Endothelium-derived Nitric Oxide Research Articles

P-321 Uterine fibroids secrete vasodilatory factors that induce their response via the opioid pathway

Abstract Study question Do uterine fibroids produce vasoactive factors that could affect vasculature and induce fibroid associated symptoms? Summary answer Ex vivo secretions from uterine fibroids induce a vasodilatory response in rat mesenteric arteries that is mediated via the opioid receptor pathway. What is known already Uterine fibroids (leiomyoma) are a common but clinically neglected gynaecological condition, where benign smooth muscle tumours arise from the myometrium and cause heavy menstrual bleeding (HMB), pain and infertility. Fibroids affect up to one in two women of reproductive age and are the main reason for failure of both medical and surgical treatments in women with HMB. Fibroids are proposed to affect the systemic vasculature system, with angiogenic growth factors and abnormal vasculature implicated in fibroid pathophysiology. However, the precise mechanism of fibroid induced HMB has yet to be fully elucidated. Study design, size, duration Fibroid and myometrium biopsies were prospectively collected from 13 patients undergoing MyoSure, myomectomy or hysterectomy for the treatment of HMB associated with fibroids at Liverpool Women’s Hospital, a tertiary referral centre. The collection and use of human tissue was ethically approved, and all participants gave informed written consent. