Intact Perivascular Adipose Tissue Research Articles

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.

Aldose Reductase (AR) Mediates and Perivascular Adipose Tissue (PVAT) Modulates Endothelial Dysfunction of Short-Term High-Fat Diet Feeding in Mice.

Increased adiposity of both visceral and perivascular adipose tissue (PVAT) depots is associated with an increased risk of diabetes and cardiovascular disease (CVD). Under healthy conditions, PVAT modulates vascular tone via the release of PVAT-derived relaxing factors, including adiponectin and leptin. However, when PVAT expands with high-fat diet (HFD) feeding, it appears to contribute to the development of endothelial dysfunction (ED). Yet, the mechanisms by which PVAT alters vascular health are unclear. Aldose reductase (AR) catalyzes glucose reduction in the first step of the polyol pathway and has been long implicated in diabetic complications including neuropathy, retinopathy, nephropathy, and vascular diseases. To better understand the roles of both PVAT and AR in HFD-induced ED, we studied structural and functional changes in aortic PVAT induced by short-term HFD (60% kcal fat) feeding in wild type (WT) and aldose reductase-null (AR-null) mice. Although 4 weeks of HFD feeding significantly increased body fat and PVAT mass in both WT and AR-null mice, HFD feeding induced ED in the aortas of WT mice but not of AR-null mice. Moreover, HFD feeding augmented endothelial-dependent relaxation in aortas with intact PVAT only in WT and not in AR-null mice. These data indicate that AR mediates ED associated with short-term HFD feeding and that ED appears to provoke 'compensatory changes' in PVAT induced by HFD. As these data support that the ED of HFD feeding is AR-dependent, vascular-localized AR remains a potential target of temporally selective intervention.

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.

BRD4 INHIBITION ATTENUATES OBESITY-RELATED MICROVASCULAR DYSFUNCTION: ROLE OF ENDOTHELIUM AND PERIVASCULAR ADIPOSE TISSUE

Objective: Bromodomain and extraterminal (BET) proteins, particularly BRD4, are epigenetic readers modulating transcriptional programs implicated in inflammation, cancer and renal disease. Though several findings suggest that BET proteins’ inhibition might be particularly relevant in the cardiometabolic field, an apparent evidence gap hinders its therapeutic potential. We aimed to define the impact of BRD4-related inflammatory response in the early stages of obesity to exploit the therapeutic potential of its inhibition. Design and method: Healthy controls (n = 13) and patients with obesity without other comorbidities (n = 13) were included in a case-control study. Small arteries from visceral fat depots were mounted on a pressurised myograph to assess vasorelaxation to acetylcholine and inhibition to L-NAME at baseline and after incubation with RVX-208 (an FDA-approved BRD4 inhibitor), canakinumab (targeting IL-1beta), tocilizumab (targeting IL-6 receptor) and infliximab (targeting TNF-alpha). The experiments were conducted, in parallel, on two vessels, one mounted with perivascular adipose tissue (PVAT) and the other without PVAT. Vascular- and PVAT-specific levels of NAPDH-derived ROS, mtROS and nitric oxide availability were assessed by confocal microscopy. Gene expression was evaluated by qPCR. Results: In patients with obesity, microvascular function and nitric oxide availability were reduced, where NADPH- and mtROS were increased. BRD4, TNF-alpha, IL-1beta and IL-6 expression levels were higher in the vessel wall and the PVAT of patients with obesity. In these patients, RVX-208 substantially attenuated microvascular dysfunction. The effect was more significant in vessels with intact PVAT, implying a restoration of the PVAT anti-contractile phenotype (Figure). The exploration of the distinct inflammatory pathways showed that the amelioration observed with BRD-4 inhibition was higher than with canakinumab, tocilizumab or infliximab. Peculiarly, anti-IL-1beta restoration of microvascular function was not affected by the presence of the PVAT, while the ones observed with the targeting of IL-6 receptor and TNF-alpha were. Conclusions: The inhibition of BRD4 restores microvascular dysfunction in patients with obesity by modulating pan-inflammatory response involving distinct IL-1beta, IL-6 and TNF-alpha pathways and restoring PVAT anti-contractile properties. Epigenetic drugs might represent a promising therapeutic strategy to rescue microvascular dysfunction, a hallmark of early cardiometabolic disease.

Lipoprotein lipase underlies the vascular anti-contractile effect of perivascular adipose tissue (PVAT)

Physiologically, perivascular adipose tissue (PVAT) exerts an anti-contractile effect on the vasculature. This effect is mediated by the biosynthesis and liberation of a myriad of anti-contractile molecules (e.g., adipokines). However, this anti-contractile function generally is lost with aging and cardiovascular diseases such as hypertension and diabetes. Lipoprotein lipase (LPL) is an enzyme classically known for hydrolyzing triglycerides into free fatty acids and glycerol. However, it is unknown whether LPL underlies the anti-contractile effect of PVAT. Specifically, we hypothesized that by inhibiting lipoprotein lipase (LPL) activity with Atglistatin, we would abolish the anti-contractile effect of PVAT. To test this hypothesis, mesenteric resistance arteries (MRA), with and without PVAT, were isolated from 3-month-old male Wistar rats and mounted on a wire myograph. Phenylephrine concentration-response curves were performed with and without Atglistatin (10 -5 M). As expected, PVAT exerted a significant anti-contractile effect on phenylephrine-induced contraction [Emax (mN), -PVAT-vehicle: 18.1±3.0 vs. +PVAT-vehicle: 7.1±1.1, p<0.05]. Incubation of MRA with intact PVAT with Atglistatin significantly increased contraction [Emax (mN), +PVAT-vehicle: 7.1±1.1vs. +PVAT-Atglistatin: 22.6±4.2, p<0.05], suggesting that LPL is indeed important for the anti-contractile effect of PVAT. Crucially, no difference was observed with Atglistatin in MRA lacking PVAT [Emax (mN), -PVAT-vehicle: 18.1±3.0 vs. -PVAT-Atglistatin: 18.5±2.9, p>0.05], indicating that Atglistatin was not increasing contraction via non-PVAT-dependent mechanisms. Overall, these data suggest that LPL is an important mechanism mediating the anti-contractile effect of PVAT and that reduced LPL activity could be contributing to pro-contractile PVAT in aging and cardiovascular diseases. NHLBI (R00HL151889), NIGMS (1P20GM103641 - Pilot Project), 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.

Abstract 14157: Effects of Follistatin on the Regulation of Vascular Function by Perivascular Adipose Tissue in a Model of Essential Hypertension

Introduction: We previously showed that follistatin (FST), a potent activin inhibitor, lowers BP in the spontaneously hypertensive rat (SHR), a model of essential HTN. This was associated with improved vessel structure (reduced collagen) and function (reduced hypercontractility and improved endothelium-dependent relaxation). FST also reduced vascular oxidative stress (ROS). Perivascular adipose tissue (PVAT) is known to regulate vascular tone. FST induces browning and thus increased thermogenic activity of peripheral adipose tissue. Transition from white to brown adipose tissue is associated with decreased risk of CVD. Here, we assess FST effects on PVAT and its regulation of SHR vascular function. Methods: SHR were treated with FST from 12-20 weeks of age. Normotensive WKY rats served as controls. Mesenteric resistance arteries were isolated with or without intact PVAT. Vessel function was assessed using wire myography. In separate studies, white mesenteric and brown aortic PVAT, or peripheral fat depots, were isolated from WKY rats. These were introduced to WKY mesenteric vessels to study PVAT-isolated function. Results: Intact PVAT augmented constriction to KCl in SHR, but not WKY arteries, an effect prevented by FST. Improvement in endothelium-dependent vasodilation in FST-treated SHR was PVAT-dependent. SHR PVAT had increased adipocyte size and ROS (by DHE), both reversed by FST. Isolated PVAT from WKY incubated with pyocyanin (30’) to induce ROS augmented the KCl response, suggesting that FST inhibition of PVAT oxidative stress in HTN restores beneficial vascular effects. Incubation of WKY vessels with brown PVAT inhibited constriction to KCl more effectively than did white PVAT. Interestingly, peripheral white adipose (inguinal) did not have any anticontractile effect. Brown adipose (interscapular) effects were intermediate. Conclusions: Improvement in anticontractile effects of PVAT by FST in resistance vessels is associated with reduced PVAT oxidative stress. Brown PVAT is more effective than white PVAT in inhibiting vessel contraction, and unlike white PVAT, peripheral white adipose does not have anticontractile effects. Future work will assess effects of FST on PVAT browning, and whether browning itself can improve BP.

The Vasoactive Role of Perivascular Adipose Tissue and the Sulfide Signaling Pathway in a Nonobese Model of Metabolic Syndrome.

The aim of this study was to evaluate the mutual relationship among perivascular adipose tissue (PVAT) and endogenous and exogenous H2S in vasoactive responses of isolated arteries from adult normotensive (Wistar) rats and hypertriglyceridemic (HTG) rats, which are a nonobese model of metabolic syndrome. In HTG rats, mild hypertension was associated with glucose intolerance, dyslipidemia, increased amount of retroperitoneal fat, increased arterial contractility, and endothelial dysfunction associated with arterial wall injury, which was accompanied by decreased nitric oxide (NO)-synthase activity, increased expression of H2S producing enzyme, and an altered oxidative state. In HTG, endogenous H2S participated in the inhibition of endothelium-dependent vasorelaxation regardless of PVAT presence; on the other hand, aortas with preserved PVAT revealed a stronger anticontractile effect mediated at least partially by H2S. Although we observed a higher vasorelaxation induced by exogenous H2S donor in HTG rats than in Wistar rats, intact PVAT subtilized this effect. We demonstrate that, in HTG rats, endogenous H2S could manifest a dual effect depending on the type of triggered signaling pathway. H2S within the arterial wall contributes to endothelial dysfunction. On the other hand, PVAT of HTG is endowed with compensatory vasoactive mechanisms, which include stronger anti-contractile action of H2S. Nevertheless, the possible negative impact of PVAT during hypertriglyceridemia on the activity of exogenous H2S donors needs to be taken into consideration.

Dysfunction of perivascular adipose tissue in mesenteric artery is restored by aerobic exercise in high‐fat diet induced obesity

This study investigated the function of perivascular adipose tissue (PVAT) on vascular contractility within resistant arteries in high-fat diet induced obese rats after long-term aerobic exercise. Male Sprague-Dawley rats were subjected to normal diet control group (N-CTRL), normal diet exercise group (N-EX), high-fat diet control group (H-CTRL), and high-fat diet exercise group (H-EX) (n=8 in each group). After intervention, adipose tissues morphology was observed. Vasomotor function of mesenteric arteries with or without PVAT were assessed; mesenteric PVAT isolated from each group were transferred to chambers bath with untreated vessels (without PVAT) to evaluate the independent effect. Isolated PVAT was further pre-treated with inhibitor of cystathionine-γ-lyase (CSE), a key hydrogen sulphide (H2 S) enzyme. Results showed that the size of lipid droplet around mesenteric arteries from H-EX was significantly reduced (P<.05); uncoupling protein1 (UCP1) in PVAT from H-EX was enhanced. In N-CTRL, N-EX, and H-EX, vessels without PVAT showed higher sensitivity to serotonin (5-HT) than that with intact PVAT. Vascular tension by 5-HT was significantly reduced in H-EX than H-CTRL (P<.05) in vessels with PVAT. Transferred PVAT from H-EX compared with H-CTRL significantly reduced vascular sensitivity to 5-HT (P<.05), and this effect was eliminated through inhibiting CSE. In summary, the anti-contractile effect of PVAT on resistance artery was impaired in obesity but restored by long-term aerobic exercise. The function of PVAT modified by obesity or by exercise has an independent influence on vascular reactivity, and PVAT derived H2 S may participate in this process.

Expression of Melatonin Receptor 1 in Rat Mesenteric Artery and Perivascular Adipose Tissue and Vasoactive Action of Melatonin

Melatonin is released by the pineal gland and can modulate cardiovascular system function via the G protein-coupled melatonin receptors MT1 and MT2. Most vessels are surrounded by perivascular adipose tissue (PVAT), which affects their contractility. The aim of our study was to evaluate mRNA and protein expression of MT1 and MT2 in the mesenteric artery (MA) and associated PVAT of male rats by RT-PCR and Western blot. Receptor localization was further studied by immunofluorescence microscopy. Effects of melatonin on neurogenic contractions were explored in isolated superior MA ex vivo by measurement of isometric contractile tension. MT1, but not MT2, was present in MA, and MT1 was localized mainly in vascular smooth muscle. Moreover, we proved the presence of MT1, but not MT2 receptors, in MA-associated PVAT. In isolated superior MA with intact PVAT, neuro-adrenergic contractile responses were significantly smaller when compared to arteries with removed PVAT. Pre-treatment with melatonin of PVAT-stripped arterial rings enhanced neurogenic contractions, while the potentiating effect of melatonin was not detected in preparations with preserved PVAT. We hypothesize that melatonin can stimulate the release of PVAT-derived relaxing factor(s) via MT1, which can override the direct pro-contractile effect of melatonin on vascular smooth muscle. Our results suggest that melatonin is involved in the control of vascular tone in a complex way, which is vessel specific and can reflect a sum of action on different layers of the vessel wall and surrounding PVAT.

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.

Influence of Age on Anticontractile Effect of Perivascular Adipose Tissue in Normotensive and Hypertensive Rats.

Perivascular adipose tissue (PVAT) and its vasomodulatory effects play an important role in the physiology and pathophysiology of blood vessels. Alterations in PVAT associated with reduction in its anticontractile influence are proven to contribute to vascular dysfunction in hypertension. The aim of this study was to examine whether the changes in PVAT properties could participate in progression of vascular abnormalities in developing spontaneously hypertensive rats (SHR). Normotensive Wistar-Kyoto (WKY) rats and SHR, both in 5th and in 12th week of age, were used. Systolic blood pressure was similar between WKY rats and SHR in 5th week of age; however, in 12th week, it was significantly increased in SHR comparing to WKY rats. The amount of retroperitoneal fat was higher in WKY rats in both age groups, whereas body weight was higher in WKY rats only in 12th week, when compared to age-matched SHR. From isolated superior mesenteric arteries, two ring preparations were prepared for isometric tension recording, one with PVAT intact and other with PVAT removed. In WKY rats as well as in SHR, arterial contractile responses to noradrenaline, applied cumulatively on rings, were significantly inhibited in the presence of intact PVAT. In both age groups, anticontractile effect of PVAT was higher in WKY rats than in SHR. Neurogenic contractions, induced by electrical stimulation of perivascular sympathoadrenergic nerves, were significantly attenuated in the presence of PVAT in WKY mesenteric arteries from both age groups; however, in arteries from SHR, intact PVAT had no influence on this type of contractile responses. The results suggest that in SHR impairment of anticontractile effect of PVAT precedes hypertension and might contribute to its development.

Maternal separation enhances anticontractile perivascular adipose tissue function in male rats on a high-fat diet.

Clinical studies have shown that obesity negatively impacts large arteries' function. We reported that rats exposed to maternal separation (MatSep), a model of early life stress, display enhanced angiotensin II (ANG II)-induced vasoconstriction in aortic rings cleaned of perivascular adipose tissue (PVAT) under normal diet (ND) conditions. We hypothesized that exposure to MatSep promotes a greater loss of PVAT-mediated protective effects on vascular function and loss of blood pressure (BP) rhythm in rats fed a high-fat diet (HFD) when compared with controls. MatSep was performed in male Wistar-Kyoto rats from days 2 to 14 of life. Normally reared littermates served as controls. On ND, aortic rings from MatSep rats with PVAT removed showed increased ANG II-mediated vasoconstriction versus controls; however, rings from MatSep rats with intact PVAT displayed blunted constriction. This effect was exacerbated by an HFD in both groups; however, the anticontractile effect of PVAT was greater in MatSep rats. Acetylcholine-induced relaxation was similar in MatSep and control rats fed an ND, regardless of the presence of PVAT. HFD impaired aortic relaxation in rings without PVAT from MatSep rats, whereas the presence of PVAT improved relaxation in both groups. On an HFD, immunolocalization of vascular smooth muscle-derived ANG-(1-7) and PVAT-derived adiponectin abundances were increased in MatSep. In rats fed an HFD, 24-h BP and BP rhythms were similar between groups. In summary, MatSep enhanced the ability of PVAT to blunt the heightened ANG II-induced vasoconstriction and endothelial dysfunction in rats fed an HFD. This protective effect may be mediated via the upregulation of vasoprotective factors within the adipovascular axis.

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.

Coronary artery hypoxic vasorelaxation is augmented by perivascular adipose tissue through a mechanism involving hydrogen sulphide and cystathionine-β-synthase.

Hypoxia causes vasodilatation of coronary arteries which protects the heart from ischaemic damage through mechanisms including the generation of hydrogen sulphide (H2 S), but the influence of the perivascular adipose tissue (PVAT) and myocardium is incompletely understood. This study aimed to determine whether PVAT and the myocardium modulate the coronary artery hypoxic response and whether this involves hydrogen sulphide. Porcine left circumflex coronary arteries were prepared as cleaned segments and with PVAT intact, myocardium intact or both PVAT and myocardium intact, and contractility investigated using isometric tension recording. Immunoblotting was used to measure levels of H2 S-synthesizing enzymes: cystathionine-β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulphurtransferase (MPST). All three H2 S-synthesizing enzymes were detected in the artery and myocardium, but only CBS and MPST were detected in PVAT. Hypoxia elicited a biphasic response in cleaned artery segments consisting of transient contraction followed by prolonged relaxation. In arteries with PVAT intact, hypoxic contraction was attenuated and relaxation augmented. In arteries with myocardium intact, hypoxic contraction was attenuated, but relaxation was unaffected. In replacement experiments, replacement of dissected PVAT and myocardium attenuated artery contraction and augmented relaxation to hypoxia, mimicking the effect of insitu PVAT and indicating involvement of a diffusible factor(s). In arteries with intact PVAT, augmentation of hypoxic relaxation was reversed by amino-oxyacetate (CBS inhibitor), but not DL-propargylglycine (CSE inhibitor) or aspartate (inhibits MPST pathway). PVAT augments hypoxic relaxation of coronary arteries through a mechanism involving H2 S and CBS, pointing to an important role in regulation of coronary blood flow during hypoxia.

Interaction of perivascular adipose tissue and sympathetic nerves in arteries from normotensive and hypertensive rats.

The inhibitory action of perivascular adipose tissue (PVAT) in modulation of arterial contraction has been recently recognized and contrasted with the prohypertensive effect of obesity in humans. In this study we demonstrated that PVAT might have opposing effect on sympatho-adrenergic contractions in different rat conduit arteries. In superior mesenteric artery isolated from normotensive Wistar-Kyoto rats (WKY), PVAT exhibited inhibitory influence on the contractions to exogenous noradrenaline as well as to endogenous noradrenaline released from arterial sympathetic nerves during transmural electrical stimulation or after application of tyramine. In contrast, the abdominal aorta with intact PVAT responded with larger contractions to transmural electrical stimulation and tyramine when compared to the aorta after removing PVAT; the responses to noradrenaline were similar in both. This indicates that PVAT may contain additional sources of endogenous noradrenaline which could be responsible for the main difference in the modulatory effect of PVAT on adrenergic contractions between abdominal aortas and superior mesenteric arteries. In spontaneously hypertensive rats (SHR), the anticontractile effect of PVAT in mesenteric arteries was reduced, and the removal of PVAT completely eliminated the difference in the dose-response curves to exogenous noradrenaline between SHR and WKY. These results suggest that in mesenteric artery isolated from SHR, the impaired anticontractile influence of PVAT might significantly contribute to its increased sensitivity to adrenergic stimuli.

Wild blueberry consumption attenuates local inflammation in the perivascular adipose tissue of obese Zucker rats

Perivascular adipose tissue (PVAT) has been shown to play important roles in regulating vascular tone and linking local and systemic vascular inflammation. We examined the impact of PVAT on phenylephrine-mediated vasoconstriction in the aorta of obese Zucker rats (OZR) and their lean littermates (LZR) by comparing aortic rings with or without PVAT. Subsequently we placed OZR and LZR on a control (C) or an 8% wild blueberry (WB) diet and evaluated the effect of WB consumption on such response. PVAT-released adipokine concentrations were also measured as a function of WB diet. Maximal constrictor force (Fmax) in aortic rings without PVAT was significantly lower in OZR-C compared with LZR-C (0.41 ± 0.05 and 0.71 ± 0.06 g, respectively). Following WB diet, Fmax significantly increased in OZR (0.54 ± 0.06 g). In aortas with intact PVAT, Fmax was significantly lower in all groups (0.31 ± 0.06 OZR-C, 0.30 ± 0.05 OZR-WB, 0.29 ± 0.03 LZR-C, and 0.30 ± 0.04 g LZR-WB), but no difference was observed between treatments. PVAT concentrations of monocyte chemoactractant protein 1 (MCP-1), tumor necrosis factor alpha, and adiponectin were significantly higher in OZR compared with LZR (+102%, +108%, and +45%, respectively). Following WB diet, PVAT concentrations of interleukin-8 were significantly lower in both OZR (-37%) and LZR (-30%), while adiponectin concentrations significantly increased in both OZR (+11%) and LZR (+16%). MCP-1 concentrations significantly decreased (-31%) in the PVAT of OZR with the WB diet. WB consumption appears to attenuate local inflammation in PVAT, which may impact systemic vascular inflammation and endothelial function.

Effect of long-term treatment with melatonin on vascular markers of oxidative stress/inflammation and on the anticontractile activity of perivascular fat in aging mice.

Some reports have suggested that inflammation in perivascular adipose tissue (PVAT) may be implicated in vascular dysfunction by causing the disappearance of an anticontractile effect. The aim of this study was to investigate the effects of chronic melatonin treatment on the functional responses of the small mesenteric arteries and on the expression of markers of inflammation/oxidative stress in the aortas of senescence-accelerated prone mice (SAMP8), a model of age-related vascular dysfunction. We investigated seven SAMP8 and seven control senescence-accelerated resistant mice (SAMR1) treated for 10 months with melatonin, as well as equal numbers of age-matched untreated SAMP8 and SAMR1. The mesenteric small resistance arteries were dissected and mounted on a wire myograph, and the concentration-response to norepinephrine was evaluated in vessels with intact PVAT and after the removal of the PVAT. The expression of markers of oxidative stress, inflammation and aging in the aortas was evaluated by immunostaining. In addition, the adiponectin content and the expression of adiponectin receptor 1 were evaluated in the visceral adipose tissue. In untreated SAMP8 mice, we observed an overexpression of oxidative stress and inflammatory markers in the vasculature compared with the controls. No anticontractile effect of the PVAT was observed in untreated SAMP8 mice. Long-term treatment of SAMP8 mice with melatonin increased the expression of some markers of vasoprotection, decreased oxidative stress and inflammation and restored the anticontractile effect of the PVAT. Decreased expression of adiponectin and adiponectin receptor 1 was also observed in visceral fat of untreated SAMP8, whereas a significant increase was observed after melatonin treatment.

Vitamin D controls resistance artery function through regulation of perivascular adipose tissue hypoxia and inflammation

Vitamin D deficiency in human subjects is associated with hypertension, metabolic syndrome and related risk factors of cardiovascular diseases. Serum 25-hydroxyvitamin D levels correlate inversely with adiposity in obese and lean individuals. Bioactive vitamin D, or calcitriol, exerts anti-inflammatory effects on adipocytes, preadipocytes and macrophages in vitro. We tested the hypothesis that vitamin D deficiency alters the phenotype of perivascular adipose tissue (PVAT) leading to impaired function in resistance artery. To examine the effects of vitamin D and PVAT on vascular reactivity, myograph experiments were performed on arteries, with or without intact PVAT, from mice maintained on vitamin D-deficient, vitamin D-sufficient or vitamin D-supplemented diet. Systolic blood pressure was significantly increased in mice on vitamin D-deficient diet. Importantly, vitamin D deficiency enhanced angiotensin II-induced vasoconstriction and impaired the normal ability of PVAT to suppress contractile responses of the underlying mesenteric resistance artery to angiotensin II and serotonin. Furthermore, vitamin D deficiency caused upregulation of the mRNA expression of tumor necrosis factor-α, hypoxia-inducible factor-1α and its downstream target lysyl oxidase in mesenteric PVAT. Incubation of mesenteric arteries under hypoxic conditions impaired the anti-contractile effects of intact PVAT on those arteries from mice on vitamin D-sufficient diet. Vitamin D supplementation protected arteries against hypoxia-induced impairment of PVAT function. The protective effects of vitamin D against vascular dysfunction, hypertension and cardiovascular diseases may be mediated, at least in part, through regulation of inflammatory and hypoxia signaling pathways in PVAT.