Impaired Endothelium-dependent Vasorelaxation Research Articles

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.

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.

Fibulin-5 Is Required for Small Artery Development and Function

Introduction: Fibulins are a family of eight extracellular matrix proteins with diverse functions. Fibulin-4 and -5 (FBLN-4 and -5), in particular, have been shown to be important for elastic fiber assembly. Mutations in each of the genes lead to cutis laxa, a syndrome characterized by loose skin with variable organ involvement, particularly in the vasculature, lungs and skeleton. We recently characterized a mouse model carrying a disease-causing mutation in Fbln4 (E57K) and, contrary to conduit arteries that had elastic fiber fragmentation and stiffness, Fbln4E57K resistance arteries were structurally intact. Functionally however, Fbln4E57K mesenteric arteries exhibited endothelial dysfunction secondary to increased shear stress from large artery stiffness. Loss of FBLN5 has been shown to lead to elastic fiber fragmentation in conduit arteries, but the consequences of its loss on resistance arteries are unknown. Therefore, the objectives of this study were to determine the structural and functional consequences of FBLN5 loss on resistance arteries. Methods: We utilized adult Fbln5 knock-out (KO) mice and examined the ultrastructure of renal and mesenteric arteries via transmission electron microscopy. Functionally, using pressure myography, we characterized the myogenic tone and reactivity of Fbln5 KO and littermate control third-order mesenteric arteries to various vasoactive substances including acetylcholine, papaverine, angiotensin II, phenylephrine and potassium chloride. Results: While the internal elastic lamina (IEL) of Fbln5KO mice resistance arteries was largely intact, the external elastic lamina (EEL) was nearly absent. Similar to Fbln4E57K mice, Fbln5KO mesenteric arteries exhibited impaired endothelial-dependent vasorelaxation, likely secondary to proximal large artery stiffness. Unlike Fbln4E57K mesenteric arteries however, Fbln5 KO mesenteric arteries exhibited increased myogenic tone as well as a hypercontractile response to angiotensin II and potassium chloride, but not to phenylephrine. Conclusions: In addition to highlighting differences in the requirements for elastic fiber assembly along the arterial tree, these data emphasize the differential contribution of FBLN4 and FBLN5 to IEL vs. EEL in resistance arteries and suggest pathway-specific changes in smooth muscle cell contractility secondary to FBLN5 deficiency. This work was supported by the Washington University Pediatric Nephrology Research Core Pilot Grant Program to CMH. Funds were also provided by The Department of Pediatrics at Washington University in St. Louis School of Medicine to CMH. 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.

Sex-specific effects of a high fat diet on aortic inflammation and dysfunction

Obesity and vascular dysfunction are independent and sexually dimorphic risk factors for cardiovascular disease. A high fat diet (HFD) is often used to model obesity in mice, but the sex-specific effects of this diet on aortic inflammation and function are unclear. Therefore, we characterized the aortic immune cell profile and function in 6-week-old male and female C57BL/6 mice fed a normal chow diet (NCD) or HFD for 10 weeks. Metabolic parameters were measured weekly and fortnightly. At end point, aortic immune cell populations and endothelial function were characterized using flow cytometry and wire myography. HFD-male mice had higher bodyweight, blood cholesterol, fasting blood glucose and plasma insulin levels than NCD mice (P < 0.05). HFD did not alter systolic blood pressure (SBP), glycated hemoglobin or blood triglycerides in either sex. HFD-females had delayed increases in bodyweight with a transient increase in fasting blood glucose at week 8 (P < 0.05). Flow cytometry revealed fewer proinflammatory aortic monocytes in females fed a HFD compared to NCD. HFD did not affect aortic leukocyte populations in males. Conversely, HFD impaired endothelium-dependent vasorelaxation, but only in males. Overall, this highlights biological sex as a key factor determining vascular disease severity in HFD-fed mice.

Gestational intermittent hypoxia induces endothelial dysfunction and hypertension in pregnant rats: role of endothelin type B receptor†.

