Endothelium-dependent Vasorelaxation Research Articles

The yeast carboxymethyl glucan reduces cardiometabolic risks by improving oxidative stress, endothelial dysfunction and platelet aggregation

The carboxymethyl glucan (CMG) obtained from Saccharomyces cerevisiae has been highlighted for its beneficial potential, especially for its high antioxidant capacity. Cardiovascular diseases are among the leading causes of death worldwide and are often associated with overweight and dietary intake. On the other hand, supplementation with bioactive foods may be important in the prevention and treatment of these diseases. In this sense, this study evaluated the effect of CMG on anthropometric, oxidative and cardiovascular variables. Wistar rats were divided into three groups: NCT - normal caloric control fed a standard diet; HCT - hypercaloric control; H-CMG - hypercaloric + CMG (20 mg/kg/day) fed a hypercaloric diet for 12 weeks. At the end of the experimental period, anthropometric variables, reactive oxygen species (ROS) production, vasorelaxation, platelet aggregation, and systolic blood pressure (SBP) were measured. The results demonstrated that hypercaloric diet induced weight gain, increased oxidative stress, and reduced endothelium-dependent vasorelaxation, as well as increased platelet aggregation levels and SBP. In addition, CMG induced a significant reduction on weight gain (68.3 ± 4.9 vs 111.1 ± 3.8) and tissue (65.3 ± 18.4 vs 176.7 ± 49.7) and platelet ROS production (464.0 ± 99.8 vs 679.0 ± 102.3), leading to improved vasorelaxation, and reduced platelet aggregation. However, CMG treatment did not significantly reduce SBP levels. In this sense, it is reasonable to conclude that CMG was effective in reducing the damage caused by a hypercaloric diet, emerging as a potential compound for the treatment of diseases characterized by these conditions, such as cardiometabolic disorders.

Time-Dependent Relationship Between Endothelial Dysfunction and High Blood Pressure in Fructose Drinking Rats.

This study aims to investigate the duration required for endothelium dysfunction to develop in the fructose drinking-induced hypertension and examine the relative contributions of endothelium-dependent relaxing factors to changes in mesenteric arterial reactivity in male Wistar Albino rats. Metabolic parameters (water intake and food consumption) and hemodynamic parameters systolic blood pressure (SBP) and diastolic blood pressure (DBP)-were monitored in vivo. Vascular reactivity was examined in the isolated organ bath. Endothelium-dependent relaxation (EDR) to acetylcholine was observed in the absence and presence of pharmacological inhibitors of endothelial nitric oxide (NO) synthase, cyclooxygenase, and KCa2.3 channels. Contractile responses to phenylephrine and relaxation of sodium nitroprusside (SNP) were also determined. A significant increase in daily water intake and decrease in food consumption were typically observed in rats treated with 10% fructose for 4 weeks (p < 0.05). SBP and DBP increased significantly as early as 2 weeks of induction and continued to rise gradually throughout the induction period (p < 0.05). Fructose consumption significantly impaired EDR at week 3 and worsened at week 4 (p < 0.05). Impairment of the KCa3.1 channel-mediated component of endothelium-dependent hyperpolarisation (EDH)-type relaxation contributed to worsening EDR, whereas the contribution of NO-mediated relaxation was not apparent compared with the controls. The reduction in EDH-type relaxation in fructose-fed rats appears to be partially compensated by increased NO sensitivity in the smooth muscle region, as fructose induction increased SNP relaxation compared with the control. These data provide evidence of early endothelial dysfunction developing concurrently with increased blood pressure in 10% fructose-fed rats. Decreased KCa3.1-mediated part of EDH-type relaxation appears to contribute to the impairment of endothelium-dependent vasorelaxation over time in this model.

Abstract P338: Impaired Endothelial-Dependent Vasorelaxation and Myogenic Response in Renal Afferent Arterioles of Diabetic mice: Role of Endothelial NOX1, and New Gene Pathways.

Diabetes increases the risk of cardiovascular disease and hypertension, in part, by decreasing arterial vasodilation through decreased NO bioavailability and increased superoxide. In the kidney, type 1 diabetes induces a decrease in renal filtration, proteinuria, inflammation, and fibrosis, increasing the incidence of kidney disease. In diabetic rats and rabbits, prone to renal disease, diabetes blunts acetylcholine (Ach)-induced vasorelaxation of afferent arterioles, in part, by enhancing endothelial superoxide. However, it is unclear whether diabetes affects afferent arteriole vasodilation and myogenic response in mice. We hypothesize that diabetes increases endothelial NOX1 mediated superoxide production in mouse afferent arterioles, decreasing Ach-induced vasodilation. We dissected and perfused afferent arterioles from male diabetic Akita mice 12-14 weeks of age and measured the effect Ach (10 -7 -10 -5 M) on luminal diameter after pre-constriction with norepinephrine. Diabetic Akita mice had a largely reduced Ach-induced dilation at all doses of Ach (80±6% average inhibition in dilation, p&lt;0.025, n=6). To study the role of NOX1, we used the inhibitor ML171 (5µM). When added to the endothelial lumen, NOX1 inhibition completely restored Ach-induced dilation of diabetic Akita mice, which was not different from WT controls. In Control C57 mice, ML171 did not change Ach-induced dilation in Aff-Art (n=6). We then examined the myogenic response in diabetic mice by measuring lumen diameter after step increases in perfusion pressure (60-140 mmHg). Afferent arterioles of diabetic Akita mice did not constrict when pressure was increased, even at 140 mmHg, whereas WT mice showed normal myogenic response (delta constriction 60-140 mmHg WT: 2.15±0.41 vs Akita: 0.37±0.32 µm, WT p&lt;0.01 from baseline, Akita n.s from baseline). To understand the mechanisms behind these effects we performed RNAseq in manually micro-dissected afferent arterioles from 12-week-old Akita and WT mice. Bioinformatics analysis showed 530 DEGs (FDR pAdj&lt;0.05, n=6). Most of the DEGs are involved in endothelial cell function/metabolism, smooth muscle cell contractility, activated inflammatory pathways, and free radical/NO production. We conclude that early diabetes has a large effect on renal afferent arteriole reactivity by affecting the expression of hundreds of genes. These effects are likely involved in hypertension and kidney damage progression in type 1 diabetes.

In vitro biological activities of Calamintha nepeta L. aqueous extracts.

This study aimed to investigate the phenolic composition, antioxidant capacity, and toxicity of aqueous extracts of Calamintha nepeta L. leaves and their potential vasorelaxant effects. Aqueous extracts of Calamintha nepeta L. were prepared by three extraction methods: decoction, infusion, and maceration. The total phenolic contents of the extracts and their antioxidant properties were investigated. The toxicity was evaluated by Artemia salina lethality bioassay. The decoction extract was analyzed by HPLC for its chemical profile and was also used to evaluate the vasorelaxant effect on thoracic aortic rings isolated from healthy Sprague Dawley rats. Pre-contraction was induced by phenylephrine, followed by cumulative doses of the extract (0.001 up to 250 µg/ml). Aqueous extracts of Calamintha nepeta L. showed noticeable radical scavenging and chelating activities. However, the decoction extract exhibited the most powerful antioxidant capacity. No toxicity was recorded for the extracts obtained by decoction and infusion. Caffeic acid, quercetin, and rosmarinic acid were the main identified compounds. Notably, the aqueous extract obtained by decoction induced significant relaxation in endothelium-intact aortic rings at lower concentrations, and at higher concentrations in denuded aortic rings. This study reveals that Calamintha nepeta L. extracted with a decoction method possesses potent antioxidant capacity and has an endothelium-dependent vasorelaxant effect.

Abstract Tu028: NADPH Oxidase Inhibition Antagonizes Endothelial Pro-inflammatory and Pro-oxidant Signaling Resulting in Enhanced Coronary Vasorelaxation in Diabetic Models

Introduction: NADPH oxidase (NOX) overactivation and reactive oxygen species amplification may underlie worse cardiac outcomes in patients with diabetes. Our group has shown impaired endothelium-dependent coronary vasodilation in diabetes, with restored endothelial function following NOX inhibition. Persistently high levels of oxidative stress may be driving this impaired coronary microvascular reactivity by altering small-conductance potassium channel activity. We hypothesized that NADPH oxidase inhibition would be vasoprotective through reductions in pro-oxidant and pro-inflammatory signaling. Methods: Human coronary arterial endothelial cells (HCAECs) were obtained from diabetic (D) and nondiabetic (C) patients (N = 4 per group). HCAECs were cultured in normo- or hyperglycemic conditions, with one hyperglycemic group receiving NADPH oxidase inhibitor apocynin (DT). Molecular signaling was assessed using immunoblotting. Results: Diabetes resulted in significantly increased pro-oxidant markers, including NOX 4 (p = 0.005), phosphorylated endothelial nitric oxide synthase (p-eNOS) (p = 0.007), and eNOS (p = 0.003). Pro-inflammatory Nrf2 was also increased (p = 0.0003), with a trend toward increased NFkB (p = 0.09) in D compared to C. Antioxidant peroxiredoxin (PRDX) 1 was significantly reduced in D compared to C (p = 0.02). NOX inhibition rescued these effects, with reversed trends in NOX 4 (p = 0.008), p-eNOS (p = 0.03), and PRDX 1 (p = 0.02) following apocynin treatment. NOX inhibition resulted in de novo decrease in NOX 1 in DT when compared to D (p = 0.008) and C (p = 0.02), and decrease in phosphorylated NFkB (p-NFkB) when compared to D (p = 0.02). There was a trend toward decreased total NFkB (p = 0.07) and p-NFkB:NFkB ratio (p = 0.06) and increased antioxidant thioredoxin (TXN) 2 (p = 0.05) in diabetic cells following treatment. Antioxidant superoxide dismutase (SOD) 2 (p = 0.01) and TXN 1 (p = 0.02) were increased in D when compared to C, while apocynin treatment reversed these changes (SOD 2, p = 0.0006; TXN 1, p = 0.04). There were no significant differences in NOX 2, SOD 1, catalase, PRDX 2, or PRDX 3 between the groups. Conclusions: Apocynin overall suppressed pro-oxidant and pro-inflammatory signaling in human diabetic coronary endothelial cells, resulting in improved coronary endothelium-dependent vasorelaxation. Cardioprotective strategies that incorporate NADPH oxidase inhibition in the setting of diabetes warrant further investigation.

Deacetylation mimetic mutation of mitochondrial SOD2 attenuates ANG II-induced hypertension by protecting against oxidative stress and inflammation.

Almost one-half of adults have hypertension, and blood pressure is poorly controlled in a third of patients despite the use of multiple drugs, likely because of mechanisms that are not affected by current treatments. Hypertension is linked to oxidative stress; however, common antioxidants are ineffective. Hypertension is associated with inactivation of key intrinsic mitochondrial antioxidant, superoxide dismutase 2 (SOD2), due to hyperacetylation, but the role of specific SOD2 lysine residues has not been defined. Hypertension is associated with SOD2 acetylation at lysine 68, and we suggested that deacetylation mimetic mutation of K68 to arginine in SOD2 inhibits vascular oxidative stress and attenuates hypertension. To test this hypothesis, we have developed a new deacetylation mimetic SOD2-K68R mice. We performed in vivo studies in SOD2-K68R mice using angiotensin II (ANG II) model of vascular dysfunction and hypertension. ANG II infusion in wild-type mice induced vascular inflammation and oxidative stress and increased blood pressure to 160 mmHg. SOD2-K68R mutation completely prevented increase in mitochondrial superoxide, abrogated vascular oxidative stress, preserved endothelial nitric oxide production, protected vasorelaxation, and attenuated ANG II-induced hypertension. ANG II and cytokines contribute to vascular oxidative stress and hypertension. Treatment of wild-type aortas with ANG II and cytokines in organoid culture increased mitochondrial superoxide twofold, which was completely prevented in aortas isolated from SOD2-K68R mice. These data support the important role of SOD2-K68 acetylation in vascular oxidative stress and pathogenesis of hypertension. We conclude that strategies to reduce SOD2 acetylation may have therapeutic potential in the treatment of vascular dysfunction and hypertension.NEW & NOTEWORTHY Essential hypertension is associated with hyperacetylation of key mitochondrial antioxidant SOD2; however, the pathophysiological role of SOD2 acetylation has not been defined. Our animal study of angiotensin II hypertension model shows that deacetylation mimetic SOD2-K68R mutation prevents pathogenic increase in vascular mitochondrial superoxide, abrogates vascular oxidative stress, preserves endothelial nitric oxide, protects endothelial-dependent vasorelaxation, and attenuates hypertension. These data support the important role of SOD2-K68 acetylation in vascular oxidative stress and the pathogenesis of hypertension.

2,7-Phloroglucinol-6,6′-bieckol from Ecklonia cava ameliorates nanoplastics-induced premature endothelial senescence and dysfunction

Nanoplastics (NPs), plastic particles ranging from 1 to 100 nm are ubiquitous environmental pollutants infiltrating ecosystems. Their small size and widespread use in various products raise concerns for human health, particularly their association with cardiovascular diseases (CVD). NPs can enter the human body through multiple routes, causing oxidative stress, and leading to the senescence and dysfunction of endothelial cells (ECs). Although there are potential natural compounds for treating CVD, there is limited research on preventing CVD induced by NPs. This study investigates the efficacy of Ecklonia cava extract (ECE) in preventing NPs-induced premature vascular senescence and dysfunction. Exposure of porcine coronary arteries (PCAs) and porcine coronary ECs to NPs, either alone or in combination with ECE, demonstrated that ECE mitigates senescence-associated β-galactosidase (SA-β-gal) activity induced by NPs, thus preventing premature endothelial senescence. ECE also improved NPs-induced vascular dysfunction. The identified active ingredient in Ecklonia cava, 2,7’-Phloroglucinol-6,6′-bieckol (PHB), a phlorotannin, proved to be pivotal in these protective effects. PHB treatment ameliorated SA-β-gal activity, reduced oxidative stress, restored cell proliferation, and decreased the expression of cell cycle regulatory proteins such as p53, p21, p16, and angiotensin type 1 receptor (AT1), well known triggers for EC senescence. Moreover, PHB also improved NPs-induced vascular dysfunction by upregulating endothelial nitric oxide synthase (eNOS) expression and restoring endothelium-dependent vasorelaxation. In conclusion, Ecklonia cava and its active ingredient, PHB, exhibit potential as therapeutic agents against NPs-induced premature EC senescence and dysfunction, indicating a protective effect against environmental pollutants-induced CVDs associated with vascular dysfunction.

Phylloquinone improves endothelial function, inhibits cellular senescence, and vascular inflammation

Phylloquinon (PK) and menaquinones (MK) are both naturally occurring compounds belonging to vitamin K group. Present study aimed to comprehensively analyze the influence of PK in several models of vascular dysfunction to determine whether PK has vasoprotective properties, similar to those previously described for MK. Effects of PK and MK on endothelial dysfunction were studied in ApoE/LDLR−/− mice in vivo, in the isolated aorta incubated with TNF, and in vascular cells as regard inflammation and cell senescence (including replicative and stress-induced models of senescence). Moreover, the vascular conversion of exogenous vitamins to endogenous MK-4 was analyzed. PK, as well as MK, given for 8 weeks in diet (10 mg/kg) resulted in comparable improvement in endothelial function in the ApoE/LDLR−/− mice. Similarly, PK and MK prevented TNF-induced impairment of endothelium-dependent vasorelaxation in the isolated aorta. In in vitro studies in endothelial and vascular smooth muscle cells, we identified that both PK and MK displayed anti-senescence effects via decreasing DNA damage while in endothelial cells anti-inflammatory activity was ascribed to the modulation of NFκB activation. The activity of PK and MK was comparable in terms of their effect on senescence and inflammation. Presence of endogenous synthesis of MK-4 from PK in aorta and endothelial and smooth muscle cells suggests a possible involvement of MK in vascular effects of PK. In conclusion, PK and MK display comparable vasoprotective effects, which may be ascribed, at least in part, to the inhibition of cell senescence and inflammation. The vasoprotective effect of PK in the vessel wall can be related to the direct effects of PK, as well as to the action of MK formed from PK in the vascular wall.

Pulmonary Vascular Responses to Chronic Intermittent Hypoxia in a Guinea Pig Model of Obstructive Sleep Apnea.

Experimental evidence suggests that chronic intermittent hypoxia (CIH), a major hallmark of obstructive sleep apnea (OSA), boosts carotid body (CB) responsiveness, thereby causing increased sympathetic activity, arterial and pulmonary hypertension, and cardiovascular disease. An enhanced circulatory chemoreflex, oxidative stress, and NO signaling appear to play important roles in these responses to CIH in rodents. Since the guinea pig has a hypofunctional CB (i.e., it is a natural CB knockout), in this study we used it as a model to investigate the CB dependence of the effects of CIH on pulmonary vascular responses, including those mediated by NO, by comparing them with those previously described in the rat. We have analyzed pulmonary artery pressure (PAP), the hypoxic pulmonary vasoconstriction (HPV) response, endothelial function both in vivo and in vitro, and vascular remodeling (intima-media thickness, collagen fiber content, and vessel lumen area). We demonstrate that 30 days of the exposure of guinea pigs to CIH (FiO2, 5% for 40 s, 30 cycles/h) induces pulmonary artery remodeling but does not alter endothelial function or the contractile response to phenylephrine (PE) in these arteries. In contrast, CIH exposure increased the systemic arterial pressure and enhanced the contractile response to PE while decreasing endothelium-dependent vasorelaxation to carbachol in the aorta without causing its remodeling. We conclude that since all of these effects are independent of CB sensitization, there must be other oxygen sensors, beyond the CB, with the capacity to alter the autonomic control of the heart and vascular function and structure in CIH.

SARS-CoV-2 S Protein Reduces Cytoprotective Defenses and Promotes Human Endothelial Cell Senescence.

Premature vascular aging and endothelial cell senescence are major risk factors for cardiovascular diseases and atherothrombotic disturbances, which are main complications of both acute and long COVID-19. The S protein of SARS-CoV2, which acts as the receptor binding protein for the viral infection, is able to induce endothelial cells inflammation and it has been found as an isolated element in the circulation and in human tissues reservoirs months after infection. Here, we investigated whether the S protein is able to directly induce endothelial cell senescence and deciphered some of the mechanisms involved. In primary cultures of human umbilical vein endothelial cells (HUVEC), SARS-CoV-2 S protein enhanced in a concentration-dependent manner the cellular content of senescence and DNA damage response markers (senescence-associated-β galactosidase, γH2AX), as well as growth-arrest effectors (p53, p21, p16). In parallel, the S protein reduced the availability of cytoprotective proteins, such as the anti-aging protein klotho, Nrf2 or heme oxygenase-1, and caused functional harm by impairing ex vivo endothelial-dependent vasorelaxation in murine microvessels. These effects were prevented by the pharmacological inhibition of the NLRP3 inflammasome with MCC950. Furthermore, the supplementation with either recombinant klotho or angiotensin-(1-7), equally protected against the pro-senescence, pro-inflammatory and pro-oxidant action of the S protein. Globally, this study proposes novel mechanisms of disease in the context of COVID-19 and its vascular sequelae and provides pharmacological clues in order to prevent such complications.

