Impaired Endothelium-dependent Relaxation Research Articles

MiR-210 as a therapeutic target in diabetes-associated endothelial dysfunction.

MicroRNA (miR)-210 function in endothelial cells and its role in diabetes-associated endothelial dysfunction are not fully understood. We aimed to characterize the miR-210 function in endothelial cells and study its therapeutic potential in diabetes. Two different diabetic mouse models (db/db and Western diet-induced), miR-210 knockout and transgenic mice, isolated vessels and human endothelial cells were used. miR-210 levels were lower in aortas isolated from db/db than in control mice. Endothelium-dependent relaxation (EDR) was impaired in aortas from miR-210 knockout mice, and this was restored by inhibiting miR-210 downstream protein tyrosine phosphatase 1B (PTP1B), mitochondrial glycerol-3-phosphate dehydrogenase 2 (GPD2), and mitochondrial oxidative stress. Inhibition of these pathways also improved EDR in both diabetic mouse models. High glucose reduced miR-210 levels in endothelial cells and impaired EDR in mouse aortas, effects that were reversed by overexpressing miR-210. However, plasma miR-210 levels were not affected in individuals with type 2 diabetes (T2D) following improved glycaemic status. Of note, genetic overexpression using miR-210 transgenic mice and pharmacological overexpression using miR-210 mimic in vivo ameliorated endothelial dysfunction in both diabetic mouse models by decreasing PTP1B, GPD2 and oxidative stress. Genetic overexpression of miR-210 altered the aortic transcriptome, decreasing genes in pathways involved in oxidative stress. miR-210 mimic restored decreased nitric oxide production by high glucose in endothelial cells. This study unravels the mechanisms by which down-regulated miR-210 by high glucose induces endothelial dysfunction in T2D and demonstrates that miR-210 serves as a novel therapeutic target.

Asperuloside as a Novel NRF2 Activator to Ameliorate Endothelial Dysfunction in High Fat Diet-Induced Obese Mice.

Aims: Current treatments are inadequate in alleviating obesity-associated vascular diseases. The development of effective therapies to ameliorate endothelial dysfunction and attenuate oxidative stress is of utmost importance. Asperuloside (ASP), a bioactive compound extracted from Eucommia species, exhibits antiobesity properties. However, the effects of ASP on vasculopathy have not been investigated. Therefore, the effects of ASP on vascular dysfunction and related mechanisms were elucidated. Results: ASP significantly reversed the impaired endothelium-dependent relaxations (EDRs) in obese mice and interleukin (IL)-1β-treated aortas. ASP suppressed endothelial activation in obese mice aortas and IL-1β-treated endothelial cells. ASP attenuated oxidative stress, scavenged mitochondrial reactive oxygen species (ROS), and upregulated heme oxygenase-1 (HO-1) expression in endothelium, independent of its anti-inflammatory properties. HO-1 knockdown diminished the protective effects of ASP against impaired EDRs, ROS overproduction, and endothelial activation. Endothelial cell-specific nuclear factor erythroid 2-related factor 2 (Nrf2) knockdown eliminated the ASP-mediated vascular protective effects and endothelial HO-1 upregulation, emphasizing that ASP improves endothelial function by activating Nrf2/HO-1 signaling. ASP facilitated Nrf2 nuclear translocation and the direct binding of Nrf2 to antioxidant response element, thereby enhancing HO-1 transcription and scavenging ROS. The cellular thermal shift assay results provide the first experimental characterization of the direct binding of ASP to Nrf2. Conclusions: These findings demonstrate that ASP ameliorates obesity-associated endothelial dysfunction by activating Nrf2/HO-1 signaling and thereby maintaining redox hemostasis, suggesting its potential as a novel Nrf2-targeted therapeutic agent and dietary supplement for vasculopathy.

H2S donor GYY4137 mitigates sFlt-1-induced hypertension and vascular dysfunction in pregnant rats†.

Gestational hypertension, often associated with elevated soluble Fms-related receptor tyrosine kinase 1 (sFlt-1), poses significant risks to both maternal and fetal health. Hydrogen sulfide (H2S), a gasotransmitter, has demonstrated blood pressure-lowering effects in hypertensive animals and humans. However, its role in pregnancy-induced hypertension remains unclear. This study investigated the impact of GYY4137, a slow-release H2S donor, on sFlt-1-induced hypertension in pregnant rats . Pregnant rats were administered sFlt-1 (6μg/kg/day, intravenously) or vehicle from gestation day (GD) 12-20. A subset of these groups received GYY4137 ( 50mg/kg/day, intraperitoneal) from GD 16-20. Serum H2S levels, mean arterial blood pressure, uterine artery blood flow, and vascular reactivity were assessed. Elevated sFlt-1 reduced both maternal weight gain and serum H2S levels. GYY4137 treatment restored both weight gain and H2S levels in sFlt-1 dams. sFlt-1 increased mean arterial pressure and decreased uterine artery blood flow in pregnant rats. However, treatment with GYY4137 normalized blood pressure and restored uterine blood flow in sFlt-1 dams. sFlt-1 dams exhibited heightened vasoconstriction to phenylephrine and GYY4137 significantly mitigated the exaggerated vascular contraction. Notably, sFlt-1 impaired endothelium-dependent relaxation, while GYY4137 attenuated this impairment by upregulating eNOS protein levels and enhancing vasorelaxation in uterine arteries. GYY4137 mitigated sFlt-1-induced fetal growth restriction. In conclusion, sFlt-1 mediated hypertension is associated with decreased H2S levels. Replenishing H2S with the donor GYY4137 mitigates hypertension and improves vascular function and fetal growth outcomes. This suggests modulation of H2S could offer a novel therapeutic strategy for managing gestational hypertension and adverse fetal effects.