Obstructive sleep apnea is a recognized risk factor for gestational hypertension, yet the exact mechanism behind this association remains unclear. Here, we tested the hypothesis that intermittent hypoxia, a hallmark of obstructive sleep apnea, induces gestational hypertension through perturbed endothelin-1 signaling. Pregnant Sprague-Dawley rats were subjected to normoxia (control), mild intermittent hypoxia (10.5% O2), or severe intermittent hypoxia (6.5% O2) from gestational days 10-21. Blood pressure was monitored. Plasma was collected and mesenteric arteries were isolated for myograph and protein analyses. The mild and severe intermittent hypoxia groups demonstrated elevated blood pressure, reduced plasma nitrate/nitrite, and unchanged endothelin-1 levels compared to the control group. Western blot analysis revealed decreased expression of endothelin type B receptor and phosphorylated endothelial nitric oxide synthase, while the levels of endothelin type A receptor and total endothelial nitric oxide synthase remained unchanged following intermittent hypoxia exposure. The contractile responses to potassium chloride, phenylephrine, and endothelin-1 were unaffected in endothelium-denuded arteries from mild and severe intermittent hypoxia rats. However, mild and severe intermittent hypoxia rats exhibited impaired endothelium-dependent vasorelaxation responses to endothelin type B receptor agonist IRL-1620 and acetylcholine compared to controls. Endothelium denudation abolished IRL-1620-induced vasorelaxation, supporting the involvement of endothelium in endothelin type B receptor-mediated relaxation. Treatment with IRL-1620 during intermittent hypoxia exposure significantly attenuated intermittent hypoxia-induced hypertension in pregnant rats. This was associated with elevated circulating nitrate/nitrite levels, enhanced endothelin type B receptor expression, increased endothelial nitric oxide synthase activation, and improved vasodilation responses. Our data suggested that intermittent hypoxia exposure during gestation increases blood pressure in pregnant rats by suppressing endothelin type B receptor-mediated signaling, providing a molecular mechanism linking intermittent hypoxia and gestational hypertension.

Dapagliflozin impedes endothelial cell senescence by activating the SIRT1 signaling pathway in type 2 diabetes

BackgroundSodium-glucose cotransporter 2 inhibitors (SGLT2i) clinically reduce atherosclerosis and lower blood pressure. However, their impact on endothelial dysfunction in type 2 diabetes (T2D) remains unclear. In this study, we investigated the protective effect and underlying mechanism of the SGLT2 inhibitor dapagliflozin in diabetes. MethodsVascular reactivity was measured to assess the vasoprotective effect of dapagliflozin in a mouse model of high glucose (HG)-induced T2D. Pulse wave velocity was measured to quantify arterial stiffness. Protein expression was assessed by western blotting and immunofluorescence, oxidative stress was evaluated using dihydroethidium, nitric oxide was evaluated using the Griess reaction, and cellular senescence was assessed based on senescence-associated beta-galactosidase (SA‐β‐gal) activity and the expression of senescence markers. Furthermore, the endothelial nitric oxide synthase (eNOS) acetylation status was determined and eNOS interactions with SIRT1 were evaluated by coimmunoprecipitation assays. ResultsDapagliflozin protected against impaired endothelium-dependent vasorelaxation and improved arterial stiffness in the mouse model of T2D; mouse aortas had significantly reduced levels of senescence activity and senescence-associated inflammatory factors. HG-induced increases in senescence activity, protein marker levels, and oxidative stress in vitro were all ameliorated by dapagliflozin. The decreases in eNOS phosphorylation and nitric oxide (NO) production in senescent endothelial cells were restored by dapagliflozin. SIRT1 expression was reduced in HG-induced senescent endothelial cells, and dapagliflozin restored SIRT1 expression. SIRT1 inhibition diminished the antisenescence effects of dapagliflozin. Coimmunoprecipitation showed that SIRT1 was physically associated with eNOS, suggesting that the effects of dapagliflozin are dependent on SIRT1 activation. ConclusionThese findings indicate that dapagliflozin protects against endothelial cell senescence by regulating SIRT1 signaling in diabetic mice.

GYY4137, a hydrogen sulfide donor, protects against endothelial dysfunction in porcine coronary arteries exposed to myeloperoxidase and hypochlorous acid

Background and aimsMyeloperoxidase (MPO) and its principal reaction product hypochlorous acid (HOCl) are part of the innate immune response but are also associated with endothelial dysfunction, thought to involve a reduction in nitric oxide (NO) bioavailability. We aimed to investigate the effect of MPO and HOCl on vasorelaxation of coronary arteries and to assess directly the involvement of NO. In addition, we hypothesised that the slow release hydrogen sulfide (H2S) donor GYY4137 would salvage coronary artery endothelial function in the presence of MPO and HOCl. Methods and resultsContractility of porcine coronary artery segments was measured using isometric tension recording. Incubation with MPO (50 ng/ml) plus hydrogen peroxide (H2O2) (30 μM; substrate for MPO) impaired endothelium-dependent vasorelaxation to bradykinin in coronary arteries. HOCl (10–500 μM) also impaired endothelium-dependent relaxations. There was no effect of MPO plus H2O2, or HOCl, on endothelium-independent relaxations to 5′-N-ethylcarboxamidoadenosine and sodium nitroprusside. L-NAME (300 μM), a NO synthase inhibitor, attenuated bradykinin relaxations, leaving L-NAME-resistant relaxations to bradykinin mediated by endothelium-dependent hyperpolarization. In the presence of L-NAME, MPO plus H2O2 largely failed to impair endothelium-dependent relaxations to bradykinin. Similarly, HOCl failed to inhibit endothelium-dependent relaxations to bradykinin in the presence of L-NAME. GYY4137 (1–100 μM) protected endothelium-dependent relaxations to bradykinin from dysfunction caused by MPO plus H2O2, and HOCl, with no effect alone on bradykinin relaxation responses. The specific MPO inhibitor aminobenzoic acid hydrazide (ABAH) (1 and 10 μM) also protected against MPO plus H2O2-induced endothelial dysfunction (at 10 μM ABAH), but was less potent than GYY4137. ConclusionsMPO plus H2O2, and HOCl, impair coronary artery endothelium-dependent vasorelaxation via inhibition of NO. GYY4137 protects against endothelial dysfunction in arteries exposed to MPO plus H2O2, and HOCl. H2S donors such as GYY4137 are possible therapeutic options to control excessive MPO activity in cardiovascular diseases.

Environmentally persistent free radicals: Methods for combustion generation, whole-body inhalation and assessing cardiopulmonary consequences

Particulate matter (PM) containing environmentally persistent free radicals (EPFRs) results from the incomplete combustion of organic wastes which chemisorb to transition metals. This process generates a particle-pollutant complex that continuously redox cycles to produce reactive oxygen species. EPFRs are well characterized, but their cardiopulmonary effects remain unknown. This publication provides a detailed approach to evaluating these effects and demonstrates the impact that EPFRs have on the lungs and vasculature. Combustion-derived EPFRs were generated (EPFR lo: 2.1e−16 radical/g, EPFR hi: 5.5e−17 radical/g), characterized, and verified as representative of those found in urban areas. Dry particle aerosolization and whole-body inhalation were established for rodent exposures. To verify that these particles and exposures recapitulate findings relevant to known PM-induced cardiopulmonary effects, male C57BL6 mice were exposed to filtered air, ∼280 μg/m3 EPFR lo or EPFR hi for 4 h/d for 5 consecutive days. Compared to filtered air, pulmonary resistance was increased in mice exposed to EPFR hi. Mice exposed to EPFR hi also exhibited increased plasma endothelin-1 (44.6 vs 30.6 pg/mL) and reduced nitric oxide (137 nM vs 236 nM), suggesting vascular dysfunction. Assessment of vascular response demonstrated an impairment in endothelium-dependent vasorelaxation, with maximum relaxation decreased from 80% to 62% in filtered air vs EPFR hi exposed mice. Gene expression analysis highlighted fold changes in aryl hydrocarbon receptor (AhR) and antioxidant response genes including increases in lung Cyp1a1 (8.7 fold), Cyp1b1 (9 fold), Aldh3a1 (1.7 fold) and Nqo1 (2.4 fold) and Gclc (1.3 fold), and in aortic Cyp1a1 (5.3 fold) in mice exposed to EPFR hi vs filtered air. We then determined that lung AT2 cells were the predominate locus for AhR activation. Together, these data suggest the lung and vasculature as particular targets for the health impacts of EPFRs and demonstrate the importance of additional studies investigating the cardiopulmonary effects of EPFRs.

Moderate-Intensity Exercise Improves Mesenteric Arterial Function in Male UC Davis Type-2 Diabetes Mellitus (UCD-T2DM) Rats: A Shift in the Relative Importance of Endothelium-Derived Relaxing Factors (EDRF)

The beneficial cardiovascular effects of exercise are well documented, however the mechanisms by which exercise improves vascular function in diabetes are not fully understood. This study investigates whether there are (1) improvements in blood pressure and endothelium-dependent vasorelaxation (EDV) and (2) alterations in the relative contribution of endothelium-derived relaxing factors (EDRF) in modulating mesenteric arterial reactivity in male UC Davis type-2 diabetes mellitus (UCD-T2DM) rats, following an 8-week moderate-intensity exercise (MIE) intervention. EDV to acetylcholine (ACh) was measured before and after exposure to pharmacological inhibitors. Contractile responses to phenylephrine and myogenic tone were determined. The arterial expressions of endothelial nitric oxide (NO) synthase (eNOS), cyclooxygenase (COX), and calcium-activated potassium channel (KCa) channels were also measured. T2DM significantly impaired EDV, increased contractile responses and myogenic tone. The impairment of EDV was accompanied by elevated NO and COX importance, whereas the contribution of prostanoid- and NO-independent (endothelium-derived hyperpolarization, EDH) relaxation was not apparent compared to controls. MIE 1) enhanced EDV, while it reduced contractile responses, myogenic tone and systolic blood pressure (SBP), and 2) caused a shift away from a reliance on COX toward a greater reliance on EDH in diabetic arteries. We provide the first evidence of the beneficial effects of MIE via the altered importance of EDRF in mesenteric arterial relaxation in male UCD-T2DM rats.

Procyanidin B2 ameliorates endothelial dysfunction induced by nicotine via the induction of tetrahydrobiopterin synthesis

• Nicotine decreases the expression of DHFR and GCH1 to inhibit BH 4 and NO production. • PCB2 prevents nicotine-induced DHFR and GCH1 loss. • PCB2 improves endothelium-dependent vasorelaxation impaired by nicotine. • PCB2 induces vasorelaxation through a PPARδ-BH 4 dependent manner. Smoking is an independent risk factor for the cardiovascular diseases. Nicotine, a major component of tobacco, is responsible for the impaired endothelial-dependent vasorelaxation in smokers. Procyanidin B2 (PCB2), a natural flavonoid, has been reported to possess several potential beneficial effects on the cardiovascular system. However, whether PCB2 prevents nicotine-induced endothelial dysfunction remains unknown. In this study, we demonstrated that PCB2 improved nicotine-impaired endothelium-dependent vasorelaxation in mouse thoracic aortas. Mechanistically, PCB2 increased the expression levels of dihydrofolate reductase (DHFR) and GTP cyclohydrolase 1 (GCH1), two important enzymes in the synthesis of tetrahydrobiopterin (BH 4 ), in mouse aortas and cultured ECs. GSK0660, a selective antagonist of peroxisome proliferator-activated receptor δ (PPARδ), abolished the PCB2-induced effects on BH 4 synthesis, nitric oxide (NO) production and vasodilation. Collectively, we demonstrated that PCB2 mitigates nicotine-induced endothelial dysfunction through a PPARδ-BH 4 dependent manner, thereby suggesting a novel role of PCB2 in preventing vascular dysfunction caused by nicotine exposure.

Bone Marrow Culture-Derived Conditioned Medium Recovers Endothelial Function of Vascular Grafts following In Vitro Ischemia/Reperfusion Injury in Diabetic Rats.

Ischemia/reperfusion injury (IRI) remains a challenge in coronary artery bypass grafting (CABG). Diabetic patients with coronary artery disease are more likely to require CABG and therefore run a high risk for cardiovascular complications. Conditioned medium (CM) from bone marrow-derived mesenchymal stem cells has been shown to have beneficial effects against IRI. We hypothesized that adding CM to physiological saline protects vascular grafts from IRI in diabetic rats. Bone-marrow derived cells were isolated from nondiabetic rat femurs/tibias, and CM was generated. As we previously reported, CM contains 23 factors involved in inflammation, oxidative stress, and apoptosis. DM was induced by streptozotocin administration. Eight weeks later, to measure vascular function, aortic rings were isolated and mounted in organ bath chambers (DM group) or stored in 4°C saline, supplemented either with a vehicle (DM-IR group) or CM (DM-IR+CM group). Although DM was associated with structural changes compared to controls, there were no functional alterations. However, compared to the DM group, in the DM-IR aortas, impaired maximum endothelium-dependent vasorelaxation in response to acetylcholine (DM 86.7 ± 0.1% vs. DM-IR 42.5 ± 2.5% vs. DM-IR+CM 61.9 ± 2.0%, p < 0.05) was improved, caspase-3, caspase-8, caspase-9, and caspase-12 immunoreactivity was decreased, and DNA strand breakage, detected by the TUNEL assay, was reduced by CM. We present the experimental finding that the preservation of vascular grafts with CM prevents endothelial dysfunction after IRI in diabetic rats. Targeting apoptosis by CM may contribute to its protective effect.

Procalcitonin mediates vascular dysfunction in obesity

AimsObesity is accompanied by a chronic low-grade inflammation associated with endothelial dysfunction and vascular complications. Procalcitonin is a marker of inflammation, secreted by adipose tissue and elevated in obese subjects. We here investigated whether visceral or perivascular fat-derived procalcitonin is a target to improve obesity-induced endothelial dysfunction. Materials and methodsProcalcitonin expression was identified by Western blot. Murine endothelial cells were isolated using CD31-antibody-coated magnetic beads and reactive oxygen species and nitric oxide (NO) determined by H2DCF- or DAF-FM diacetate loading. Endothelium-dependent vasorelaxation was analyzed using pressure myography of murine arterioles. Calcitonin gene-related peptide (CGRP) was used to activate the calcitonin receptor-like receptor (CRLR)/RAMP1 complex and olcegepant or the dipeptidyl-peptidase 4 (DPP4) inhibitor sitagliptin to block procalcitonin signaling or activation. Key findingsIn addition to visceral adipose tissue, procalcitonin was present in perivascular and epicardial tissue. In concentrations typical for obesity, procalcitonin doubled reactive oxygen species formation and decreased endothelial nitric oxide production in murine endothelial cells. Intravenous delivery of procalcitonin to mice in obesity-associated concentrations impaired endothelium-dependent vasorelaxation in a CRLR/RAMP1-dependent manner and antagonized CGRP-induced endothelial NO release in vitro. Use of CRLR/RAMP1-receptor antagonist olcegepant counteracted procalcitonin effects on vasodilation, nitric oxide production and reactive oxygen species formation. Similarly, blocking procalcitonin activation by the DPP4 inhibitor sitagliptin antagonized endothelial procalcitonin effects. SignificanceProcalcitonin, liberated either from visceral or perivascular adipose tissue, contributes to endothelial dysfunction by antagonizing CGRP signaling in obesity. Targeting hyperprocalcitonemia may be a means to preserve endothelial function and reduce comorbidity burden in obese subjects.

Bread crust extract protects rats’ vascular grafts from in vitro ischemia/reperfusion injury

Ischemia/reperfusion (IR) injury affects harvested vascular grafts during coronary artery bypass surgery. We hypothesized that bread crust extract (BCE) protects vascular grafts from IR injury. Thoracic aortic rings from Wistar rats were mounted on organ bath chambers (control) or subjected to in vitro IR injury either in saline (IR) or saline-supplemented with BCE (IR + BCE). Impaired maximum endothelium-dependent vasorelaxation to acetylcholine (control 73 ± 2 % vs IR 40 ± 2 % vs IR + BCE 63 ± 3 %, p < 0.05), altered maximal phenylephrine-induced contractile responses and high K+-induced depolarization in the IR-group compared to controls were improved by BCE. Computer-assisted artificial intelligence methods, applied on gene expression data, showed that increased Ccl2 expression (>-7.23) with a decrease in the anti-oxidant gene Gclc (≤-2.947) and Stab1 (≤-6.144) levels were reflecting IR-group, whereas increased Ccl2 expression (>-7.23) with an increased Gclc (>-2.947) level were indicatory for IR + BCE-rings. Furthermore, BCE lowered nitrotyrosine and poly(ADP-ribose)-polymerase-1 immunoreactivity. BCE protects vascular grafts from in vitro IR injury.