Short-Term Statin Treatment Reduces, and Long-Term Statin Treatment Abolishes, Chronic Vascular Injury by Radiation Therapy.

The incidental use of statins during radiation therapy has been associated with a reduced long-term risk of developing atherosclerotic cardiovascular disease. We examined whether irradiation causes chronic vascular injury and whether short-term administration of statins during and after irradiation is sufficient to prevent chronic injury compared with long-term administration. C57Bl/6 mice were pretreated with pravastatin for 72 hours and then exposed to 12 Gy X-ray head-and-neck irradiation. Pravastatin was then administered either for an additional 24 hours or for 1 year. Carotid arteries were tested for vascular reactivity, altered gene expression, and collagen deposition 1 year after irradiation. Treatment with pravastatin for 24 hours after irradiation reduced the loss of endothelium-dependent vasorelaxation and protected against enhanced vasoconstriction. Expression of markers associated with inflammation (NFκB p65 [phospho-nuclear factor kappa B p65] and TNF-α [tumor necrosis factor alpha]) and with oxidative stress (NADPH oxidases 2 and 4) were lowered and subunits of the voltage and Ca2+ activated K+ BK channel (potassium calcium-activated channel subfamily M alpha 1 and potassium calcium-activated channel subfamily M regulatory beta subunit 1) in the carotid artery were modulated. Treatment with pravastatin for 1 year after irradiation completely reversed irradiation-induced changes. Short-term administration of pravastatin is sufficient to reduce chronic vascular injury at 1 year after irradiation. Long-term administration eliminates the effects of irradiation. These findings suggest that a prospective treatment strategy involving statins could be effective in patients undergoing radiation therapy. The optimal duration of treatment in humans has yet to be determined.

In a rat model of bypass DuraGraft ameliorates endothelial dysfunction of arterial grafts

Coronary artery bypass surgery can result in endothelial dysfunction due to ischemia/reperfusion (IR) injury. Previous studies have demonstrated that DuraGraft helps maintain endothelial integrity of saphenous vein grafts during ischemic conditions. In this study, we investigated the potential of DuraGraft to mitigate endothelial dysfunction in arterial grafts after IR injury using an aortic transplantation model. Lewis rats (n = 7–9/group) were divided in three groups. Aortic arches from the control group were prepared and rings were immediately placed in organ baths, while the aortic arches of IR and IR + DuraGraft rats were preserved in saline or DuraGraft, respectively, for 1 h before being transplanted heterotopically. After 1 h after reperfusion, the grafts were explanted, rings were prepared, and mounted in organ baths. Our results demonstrated that the maximum endothelium-dependent vasorelaxation to acetylcholine was significantly impaired in the IR group compared to the control group, but DuraGraft improved it (control: 89 ± 2%; IR: 24 ± 1%; IR + DuraGraft: 48 ± 1%, p < 0.05). Immunohistochemical analysis revealed decreased intercellular adhesion molecule-1, 4-hydroxy-2-nonenal, caspase-3 and caspase-8 expression, while endothelial cell adhesion molecule-1 immunoreactivity was increased in the IR + DuraGraft grafts compared to the IR-group. DuraGraft mitigates endothelial dysfunction following IR injury in a rat bypass model. Its protective effect may be attributed, at least in part, to its ability to reduce the inflammatory response, oxidative stress, and apoptosis.

N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine induces endothelium-dependent hyperpolarization-mediated vasorelaxation via store-operated calcium entry mechanism in healthy and intestinal inflammatory mice

Although the store-operated Ca2+ entry (SOCE) plays a critical role in maintaining Ca2+ homeostasis in vascular endothelial cells (VECs), its role in regulating endothelium-dependent hyperpolarization (EDH)-mediated vasorelaxation is largely unknown. Inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) are the most common gastrointestinal disorders with no effective cures. The present study applied N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) as a Ca2+ chelator in the endoplasmic reticulum (ER) to study the SOCE/EDH-mediated vasorelaxation of micro-arteries and their involvements in the pathogenesis of IBD and IBS. Human submucosal arterioles and the second-order branch of 6–8 weeks male C57BL/6 mouse mesenteric arterioles were used, and TPEN-induced vasorelaxation was recorded by Danish DMT520A microvascular measuring system. The mice were fed water with 2.5 % dextran sulfate sodium for 7 days to induce mouse model of ulcerative colitis, and water avoidance stress was used to induce mouse model of IBS. The statistical significance of differences in the means of experimental groups was determined using a t-test for two groups or one-way ANOVA for more than two groups. TPEN concentration-dependently induced vasorelaxation of human colonic submucosal arterioles and the second-order branch of murine mesenteric arteries in endothelium-dependent manner. TPEN-induced vasorelaxation was much greater in the arteries pre-constricted by noradrenaline than those by high K+. While TPEN-induced vasorelaxation was unaffected by inhibitors of NO and PGI2, it was significantly inhibited by the selective inhibitors of IKCa and SKCa channels but was potentiated by their activator. Moreover, TPEN-induced vasorelaxation was attenuated by selective inhibitors of NCX, NKA, SOCE, STIM translocation and Orai transportation. Finally, TPEN-induced vasorelaxation via SOCE/EDH was impaired in colitic mice but remained intact in IBS mice. Interestingly, TPEN could rescue vagus neurotransmitter ACh-induced vasorelaxation that was impaired in IBS mice. Therefore, since TPEN-induced SOCE/EDH-mediated vasorelaxation of mesenteric arteries is well-preserved to be able to rescue ACh-induced vasorelaxation impaired in IBS, TPEN has therapeutic potentials for IBS.

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

#1131 Significant reno-cardio-metabolic protection by the food additive Flexovital in a murine model combining unilateral nephrectomy and Western diet

Abstract Background and Aims Cardiovascular complications are major threats in advanced renal failure and metabolic dysfunction with several unmet medical needs. This study aimed to investigate the therapeutic effects of a special food additive (FLEXOVITAL) in a newly developed mouse model of reno-cardio-metabolic disease, induced by moderate renal failure in combination with a special Western diet. Method Male C57BL/6J mice (4 weeks old, n = 18) were subject to unilateral nephrectomy (UNX), fed a Western diet rich in fat carbohydrates and salt (WD), and were followed for 12 weeks compared with SHAM-operated mice on standard chow (n = 19). One group of UNX+WD mice (n = 8) was fed a food additive (FLEXOVITAL; FLX) containing extracts of Rhodiola rosea, beetroot, and the amino acids arginine and citrulline. Body weight (BW), blood pressure (tail-cuff), endothelial-dependent vasorelaxation (myograph), glucose metabolism (IPGTT), body fat and lean mass composition (DEXA), adipocyte area, renal function (Glomerular Filtration Rate (GFR) by plasma clearance of FITC-inulin), and mitochondrial function (Oroboros, High-resolution respirometry) were measured together with biochemical analysis of heart injury (Troponin-I), inflammation (IL6) and histological analyses of the kidney and heart. Results BW gain was seen in the UNX+WD and SHAM group, and was 25% less in the FLX group (p &amp;lt; 0.05). The fat/lean-mass ratio increased by 17% (p &amp;lt; 0.01) and the adipocyte area by 31% (p &amp;lt; 0.001) in the UNX+WD group but was virtually normalized in the FLX group (p &amp;lt; 0.01). Fasting and non-fasting glucose levels became elevated in the UNX+WD group and were reduced in the FLX group (p &amp;lt; 0.05). Impaired glucose clearance in the UNX+WD group, was partially prevented by FLX. BP significantly increased in the UNX+WD group (MAP = 78 → 90 mmHg, p &amp;lt; 0.001), and was largely prevented by FLX (MAP = 82 mmHg, p &amp;lt; 0.01). Endothelial function was significantly impaired in the UNX+WD group (p &amp;lt; 0.01) and partially preserved in the FLX group (p &amp;lt; 0.05). GFR was reduced by almost 60% in the UNX+WD group but improved with a 75% protective effect in the FLX group (p &amp;lt; 0.05). Significant glomerular injuries with mesangial proliferation were observed in the UNX+WD group (p &amp;lt; 0.001) but were less pronounced in the FLX group (p &amp;lt; 0.01). Tubular injury score (1-10) increased from 1 in SHAM to 6.5 in the UNX+WD group and was partly protected (4.5) in the FLX group (p &amp;lt; 0.05). Troponin-I levels were 15 pg/ml in the SHAM group, markedly increased in the UNX+WD group (p &amp;lt; 0.001), whereas completely reversed in the FLX group (p &amp;lt; 0.01). No significant histological cardiac injuries or deviations could be demonstrated. Inflammatory activity (IL6) increased by 88% in the UNX+WD there was a trend of reduction in mice with FLX. Mitochondrial oxygen efficiency (P/O ratio) was improved in the kidneys of the FLX group compared to the UNX+WD group (p &amp;lt; 0.05). Conclusion In the present multiorgan disease model, significant renal, cardiovascular, and metabolic dysfunction/injuries emerge in mice with a moderate reduction of renal function when fed a Western diet rich in fat, carbohydrates, and salt. Protective effects were noted by dietary FLX treatment in most functional assessments made in the model. This indicates FLX is a potential new treatment in patients with reno-cardio-metabolic disease. The next phase involves confirming these findings in the clinical setting.

MiR-486-5p protects against rat ischemic kidney injury and prevents the transition to chronic kidney disease and vascular dysfunction.

Acute kidney injury (AKI) increases the risk for progressive chronic kidney disease (CKD). MicroRNA (miR)-486-5p protects against kidney ischemia-reperfusion (IR) injury in mice, although its long-term effects on the vasculature and development of CKD are unknown. We studied whether miR-486-5p would prevent the AKI to CKD transition in rat, and affect vascular function. Adult male rats were subjected to bilateral kidney IR followed by i.v. injection of liposomal-packaged miR-486-5p (0.5 mg/kg). Kidney function and histologic injury were assessed after 24 h and 10 weeks. Kidney endothelial protein levels were measured by immunoblot and immunofluorescence, and mesenteric artery reactivity was determined by wire myography. In rats with IR, miR-486-5p blocked kidney endothelial cell increases in intercellular adhesion molecule-1 (ICAM-1), reduced neutrophil infiltration and histologic injury, and normalized plasma creatinine (P<0.001). However, miR-486-5p attenuated IR-induced kidney endothelial nitric oxide synthase (eNOS) expression (P<0.05). At 10 weeks, kidneys from rats with IR alone had decreased peritubular capillary density and increased interstitial collagen deposition (P<0.0001), and mesenteric arteries showed impaired endothelium-dependent vasorelaxation (P<0.001). These changes were inhibited by miR-486-5p. Delayed miR-486-5p administration (96 h, 3 weeks after IR) had no impact on kidney fibrosis, capillary density, or endothelial function. In rats, administration of miR-486-5p early after kidney IR prevents injury, and protects against CKD development and systemic endothelial dysfunction. These protective effects are associated with inhibition of endothelial ICAM-1 and occur despite reduction in eNOS. miR-486-5p holds promise for the prevention of ischemic AKI and its complications.

Abstract 130: Cftr Is Essential For Endothelium-dependent Vasorelaxation

Genetic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene lead to cystic fibrosis (CF) which causes multiple health issues including lung disease and diabetes. Importantly, along with other health conditions, CF patients are long known to have impaired vascular function. Specifically, endothelium-dependent vasorelaxation is impaired in people with CF. However, the molecular mechanism(s) involved in the impairment of endothelial function in CF is not known. Since impaired endothelial function is an established risk factor for cardiovascular diseases, we investigated how CFTR regulates vascular endothelial function. We evaluated coronary arteries of CFTR -/- and littermate wild type piglets and noted profound endothelial dysfunction in coronary arteries of CFTR -/- piglets (n=10-12 rings/3 piglets). Next, we generated a mouse model with endothelium-specific deletion of CFTR (e-CFTR -/- ). Aortic rings of e-CFTR -/- mice showed a significant delay in endothelium-dependent vasorelaxation (P&lt;0.001; n=20 rings /5 mice). To determine the molecular signaling mediating this effect, we used a pharmacological inhibitor of CFTR (CFTRinh-172) and examined changes in endothelial nitric oxide synthase (eNOS). CFTRinh-172 treatment (10 μM, 5 days) led to a significant reduction in total and S1177 phosphorylated level of eNOS in human coronary artery endothelial cells (P&lt;0.01 and P&lt;0.05; n=3). In addition, CFTRinh-172 treated endothelial cells exhibited increased inflammation and altered endothelial cell metabolism (n=6; P&lt;0.001). Together, these findings suggest that CFTR regulates eNOS and cellular metabolism in endothelial cells and therefore, functional CFTR is essential for vascular endothelial function. This fundamental role of CFTR in endothelial function will have future translational value in treating people with CF.

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.

