phenylephrine was similar between MET and CTR groups in the presence and absence of PVAT and endothelium. Therefore, metformin exposure during pregnancy had no intrinsic effect on the offspring abdominal aorta PVAT and endothelial function, demonstrating it to be safe to the vascular system of the offspring.

Modulation of BAY 41-2272-induced-relaxation in normotensive and hypertensive rat aorta by perivascular adipose tissue

In spontaneously hypertensive rat (SHR), perivascular adipose tissue (PVAT) has been recently identified as the effector of vascular dysfunction. In order to restore this alteration, therapeutic strategies target the cGMP/PKG pathway which is involved in both vascular and adipose tissue functions. However, direct activation of soluble guanylate cyclase, a key enzyme of this signalling pathway has not been studied yet in arterial hypertension. This study was designed to determine whether relaxation induced by BAY 41-2272, a soluble guanylate cyclase stimulator agent, was modified in SHR aorta and whether PVAT modulates this effect. Aortic rings isolated from 12-week-old Wistar Kyoto (WKY) rats and SHR were mounted in organ baths and pre-constricted with phenylephrine. Cumulative concentration-response curves to BAY 41-2272 (0.1 nM to 30 μM) were constructed in different experimental conditions in the absence or presence of endothelium and PVAT. In the absence of PVAT, BAY 41-2272 produced concentration-dependent relaxation, which was inhibited by L-NAME (100 μM), a nitric oxide (NO) synthase inhibitor only in WKY rat aorta. Endothelium denudation enhanced the BAY 41-2272-induced-relaxation only in SHR aorta. In both rat strains, relaxant effect to BAY 41-2272 was attenuated by PVAT and this was associated with a decrease in tissue cGMP content. NO and cyclo-oxygenases (COX)-derived products do not appear to participate to this impairment since 100 μM L-NAME and 10 μM indomethacin, a non-specific COX inhibitor, do not modify the BAY 41-2272-induced-relaxation. We showed for the first time that BAY 41-2272 partially relaxes WKY rat aorta by NO-dependent-mechanism and that the endothelium negatively modulates the BAY 41-2272-induced-relaxation only in hypertensive rats. The decrease of BAY 41-2272-induced-relaxation in SHR aorta by PVAT suggests the release of adipose contracting factors that need further elucidation.

Sensory innervation of perivascular adipose tissue: a crucial role in artery vasodilatation and leptin release.

Electrical field stimulation (EFS) elicits robust sensory neurogenic relaxation responses in the rat isolated mesenteric arterial bed but these responses are absent or difficult to demonstrate in isolated arteries. We believe that this mismatch is due to the absence of perivascular adipose tissue (PVAT) as it is conventionally removed in studies on isolated vessels. We aimed to determine whether sensory nerves are expressed in PVAT, their physiological roles and their possible interactions with PVAT-derived adipokines. Using confocal imaging, enzyme immunoassay (EIA), myography, vascular perfusion, and multiplex analysis of rat mesenteric arteries, we show that PVAT is crucial for the roles of sensory nerves in control of vasomotor tone and adipokine release. Immunofluorescence double staining showed co-expression of calcitonin gene-related peptide (CGRP; sensory neurotransmitter) and PGP9.5 (neuronal marker) in PVAT of mesenteric arteries. CGRP release from dissected PVAT, measured using EIA, was increased by capsaicin which activates sensory nerves. EFS in both mesenteric arteries and perfused mesenteric arterial beds, with and without PVAT, demonstrated neurogenic relaxation in the presence of PVAT, which was greatly attenuated in preparations without PVAT. Neurogenic relaxation due to EFS was associated with release of leptin in PVAT-intact mesenteric arterial beds, which was abolished in preparations without PVAT. Exposure to low oxygen was associated with an attenuated leptin and adiponectin release, but an increase in IL-6 release, from mesenteric arterial beds. Exogenous leptin augmented relaxation to CGRP in mesenteric arteries. These data show, for the first time, expression of sensory nerves within PVAT and that PVAT is crucial for sensory neurogenic vasorelaxation and crosstalk with adipocytes leading to leptin release, which may augment CGRP-mediated relaxation; leptin release is abolished after exposure to conditions of reduced oxygenation.

The Role of DPO-1 and XE991-Sensitive Potassium Channels in Perivascular Adipose Tissue-Mediated Regulation of Vascular Tone.

The anti-contractile effect of perivascular adipose tissue (PVAT) is an important mechanism in the modulation of vascular tone in peripheral arteries. Recent evidence has implicated the XE991-sensitive voltage-gated KV (KCNQ) channels in the regulation of arterial tone by PVAT. However, until now the in vivo pharmacology of the involved vascular KV channels with regard to XE991 remains undetermined, since XE991 effects may involve Ca2+ activated BKCa channels and/or voltage-dependent KV1.5 channels sensitive to diphenyl phosphine oxide-1 (DPO-1). In this study, we tested whether KV1.5 channels are involved in the control of mesenteric arterial tone and its regulation by PVAT. Our study was also aimed at extending our current knowledge on the in situ vascular pharmacology of DPO-1 and XE991 regarding KV1.5 and BKCa channels, in helping to identify the nature of K+ channels that could contribute to PVAT-mediated relaxation. XE991 at 30 μM reduced the anti-contractile response of PVAT, but had no effects on vasocontraction induced by phenylephrine (PE) in the absence of PVAT. Similar effects were observed for XE991 at 0.3 μM, which is known to almost completely inhibit mesenteric artery VSMC KV currents. 30 μM XE991 did not affect BKCa currents in VSMCs. Kcna5−/− arteries and wild-type arteries incubated with 1 μM DPO-1 showed normal vasocontractions in response to PE in the presence and absence of PVAT. KV current density and inhibition by 30 μM XE991 were normal in mesenteric artery VSMCs isolated from Kcna5−/− mice. We conclude that KV channels are involved in the control of arterial vascular tone by PVAT. These channels are present in VSMCs and very potently inhibited by the KCNQ channel blocker XE991. BKCa channels and/or DPO-1 sensitive KV1.5 channels in VSMCs are not the downstream mediators of the XE991 effects on PVAT-dependent arterial vasorelaxation. Further studies will need to be undertaken to examine the role of other KV channels in the phenomenon.