Monocrotaline‐induced Pulmonary Hypertension Impairs Diaphragm Vasomotor Function

INTRODUCTIONPulmonary hypertension (PH) is a chronic and progressive disease characterized by high arterial blood pressure and compensatory vascular remodeling in the pulmonary circulation. Mounting evidence suggests that the effects of PH extend beyond the pulmonary circulation. In humans and animals, PH induces diaphragm contractile dysfunction and diaphragm muscle atrophy. While the mechanistic bases for the PH‐induced diaphragm dysfunction is relatively unknown, alterations in the vasomotor control of the diaphragm resistance vasculature (i.e. arterioles) may play a key role. Therefore, we tested the hypothesis that PH increases the vasoconstrictor responsiveness and impairs the vasorelaxation of diaphragm arterioles.METHODSAdult female Sprague‐Dawley rats (n=8) were divided into healthy control (HC; n=2), ovariectomized (OVX; n=3), and PH + OVX (n=3). PH + OVX animals were given a single intraperitoneal injection of monocrotaline (MCT; 50mg/kg) to induce PH. Disease progression was then monitored via echocardiography. Once animals developed PH (~3 weeks post injection), the diaphragm was removed and first‐order arterioles (1A) from the medial costal diaphragm were isolated, cannulated, and pressurized at 90 cmH2O. After a 60 min equilibration period and spontaneous tone development, vasoconstrictor responses to potassium chloride (KCl) (10‐100 mM) and endothelium‐dependent and ‐independent vasorelaxation responses to cumulative doses of acetylcholine (ACh) and sodium nitroprusside (SNP) (10‐9 to 10‐4 M) were determined.RESULTSSpontaneous tone (%) development was not different across all three groups (HC: 36 ± 9%; OVX: 33 ± 9%; PH + OVX: 39 ± 2%; P&gt;0.05). Compared to control vessels, maximal percent vasoconstriction with KCl was decreased with OVX (67 ± 2 versus 50 ± 1%; P&lt;0.05). Maximal vasoconstriction (%) was reduced with PH + OVX (36 ± 4%) compared to vessels from both healthy control and OVX groups (P&lt;0.05). Ovariectomy had no effect on endothelium‐dependent vasodilation responses to ACh versus intact control animals (P&gt;0.05). The maximal ACh‐mediated vasodilation with PH + OVX (29 ± 9%) was decreased versus both healthy (45 ± 6%; P&lt;0.05) and OVX animals (41 ± 4%; P&lt;0.05). Across all three groups, there were no differences in percent relaxation with SNP (P&gt;0.05).CONCLUSIONThese findings suggest that estrogen plays a role in the maintenance of K+ channel activity and/or the contribution of K+ channels to diaphragm vasomotor tone, and that PH further disrupts K+ channel function in diaphragm arterioles. In addition, the absence of estrogen in OVX animals had no effect on endothelial function whereas PH + OVX impaired endothelium‐dependent vasorelaxation of diaphragm resistance vessels. These findings suggest that PH, at least in the rodent model utilized herein, elicits endothelial dysfunction in the diaphragm vasculature and provide a potential mechanism for the impaired diaphragm contractile dysfunction in PH patients.

The effect of TNF-α inhibitor treatment on microRNAs and endothelial function in collagen induced arthritis

Chronic inflammation causes dysregulated expression of microRNAs. Aberrant microRNA expression is associated with endothelial dysfunction. In this study we determined whether TNF-α inhibition impacted the expression of miRNA-146a-5p and miRNA-155-5p, and whether changes in the expression of these miRNAs were related to inflammation-induced changes in endothelial function in collagen-induced arthritis (CIA). Sixty-four Sprague-Dawley rats were divided into control (n = 24), CIA (n = 24) and CIA+etanercept (n = 16) groups. CIA and CIA+etanercept groups were immunized with bovine type-II collagen, emulsified in incomplete Freund’s adjuvant. Upon signs of arthritis, the CIA+etanercept group received 10mg/kg of etanercept intraperitoneally, every three days. After six weeks of treatment, mesenteric artery vascular reactivity was assessed using wire-myography. Serum concentrations of TNF-α, C-reactive protein, interleukin-6, vascular adhesion molecule-1 (VCAM-1) and pentraxin-3 (PTX-3) were measured by ELISA. Relative expression of circulating miRNA-146a-5p and miRNA-155-5p were determined using RT-qPCR. Compared to controls, circulating miRNA-155-5p, VCAM-1 and PTX-3 concentrations were increased, and vessel relaxation was impaired in the CIA (all p<0.05), but not in the CIA+etanercept (all p<0.05) groups. The CIA group had greater miRNA-146a-5p expression compared to the CIA+etanercept group (p = 0.005). Independent of blood pressure, miRNA-146a-5p expression was associated with increased PTX-3 concentrations (p = 0.03), while miRNA-155-5p expression was associated with impaired vessel relaxation (p = 0.01). In conclusion, blocking circulating TNF-α impacted systemic inflammation-induced increased expression of miRNA-146a-5p and miRNA-155-5p, which were associated with endothelial inflammation and impaired endothelial dependent vasorelaxation, respectively.