Myeloid cell-derived interleukin-6 induces vascular dysfunction and vascular and systemic inflammation.

The cytokine interleukin-6 (IL-6) plays a central role in the inflammation cascade as well as cardiovascular disease progression. Since myeloid cells are a primary source of IL-6 formation, we aimed to generate a mouse model to study the role of myeloid cell-derived IL-6 in vascular disease. Interleukin-6-overexpressing (IL-6OE) mice were generated and crossed with LysM-Cre mice, to generate mice (LysM-IL-6OE mice) overexpressing the cytokine in myeloid cells. Eight- to 12-week-old LysM-IL-6OE mice spontaneously developed inflammatory colitis and significantly impaired endothelium-dependent aortic relaxation, increased aortic reactive oxygen species (ROS) formation, and vascular dysfunction in resistance vessels. The latter phenotype was associated with decreased survival. Vascular dysfunction was accompanied by a significant accumulation of neutrophils, monocytes, and macrophages in the aorta, increased myeloid cell reactivity (elevated ROS production), and vascular fibrosis associated with phenotypic changes in vascular smooth muscle cells. In addition to elevated Mcp1 and Cxcl1 mRNA levels, aortae from LysM-IL-6OE mice expressed higher levels of inducible NO synthase and endothelin-1, thus partially accounting for vascular dysfunction, whereas systemic blood pressure alterations were not observed. Bone marrow (BM) transplantation experiments revealed that vascular dysfunction and ROS formation were driven by BM cell-derived IL-6 in a dose-dependent manner. Mice with conditional overexpression of IL-6 in myeloid cells show systemic and vascular inflammation as well as endothelial dysfunction. A decrease in circulating IL-6 levels by replacing IL-6-producing myeloid cells in the BM improved vascular dysfunction in this model, underpinning the relevant role of IL-6 in vascular disease.

XX Chromosome Complement Predisposes Females to Obesity-induced, Leptin-mediated Aldosterone Production and Hypertension

Despite the historical dogma that premenopausal women are protected from cardiovascular disease, clinical and experimental evidence indicate that obesity abolishes this protection, placing women at increased risk for hypertension. Previously, our laboratory demonstrated that the adipokine leptin contributes to obesity-associated hypertension in both males and females but via sex specific mechanisms: leptin-mediated sympatho-activation in males and leptin-induced aldosterone production in females. However, the origin of these sex-specific mechanisms remains unknown. Therefore, we aimed to test the contribution of both sex hormones and chromosomes. Female sex hormones were preserved (sham) or depleted via ovariectomy (OVX) in lean control and obese female agouti yellow mice. Blood pressure (BP) recording via radiotelemetry indicated that obesity increased BP (2-way ANOVA, p<0.0001) but OVX did not alter BP in agouti mice. Vascular function measured via mesenteric wire myography indicated obesity impaired (3-way ANOVA, p<0.05) endothelial-dependent relaxation but OVX did not inflict further impairment in agouti mice. OVX did not alter plasma aldosterone levels or adrenal aldosterone synthase (CYP11B2) expression in agouti mice. Leptin receptor blockade (Allo-Aca) decreased BP (2-way ANOVA, p<0.05), restored endothelial-dependent relaxation and decreased (2-way ANOVA, p<0.05) adrenal CYP11B2 expression in both intact and OVX agouti mice indicating a preservation of leptin-mediated BP elevation in the absence of female sex hormones and ruling out sex steroids as the origin of the sex specific mechanisms. Additionally, neither estrogen, progesterone, a combination of estrogen and progesterone, or dihydrotestosterone increase CYP11B2 expression in primary isolated human adrenal cells in culture further supporting that sex hormones do not mediate aldosterone production. These data suggest female sex hormones are not the source for the sex difference prompting us to utilize the four-core genotype (FCG) mouse model to assess the role of sex chromosomes. In order to mimic obesity conditions, BP was recorded at baseline and in response to leptin infusion in OVX FCG XX and XY female mice (XXF and XYF). XX females displayed higher baseline BP than XY (XXF: 109.1±2.4 vs. XYF: 100.4±1.1 mmHg, p<0.05) but leptin infusion increased BP and concurrently ablated the difference. Similarly, XXF displayed heightened plasma aldosterone (2-way ANOVA, 183.7±27 vs. 89.4±34 pg/mL, p<0.05) but leptin infusion abolished the difference. Collectively, these results indicate that XX chromosome rather than female sex steroid hormones regulates aldosterone production, and that sex chromosomes are likely the source for the sexual dimorphism in the mechanism for hypertension associated with obesity. R01HL130301, R01HL155265 (E.J.B.) F31HL168963-01 (C.T.B.). 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.

Aged HIV-1 Tg26 Transgenic Mice display Vascular Dysfunction with No Alterations in Neurocognitive Function or Cerebral Blood Flow

Although combination antiretroviral therapy (cART) has markedly reduced HIV mortality and increased life expectancy, people living with HIV (PLWH) develop premature cognitive and brain aging, classified as HIV-associated neurocognitive disorder (HAND). Vascular dysfunction and expression of HIV-derived proteins have independently been associated with cognitive impairment without causality being established. Thus, we investigated the role of HIV viral proteins in endothelial dysfunction and cognitive impairment in an aged mouse model of HIV. We used 18-month-old Tg26 mice and their wild-type littermate controls. Tg26 mice express 7 out of the 9 HIV viral proteins under the control of the long-term repeat promoter and have previously been shown to exhibit signs of cognitive impairment at eight months of age. Conduction of concentration-response curves to acetylcholine in mesenteric arteries using wire myography revealed impaired endothelium-dependent relaxation in Tg26 mice compared to WT mice. Measurements of brain perfusion with arterial spin labeling magnetic resonance imaging showed no differences in the whole brain and hippocampal cerebral blood flow. In addition, there were no changes in the hippocampus volume, which has been reported to be reduced with cognitive impairments. Two methods were employed to assess the cognitive abilities of the mice: Morris water maze (MWM) and the IntelliCage system. MWM requires human handling while the IntelliCage system is fully automated enabling the testing of mouse cognitive function during their active period and independently of human handling. Tg26 and WT mice underwent ten days of MWM testing and eight days of IntelliCage testing to assess spatial learning and memory. Tg26 mice showed no significant differences in latency to platform, distance swam to platform, or swim velocity when compared to their WT littermates. The IntelliCage results showed no differences in total visits, total visits with nose pokes, or total licks during the reversal learning period. Additionally, there were no significant differences in percent side error or place error indicating that cognitive function is not impaired in Tg26 mice. The Y-maze test further showed no differences in spontaneous alteration, a measure of spatial working memory. Altogether, our data shows that although 18-month-old female Tg26 mice develop endothelial dysfunction, they display no alterations in cerebral blood flow and no cognitive impairment indicating that systemic viral protein expression and vascular dysfunction might minimally contribute to neurocognitive impairment in HIV. R01HL130301 & R01HL155265 (E.J.B). 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.

Abstract 1127: Erythrocyte Derived Extracellular Vesicles From Patients With Type 2 Diabetes Induce Endothelial Dysfunction Through Increased Vascular Oxidative Stress

Background: Red blood cells (RBCs) from patients with T2D (T2D-RBCs) induce endothelial dysfunction, but the mechanisms remain unclear. It is increasingly clear that extracellular vesicles (EVs) are actively secreted by RBCs and represent a novel mechanism of intercellular communication. However, the functional role of T2D-RBCs EVs in communication between RBCs and the cardiovascular system remains unknown. Purpose: This study aims to investigate whether EVs induce endothelial dysfunction in T2D and, if this is mediated via increased vascular oxidative stress. Material and Methods: EVs derived from T2D-RBCs and healthy RBCs (H-RBCs) were isolated using a membrane affinity column and co-incubated with wild-type mouse aortas for evaluation of the endothelium-dependent relaxation (EDR) using the wire myograph in the presence or absence of non-selective reactive oxygen species scavenger N-acetyl cysteine (NAC; 100 μM) applied to the aortas following the 18h EV incubation. The levels of the oxidative stress marker 4-hydroxynonenal (4-HNE) were quantified by immunohistochemistry in mouse aortas incubated with EVs. Additionally, T2D-RBCs EVs and H-RBCs EVs were co-incubated with human carotid artery endothelial cells (HCtAEC) for 8h and 24h to study the effects of EVs on vascular mRNA expression levels of eNOS, NOX1, and NOX4. Results: T2D-RBCs EVs but not H-RBCs EVs impaired EDR in aortas isolated from mice ( Fig. 1A ). This impairment was reversed by inhibiting oxidative stress in the vessel by NAC ( Fig. 1B ). Immunohistochemistry showed a significant increase in 4-HNE levels in aortas incubated with T2D-RBCs EVs ( Fig. 1C, D ). The T2D-RBCs EVs also induced a significant increase in NOX4 mRNA levels in HCtAEC after 24h but not 8h ( Fig. 1E, F ). However, T2D-RBCs EVs did not affect eNOS or NOX1 in endothelial cells. Conclusion: EVs derived from T2D-RBCs induce endothelial dysfunction through increased vascular oxidative stress.

Deficiency of neutral cholesterol ester hydrolase 1 (NCEH1) impairs endothelial function in diet-induced diabetic mice

BackgroundNeutral cholesterol ester hydrolase 1 (NCEH1) plays a critical role in the regulation of cholesterol ester metabolism. Deficiency of NCHE1 accelerated atherosclerotic lesion formation in mice. Nonetheless, the role of NCEH1 in endothelial dysfunction associated with diabetes has not been explored. The present study sought to investigate whether NCEH1 improved endothelial function in diabetes, and the underlying mechanisms were explored.MethodsThe expression and activity of NCEH1 were determined in obese mice with high-fat diet (HFD) feeding, high glucose (HG)-induced mouse aortae or primary endothelial cells (ECs). Endothelium-dependent relaxation (EDR) in aortae response to acetylcholine (Ach) was measured.ResultsResults showed that the expression and activity of NCEH1 were lower in HFD-induced mouse aortae, HG-exposed mouse aortae ex vivo, and HG-incubated primary ECs. HG exposure reduced EDR in mouse aortae, which was exaggerated by endothelial-specific deficiency of NCEH1, whereas NCEH1 overexpression restored the impaired EDR. Similar results were observed in HFD mice. Mechanically, NCEH1 ameliorated the disrupted EDR by dissociating endothelial nitric oxide synthase (eNOS) from caveolin-1 (Cav-1), leading to eNOS activation and nitric oxide (NO) release. Moreover, interaction of NCEH1 with the E3 ubiquitin-protein ligase ZNRF1 led to the degradation of Cav-1 through the ubiquitination pathway. Silencing Cav-1 and upregulating ZNRF1 were sufficient to improve EDR of diabetic aortas, while overexpression of Cav-1 and downregulation of ZNRF1 abolished the effects of NCEH1 on endothelial function in diabetes. Thus, NCEH1 preserves endothelial function through increasing NO bioavailability secondary to the disruption of the Cav-1/eNOS complex in the endothelium of diabetic mice, depending on ZNRF1-induced ubiquitination of Cav-1.ConclusionsNCEH1 may be a promising candidate for the prevention and treatment of vascular complications of diabetes.

Metformin reverses the effect of high glucose on mesenteric artery relaxation in normal rats

Elevated glucose produces alterations in the cardiovascular system, which affect the health of subjects with these increases, which affect systems such as the gastrointestinal, since the mesenteric arteries play an important role for the proper development of the individual. These diseases are related and cause great morbidity and mortality worldwide. Metformin is a biguanide used to control blood glucose levels. This study assessed the effect of metformin on arterial tension and endothelial function on rat normal mesenteric arteries. Arteries incubated in glucose (30 mM) demonstrated a profoundly impaired endothelium-dependent relaxation to acetylcholine. The treatment with metformin (1 mM) produced the change in the vascular activity, like the control group. These results indicated that the action of metformin in the vascular activity is recovered to normal condition, which is important in patients with diabetes mellitus type 2, already the cardiovascular alterations were presents in these patients. Glucose modified the effect of Nitric oxide synthase and CYTP450, decreasing the production of Nitric Oxide and hyperpolarizing factor, in arteries relaxed with acetylcholine, where metformin reversed to normal conditions. On the other hand, the used of metformin plus indomethacin produced inhibition on metformin, effects inducing a mayor inhibition on the vascular relaxation to acetylcholine. Also, metformin presented a minimum effect on K+ channels, when are inactivated with TEA, recovering of manner partial the action of this channel, increased relaxation effect by acetylcholine. Metformin effect is not due to osmotic control or oxidative stress. An important effect is the use of analgesics such as indomethacin, which produces dysfunction of the vascular endothelium, generating the presence of diseases due to the state of hypertension obtained in the mesenteric arteries, affecting the proper functioning of the gastrointestinal system, decreasing the uptake of nutrients and presenting various diseases.

DHCR24 Insufficiency Promotes Vascular Endothelial Cell Senescence and Endothelial Dysfunction via Inhibition of Caveolin-1/ERK Signaling.

Endothelial cells (ECs) senescence is critical for vascular dysfunction, which leads to age-related disease. DHCR24, a 3β-hydroxysterol δ 24 reductase with multiple functions other than enzymatic activity, has been involved in age-related disease. However, little is known about the relationship between DHCR24 and vascular ECs senescence. We revealed that DHCR24 expression is chronologically decreased in senescent human umbilical vein endothelial cells (HUVECs) and the aortas of aged mice. ECs senescence in endothelium-specific DHCR24 knockout mice was characterized by increased P16 and senescence-associated secretory phenotype, decreased SIRT1 and cell proliferation, impaired endothelium-dependent relaxation, and elevated blood pressure. In vitro, DHCR24 knockdown in young HUVECs resulted in a similar senescence phenotype. DHCR24 deficiency impaired endothelial migration and tube formation and reduced nitric oxide (NO) levels. DHCR24 suppression also inhibited the caveolin-1/ERK signaling, probably responsible for increased reactive oxygen species production and decreased eNOS/NO. Conversely, DHCR24 overexpression enhanced this signaling pathway, blunted the senescence phenotype, and improved cellular function in senescent cells, effectively blocked by the ERK inhibitor U0126. Moreover, desmosterol accumulation induced by DHCR24 deficiency promoted HUVECs senescence and inhibited caveolin-1/ERK signaling. Our findings demonstrate that DHCR24 is essential in ECs senescence.

Portulaca Oleracea L. (Purslane) Extract Protects Endothelial Function by Reducing Endoplasmic Reticulum Stress and Oxidative Stress through AMPK Activation in Diabetic Obese Mice.

Portulaca oleracea L. (purslane) is a food and a traditional drug worldwide. It exhibits anti-inflammatory, anti-oxidative, anti-tumor, and anti-diabetic bioactivities; but its activity on diabetic-associated endothelial dysfunction is unknown. This study aimed to investigate the effect of purslane on endothelial function and the underlying mechanisms. Male C57BL/6 mice had 14-week ad libitum access to a high-fat rodent diet containing 60% kcal% fat to induce obesity and diabetes whereas purslane extract (200 mg/kg/day) was administered during the last 4 weeks via intragastric gavage. Primary rat aortic endothelial cells and isolated mouse aortas were cultured with a risk factor, high glucose or tunicamycin, together with purslane extract. By ESI-QTOF-MS/MS, flavonoids and their glycoside products were identified in the purslane extract. Exposure to high glucose or tunicamycin impaired acetylcholine-induced endothelium-dependent relaxations in aortas and induced endoplasmic reticulum (ER) stress and oxidative stress with the downregulation of 5' AMP-activated protein kinase (AMPK)/ endothelial nitric oxide synthase (eNOS) signaling. Co-incubation with purslane significantly ameliorated these impairments. The effects of purslane were abolished by Compound C (AMPK inhibitor). Four-week purslane treatment ameliorated aortic relaxations, ER stress, and oxidative stress in diabetic obese mice. This study supported that purslane protected endothelial function, and inhibited ER stress and oxidative stress in vasculature through AMPK/eNOS activation, revealing its therapeutic potential against vascular complications in diabetes.

Urocortin2 attenuates diabetic coronary microvascular dysfunction by regulating macrophage extracellular vesicles

Diabetic patients develop coronary microvascular dysfunction (CMD) and exhibit high mortality of coronary artery disease. Methylglyoxal (MGO) largely accumulates in the circulation due to diabetes. We addressed whether macrophages exposed to MGO exhibited damaging effect on the coronary artery and whether urocortin2 (UCN2) serve as protecting factors against such diabetes-associated complication. Type 2 diabetes was induced by high-fat diet and a single low-dose streptozotocin in mice. Small extracellular vesicles (sEV) derived from MGO-treated macrophages (MGO-sEV) were used to produce diabetes-like CMD. UCN2 was examined for a protective role against CMD. The involvement of arginase1 and IL-33 was tested by pharmacological inhibitor and IL-33-/- mice. MGO-sEV was capable of causing coronary artery endothelial dysfunction similar to that by diabetes. Immunocytochemistry studies of diabetic coronary arteries supported the transfer of arginase1 from macrophages to endothelial cells. Mechanism studies revealed arginase1 contributed to the impaired endothelium-dependent relaxation of coronary arteries in diabetic and MGO-sEV-treated mice. UCN2 significantly improved coronary artery endothelial function, and prevented MGO elevation in diabetic mice or enrichment of arginase1 in MGO-sEV. Diabetes caused a reduction of IL-33, which was also reversed by UCN2. IL-33-/- mice showed impaired endothelium-dependent relaxation of coronary arteries, which can be mitigated by arginase1 inhibition but can’t be improved by UCN2 anymore, indicating the importance of restoring IL-33 for the protection against diabetic CMD by UCN2. Our data suggest that MGO-sEV induces CMD via shuttling arginase1 to coronary arteries. UCN2 is able to protect against diabetic CMD via modulating MGO-altered macrophage sEV cargoes.