Effects of Cross-Sex Hormone Therapy on Metabolic Parameters and Aortic Relaxation in Castrated Male Rats

With the rising awareness, the number of individuals identifying themselves as transgender in the US is increasing over the years. Cross-sex hormone therapy (CSHT) may alter numerous cardiometabolic parameters leading to the increased risk of cardiovascular diseases (CVD) in transgender population. Epidemiological studies suggest that transgender females (male to female, MtF) on estrogen therapy develop higher risk of CVD compared to transgender males (female to male, FtM) taking testosterone. Underlying mechanisms of impaired cardiovascular functions in MtF are not well documented. This study was undertaken to examine the effects of 17β-estradiol (E2) on certain cardiometabolic parameters and aortic reactivity in castrated (CAS) male Sprague Dawley (SD) rats. The 8-10-weeks old CAS rats were subcutaneously implanted with either 1.5 mg of E2 (CAS+E2) or placebo (CAS+PL) containing pellets for ~35 days. The age-matched SD intact male (IM) rats were also included in the experimental groups. After performing the glucose tolerance test, animals were sacrificed, and the blood was collected for later measurements of the HbA1C and triglyceride (TG) levels. Endothelium-dependent vasorelaxation (EDV) to acetylcholine (ACh) in aortic rings pre-contracted with phenylephrine (PE) was measured before and after treatment with indomethacin (a cyclooxygenase inhibitor), followed by L-NAME [a nonselective nitric oxide (NO) synthase (NOS) inhibitor]. Contractile responses to PE before and after L-NAME in aortic rings and sodium nitroprusside (SNP)-induced relaxation responses in endothelium-denuded rings were also measured. Here, we report the decreased body weight and visceral white adipose tissue, and improved HbA1C and glucose tolerance in E2-treated CAS rats. However, the E2-treated CAS rats exhibited elevated plasma level of TG. Furthermore, the vascular relaxation to ACh was significantly impaired in aortas from this group compared to those in CAS+PL and IM rats. The impairment of ACh responses in CAS+E2 rats was accompanied by a significant decrease in NO contribution to ACh vasorelaxation. The basal NO levels and the smooth muscle sensitivity to NO were not altered, whereas the maximum contractile responses to PE were enhanced in aortas of CAS+E2 group compared to IM. In conclusion, our data suggests that the glucose homeostasis was improved in CAS+E2 rats. Regardless, this group exhibited increased level of TG in plasma and decreased aortic vasorelaxation to ACh. The impairment of EDV in E2-treated CAS rats might be due to reduced NO-mediated vasorelaxation or increased contractile responses observed in this group. Additional studies are needed to document the underlying mechanisms for improvement of glucose homeostasis and impairment of EDV in CAS+E2 rats. NIDA/NIGMS, 1SC1DA052120-01 to MF and Bridge Grant from University of the Pacific to RR. This is the full abstract presented at the American Physiology Summit 2024 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.

Cross-sexual Effects of Testosterone on Mesenteric Arterial Reactivity in Ovariectomized Female Rats

The number of transgender individuals is on the rise in the United States. Although there are studies on the effects of sex hormones on cardiovascular function of cisgender male and female, still much is not known about the effects of cross-sex hormone therapy (CSHT) on cardiovascular function in transgender individuals. It has been shown that the risk factors of cardiovascular diseases (CVD) are increased in transgenders after they undergo CSHT. Epidemiological studies suggest that transgender females (male to female, MtF) on estrogen therapy develop higher risk of CVD compared to transgender males (female to male, FtM) taking testosterone. This study investigates the effects of testosterone or estrogen treatment on the third branch of mesenteric arterial reactivity in ovariectomized female rats. Briefly, 8-10 weeks old female Sprague Dawley (SD) rats were ovariectomized (OVX) and subcutaneously implanted with placebo (OVX + PL) or testosterone (OVX + T, 7.5 mg) or 17β- estradiol (OVX + E2, 1.5 mg) pellets for about 35 days. Age matched intact female SD rats were also included in the experimental groups. Endothelium-dependent vasorelaxation (EDV) to acetylcholine (ACh) was measured in the precontracted mesenteric arteries with phenylephrine (PE), using wire myography. Responses to sodium nitroprusside (SNP)-induced vasorelaxation, and PE- and endothelin-1 (ET-1) induced vasocontraction were also determined. We demonstrated that the responses to ACh in rat mesenteric arteries were not altered by either ovariectomy or estrogen treatment. However, testosterone treatment of OVX female rats significantly enhanced the sensitivity to ACh-induced vasorelaxation compared with those in other experimental groups. Moreover, the mesenteric arteries of OVX + T exhibited a trend of reduced sensitivity to PE responses compared to other groups. Furthermore, the maximum tension to ET-1 was significantly reduced in mesenteric arteries of OVX + T compared to OVX+E2 and OVX+PL groups. The vasorelaxation responses to SNP were not altered in any of experimental groups studied. These data suggest that elevated sensitivity to ACh in testosterone-treated ovariectomized female rats might be, in part, due to reduced vessel responses to contractile agents, but not altered sensitivity of smooth muscle to nitric oxide (NO) in this group. Additional studies are needed to investigate the underlying mechanisms and document the consequences for the improvement of EDV observed in OVX + T rats. NIDA/NIGMS, 1SC1DA052120-01 to MF, Bridge Grant from University of the Pacific to RR. This is the full abstract presented at the American Physiology Summit 2024 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.