Chronic Exposure to HIV-Derived Protein Tat Impairs Endothelial Function via Indirect Alteration in Fat Mass and Nox1-Mediated Mechanisms in Mice.

People living with human immunodeficiency virus (HIV) (PLWH) have increased risk for atherosclerosis-related cardiovascular disease (CVD), the main cause of death in this population. Notwithstanding, the mechanisms of HIV-associated vascular pathogenesis are not fully elucidated. Therefore, we sought to determine whether HIV-regulatory protein Tat mediates HIV-induced endothelial dysfunction via NADPH oxidase 1 (Nox1)-dependent mechanisms. Body weight, fat mass, leptin levels, expression of reactive oxygen species (ROS)-producing enzymes and vascular function were assessed in C57BL/6 male mice treated with Tat for 3 days and 4 weeks. Aortic rings and human endothelial cells were also treated with Tat for 2–24 h in ex vivo and in vitro settings. Chronic (4 weeks) but not acute (3 days and 2–24 h) treatment with Tat decreased body weight, fat mass, and leptin levels and increased the expression of Nox1 and its coactivator NADPH oxidase Activator 1 (NoxA1). This was associated with impaired endothelium-dependent vasorelaxation. Importantly, specific inhibition of Nox1 with GKT771 and chronic leptin infusion restored endothelial function in Tat-treated mice. These data rule out direct effects of HIV-Tat on endothelial function and imply the contribution of reductions in adipose mass and leptin production which likely explain upregulated expression of Nox1 and NoxA1. The Nox1 and leptin system may provide potential targets to improve vascular function in HIV infection-associated CVD.

Selective Inhibition of the C-Domain of ACE (Angiotensin-Converting Enzyme) Combined With Inhibition of NEP (Neprilysin): A Potential New Therapy for Hypertension.

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Periodontitis induces endothelial dysfunction in mice

The treatment of periodontitis has numerous positive effects on established chronic health conditions, including cardiovascular disease and diabetes. However, ethical considerations do limit the establishment of human trials to investigate whether periodontitis promotes the early stages of chronic conditions. Therefore, the aim of this study was to investigate whether periodontitis induces endothelial dysfunction in hyperlipidemic apolipoprotein E gene-deficient (ApoE-/-) mice. Forty-five 8-week-old ApoE-/- mice were challenged by oral lavage with Porphyromonas gingivalis and Streptococcus gordonii for 4 weeks. A subgroup of animals (n = 15–17/group) was placed in a metabolic chamber immediately before euthanasia at 4 weeks to measure VO2/CO2 concentrations and voluntary locomotion. In infected and control animals alveolar bone levels were measured by x-ray imaging and endothelial function was determined by measuring endothelial-dependent vasorelaxation of aortic rings. The mRNA expression levels of serum amyloid A and tumor necrosis factor were determined in liver tissues by qRT PCR and protein concentrations in serum by ELISA. Caecal contents were analysed by sequencing to determine changes to the gut microbiota to investigate linkages between microbiome and systemic changes. The results showed that oral lavage of P. gingivalis and S. gordonii for 4 weeks, initiated periodontitis in ApoE-/- mice, similar to the human situation. The oral inflammation was accompanied by a significant increase in mRNA expression of pro-inflammatory mediators serum amyloid A1 and tumor necrosis factor in the liver. Mice with periodontitis also exhibited impaired endothelial-dependent vasorelaxation responses to acetylcholine. This systemic response was connected to increased energy expenditure, locomotion and respiratory quotient. No differences were detected in caecal microbiota between the infected and control animals. Overall, this is the first report that provide evidence that periodontitis induces endothelial dysfunction in mice. Other systemic responses observed in response to the local reaction need further investigation. The study suggests that early prevention of periodontitis may help limit the early stages of endothelial dysfunction that is linked to atherogenesis in humans.