Follistatin-like 1 protects endothelial function in the spontaneously hypertensive rat by inhibition of endoplasmic reticulum stress through AMPK-dependent mechanism

ABSTRACT Objective Endothelial dysfunction is a critical initiating factor in the development of hypertension and related complications. Follistatin-like 1 (FSTL1) can promote endothelial cell function and stimulates revascularization in response to ischemic insult. However, it is unclear whether FSTL1 has an effect on ameliorating endothelial dysfunction in spontaneously hypertensive rats (SHRs). Methods Wistar Kyoto (WKY) and SHRs were treated with a tail vein injection of vehicle (1 mL/day) or recombinant FSTL1 (100 μg/kg body weight/day) for 4 weeks. Blood pressure was measured by tail-cuff plethysmograph, and vascular reactivity in mesenteric arteries was measured using wire myography. Results We found that treatment with FSTL1 reversed impaired endothelium-dependent relaxation (EDR) in mesenteric arteries and lowered blood pressure of SHRs. Decreased AMP-activated protein kinase (AMPK) phosphorylation, elevated endoplasmic reticulum (ER) stress markers, increased reactive oxygen species (ROS), and reduction of nitric oxide (NO) production in mesenteric arteries of SHRs were also reversed by FSTL1 treatment. Ex vivo treatment with FSTL1 improved the impaired EDR in mesenteric arteries from SHRs and reversed tunicamycin (ER stress inducer)-induced ER stress and the impairment of EDR in mesenteric arteries from WKY rats. The effects of FSTL1 were abolished by cotreatment of compound C (AMPK inhibitor). Conclusions These results suggest that FSTL1 prevents endothelial dysfunction in mesenteric arteries of SHRs through inhibiting ER stress and ROS and increasing NO production via activation of AMPK signaling.

Abstract 14657: p66Shc Mediates SUMO2-Induced Endothelial Dysfunction and ROS Production

Endothelial dysfunction is one of the earliest changes promoting vascular pathologies. Studies suggest that promoting sumoylation increases endothelial dysfunction and development of atherosclerotic plaque. We showed that overexpression of SUMO2 (one of the sumo isoforms) leads to impaired vasorelaxation. However, the molecular signaling is not well understood.P66Shc is an adaptor protein known to regulate vascular function. Studies in mice suggest that p66Shc impairs endothelial function via promoting oxidative stress. Mice with p66Shc knockout are protected against endothelial dysfunction due to hyperlipidemia, diabetes, and aging. Therefore, we asked if p66Shc mediates endothelial effects of SUMO2. We show that p66Shc is modified by SUMO2 and knockdown of p66Shc in endothelial cells inhibits SUMO2-induced reactive oxygen species production as well as inflammation. To identify the specific residue of p66Shc getting sumoylated, we generated recombinant p66Shc and SUMO2ylated it with a commercially available kit. Using mass spectroscopy, we identified that lysine-81 in CH2 domain of p66Shc is specifically modified by SUMO2. We also show that SUMO2-modificaiton of p66ShcK81 promotes oxidative activation (serine-36 phosphorylation) and mitochondrial translocation of p66Shc which are essential for ROS production. P66Shc non-sumoylatable at lysine 81 (p66ShcK81R) is resistant to SUMO2-induced serine-36 phosphorylation and mitochondrial translocation. To examine physiological significance of p66ShcK81-SUMO2, we generated a mouse model having knock in of p66ShcK81R. Using aortic rings from p66ShcK81R knock in and wild type mice, we show that overexpression of SUMO2 significantly impairs endothelium dependent relaxation in wild type mice aortic rings while aortic rings from p66ShcK81R knock are protected against it. Together, we demonstrate that SUMO2-p66Shc is a novel molecular signaling regulating vascular endothelial function.

Adding SGLT2 Cotransporter Inhibitor to PPARγ Activator Does Not Provide an Additive Effect in the Management of Diabetes-Induced Vascular Dysfunction

Introduction: Endothelial dysfunction (ED) plays a key role in the pathogenesis of diabetic vascular complications. In monotherapy, dapagliflozin (Dapa) as well as pioglitazone (Pio) prevent the progression of target organ damage in both type 1 (T1DM) and type 2 diabetes. We investigated whether the simultaneous PPAR-γ activation and SGLT2 cotransporter inhibition significantly alleviate ED-related pathological processes and thus normalize vascular response in experimental T1DM. Methods: Experimental diabetes was induced by streptozotocin (STZ; 55 mg/kg, i.p.) in Wistar rats. Dapa (10 mg/kg), Pio (12 mg/kg), or their combination were administrated to the STZ rats orally. Six weeks after STZ administration, the aorta was excised for functional studies and real-time qPCR analysis. Results: In the aorta of diabetic rats, impaired endothelium-dependent and independent relaxation were accompanied by the imbalance between vasoactive factors (eNos, Et1) and overexpression of inflammation (Tnfα, Il1b, Il6, Icam, Vcam) and oxidative stress (Cybb) markers. Pio monotherapy normalized response to vasoactive substances and restored balance between Et1-eNos expression, while Dapa treatment was ineffective. Nevertheless, Dapa and Pio monotherapy significantly reverted inflammation and oxidative stress markers to normal values. The combination treatment exhibited an additive effect in modulating Il6 expression, reaching the effect of Pio monotherapy in other measured parameters. Conclusion: Particularly, Pio exerts a vasoprotective character when used in monotherapy. When combined with Dapa, it does not exhibit an expected additive effect within modulating vasoreactivity or oxidative stress, though having a significant influence on IL6 downregulation.