Potentiation of Acetylcholine-Induced Relaxation of Aorta in Male UC Davis Type 2 Diabetes Mellitus (UCD-T2DM) Rats: Sex-Specific Responses.

Previous reports suggest that diabetes may differentially affect the vascular beds of females and males. The objectives of this study were to examine whether there were (1) sex differences in aortic function and (2) alterations in the relative contribution of endothelium-derived relaxing factors in modulating aortic reactivity in UC Davis Type 2 Diabetes Mellitus (UCD-T2DM) rats. Endothelium-dependent vasorelaxation (EDV) in response to acetylcholine (ACh) was measured in aortic rings before and after exposure to pharmacological inhibitors. Relaxation responses to sodium nitroprusside were assessed in endothelium-denuded rings. Moreover, contractile responses to phenylephrine (PE) were measured before and after incubation of aortic rings with a nitric oxide synthase (NOS) inhibitor in the presence of indomethacin. Metabolic parameters and expression of molecules associated with vascular and insulin signaling as well as reactive oxygen species generation were determined. Diabetes slightly but significantly impaired EDV in response to ACh in aortas from females but potentiated the relaxation response in males. The potentiation of EDV in diabetic male aortas was accompanied by a traces of nitric oxide (NO)- and prostanoid-independent relaxation and elevated aortic expression of small- and intermediate conductance Ca2+-activated K+ channels in this group. The smooth muscle sensitivity to NO was not altered, whereas the responsiveness to PE was significantly enhanced in aortas of diabetic groups in both sexes. Endothelium-derived NO during smooth muscle contraction, as assessed by the potentiation of the response to PE after NOS inhibition, was reduced in aortas of diabetic rats regardless of sex. Accordingly, decreases in pAkt and peNOS were observed in aortas from diabetic rats in both sexes compared with controls. Our data suggest that a decrease in insulin sensitivity via pAkt-peNOS-dependent signaling and an increase in oxidative stress may contribute to the elevated contractile responses observed in diabetic aortas in both sexes. This study demonstrates that aortic function in UCD-T2DM rats is altered in both sexes. Here, we provide the first evidence of sexual dimorphism in aortic relaxation in UCD-T2DM rats.

Conditioned Medium from Mesenchymal Stem Cells Alleviates Endothelial Dysfunction of Vascular Grafts Submitted to Ischemia/Reperfusion Injury in 15-Month-Old Rats.

In patients undergoing coronary artery bypass grafting (CABG), ischemia/reperfusion injury (IRI) is the main contributor to organ dysfunction. Aging-induced vascular damage may be further aggravated during CABG. Favorable effects of conditioned medium (CM) from mesenchymal stem cells (MSCs) have been suggested against IRI. We hypothesized that adding CM to saline protects vascular grafts from IRI in rats. We found that CM contains 28 factors involved in apoptosis, inflammation, and oxidative stress. Thoracic aortic rings from 15-month-old rats were explanted and immediately mounted in organ bath chambers (aged group) or underwent 24 h of cold ischemic preservation in saline-supplemented either with vehicle (aged-IR group) or CM (aged-IR+CM group), prior to mounting. Three-month-old rats were used as referent young animals. Aging was associated with an increase in intima-to-media thickness, an increase in collagen content, higher caspase-12 mRNA levels, and immunoreactivity compared to young rats. Impaired endothelium-dependent vasorelaxation to acetylcholine in the aged-IR group compared to the aged-aorta was improved by CM (aged 61 ± 2% vs. aged-IR 38 ± 2% vs. aged-IR+CM 50 ± 3%, p < 0.05). In the aged-IR group, the already high mRNA levels of caspase-12 were decreased by CM. CM alleviates endothelial dysfunction following IRI in 15-month-old rats. The protective effect may be related to the inhibition of caspase-12 expression.