Varied effects of tobacco smoke and e-cigarette vapor suggest that nicotine does not affect endothelium-dependent relaxation and nitric oxide signaling

Chronic smoking causes dysfunction of vascular endothelial cells, evident as a reduction of flow-mediated dilation in smokers, but the role of nicotine is still controversial. Given the increasing use of e-cigarettes and other nicotine products, it appears essential to clarify this issue. We studied extracts from cigarette smoke (CSE) and vapor from e-cigarettes (EVE) and heated tobacco (HTE) for their effects on vascular relaxation, endothelial nitric oxide signaling, and the activity of soluble guanylyl cyclase. The average nicotine concentrations of CSE, EVE, and HTE were 164, 800, and 85 µM, respectively. At a dilution of 1:3, CSE almost entirely inhibited the relaxation of rat aortas and porcine coronary arteries to acetylcholine and bradykinin, respectively, while undiluted EVE, with a 15-fold higher nicotine concentration, had no significant effect. With about 50% inhibition at 1:2 dilution, the effect of HTE was between CSE and EVE. Neither extract affected endothelium-independent relaxation to an NO donor. At the dilutions tested, CSE was not toxic to cultured endothelial cells but, in contrast to EVE, impaired NO signaling and inhibited NO stimulation of soluble guanylyl cyclase. Our results demonstrate that nicotine does not mediate the impaired endothelium-dependent vascular relaxation caused by smoking.

Short-term Effects of Cadmium Exposure on Blood Pressure and Vascular Function in Wistar Rats.

Chronic cadmium exposure is known to be associated with vascular changes and increased blood pressure, but its short-term effects on the cardiovascular system remain poorly understood. This study aimed to investigate the pressoric and vascular effects of a 7-day exposure to CdCl2 in Wistar rats. The rats were divided in control group (Ct), which received tap water, and the Cd group, which received a 100mg/L CdCl2 solution via drinking water for 7days. We analyzed body weight, plasma Cadmium concentration, systolic blood pressure (SBP), and vascular responses. Despite relatively low plasma Cadmium concentration, the Cd group exhibited elevated SBP and increased contractile response to phenylephrine. Endothelium removal and NOS inhibition increased contractions in both groups. In the Cd group's aorta, we observed enhanced levels of phospho-eNOS (Ser1177) and basal NO release. Cd group showed reduced Catalase expression and increased basal release of H2O2, with catalase reducing the contractile response. In arteries pre-contracted with phenylephrine, Cd group showed impaired endothelium-dependent (Acetylcholine) and independent (sodium nitroprussiate-SNP) relaxation responses. However, responses to SNP were similar after pre-contraction with KCl in both groups. These data suggest early effects of Cadmium on blood pressure and aortic function, indicating impaired H2O2-scavenging by catalase. Increased H2O2 due to Cadmium exposure might explain heightened responses to phenylephrine and weakened relaxation responses mediated by the NO-K+-channels pathway. Our findings shed light on Cadmium's short-term impact on the cardiovascular system, providing insights into potential mechanisms underlying its effects on blood pressure regulation and vascular function.

Abstract P181: Tcell Derived Hiv Proteins Promote Oxidative Stress, Endothelial Dysfunction And Hypertension Via Interleukin 1a-mediated Mechanisms In Mice.

Antiretroviral therapy (ART) has markedly extended the life of people living with HIV (PLWH). However, PLWH now experience accelerated development of hypertension and cardiovascular disease (CVD), the etiology of which, including the individual contributions of ART and repressed viral infection, remain ill-defined. While ART prevents viral replication, viral reservoirs persist, and viral proteins remain in the circulation leading us to hypothesize that Tcell-derived HIV proteins contribute to endothelial dysfunction and hypertension. We utilized the Tg26 transgenic mouse model which ubiquitously express 7/9 viral proteins and reported increased blood pressure (BP) and impaired endothelium-dependent relaxation (EDR) in aorta and mesenteric arteries in both sexes. Bone marrow transplant (BMT) from Tg26 mice into WT (Tg26->WT) increased BP (WT->WT: 141.6 Tg26->WT 160.3 mmHg; P<0.05) and impaired EDR in conduit and resistance vessels (P<0.05). To determine whether Tcells, the main target of HIV, are responsible for CVD in Tg26 mice, thoracic aortas from WT male and female mice were co-cultured with either WT or Tg26 primary CD3+ Tcells, and yielded endothelial dysfunction. Moreover, inhibition of Tcell activation via Abatacept treatment in intact Tg26 mice lowered BP, restored vasorelaxation and reduced sympatho-activation to WT levels in Tg26 mice (P<0.05). A cytokine panel revealed increased production of interleukin 1α (IL-1α) in Tg26 CD3+ Tcells and plasma of Tg26->WT BMT (P<0.05). Abatacept decreased plasma IL-1α levels in intact Tg26 mice (P<0.05). Treatment with anti-IL-1α antibody abolished Tcell-mediated endothelial dysfunction and primary human microvascular endothelial cells (hMVEC) treated with IL-1α for 24 hours increased ROS mediated DNA damage (8-OHdG). IL-1α exposed hMVEC displayed increases in NOX1, NOXO1, and NOX4 while BMT aortas showed increased NOX1, NOXO1, and NOXA1. Inhibition of NOX1 via GKT771 ex vivo restored vasorelaxation. Additionally, anti-IL-1α restored vasorelaxation and BP in intact Tg26 mice. Ultimately, we found that Tcells expressing viral proteins produce high IL-1α levels which increase endothelial NOX1 expression and ROS production, leading to endothelial dysfunction and hypertension

AMPK-Dependent YAP Inhibition Mediates the Protective Effect of Metformin against Obesity-Associated Endothelial Dysfunction and Inflammation.

Hyperglycemia is a crucial risk factor for cardiovascular diseases. Chronic inflammation is a central characteristic of obesity, leading to many of its complications. Recent studies have shown that high glucose activates Yes-associated protein 1 (YAP) by suppressing AMPK activity in breast cancer cells. Metformin is a commonly prescribed anti-diabetic drug best known for its AMPK-activating effect. However, the role of YAP in the vasoprotective effect of metformin in diabetic endothelial cell dysfunction is still unknown. The present study aimed to investigate whether YAP activation plays a role in obesity-associated endothelial dysfunction and inflammation and examine whether the vasoprotective effect of metformin is related to YAP inhibition. Reanalysis of the clinical sequencing data revealed YAP signaling, and the YAP target genes CTGF and CYR61 were upregulated in aortic endothelial cells and retinal fibrovascular membranes from diabetic patients. YAP overexpression impaired endothelium-dependent relaxations (EDRs) in isolated mouse aortas and increased the expression of YAP target genes and inflammatory markers in human umbilical vein endothelial cells (HUVECs). High glucose-activated YAP in HUVECs and aortas was accompanied by increased production of oxygen-reactive species. AMPK inhibition was found to induce YAP activation, resulting in increased JNK activity. Metformin activated AMPK and promoted YAP phosphorylation, ultimately improving EDRs and suppressing the JNK activity. Targeting the AMPK-YAP-JNK axis could become a therapeutic strategy for alleviating vascular dysfunction in obesity and diabetes.

Type 1 Diabetes Impairs Endothelium-Dependent Relaxation Via Increasing Endothelial Cell Glycolysis Through Advanced Glycation End Products, PFKFB3, and Nox1-Mediated Mechanisms.

Type 1 diabetes (T1D) is a major cause of endothelial dysfunction. Although cellular bioenergetics has been identified as a new regulator of vascular function, whether glycolysis, the primary bioenergetic pathway in endothelial cells (EC), regulates vascular tone and contributes to impaired endothelium-dependent relaxation (EDR) in T1D remains unknown. Experiments were conducted in Akita mice with intact or selective deficiency in EC PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3), the main regulator of glycolysis. Seahorse analyzer and myography were employed to measure glycolysis and mitochondrial respiration, and EDR, respectively, in aortic explants. EC PFKFB3 (Ad-PFKFB3) and glycolysis (Ad-GlycoHi) were increased in situ via adenoviral transduction. T1D increased EC glycolysis and elevated EC expression of PFKFB3 and NADPH oxidase Nox1 (NADPH oxidase homolog 1). Functionally, pharmacological and genetic inhibition of PFKFB3 restored EDR in T1D, while in situ aorta EC transduction with Ad-PFKFB3 or Ad-GlycoHi reproduced the impaired EDR associated with T1D. Nox1 inhibition restored EDR in aortic rings from Akita mice, as well as in Ad-PFKFB3-transduced aorta EC and lactate-treated wild-type aortas. T1D increased the expression of the advanced glycation end product precursor methylglyoxal in the aortas. Exposure of the aortas to methylglyoxal impaired EDR, which was prevented by PFKFB3 inhibition. T1D and exposure to methylglyoxal increased EC expression of HIF1α (hypoxia-inducible factor 1α), whose inhibition blunted methylglyoxal-mediated EC PFKFB3 upregulation. EC bioenergetics, namely glycolysis, is a new regulator of vasomotion and excess glycolysis, a novel mechanism of endothelial dysfunction in T1D. We introduce excess methylglyoxal, HIF1α, and PFKFB3 as major effectors in T1D-mediated increased EC glycolysis.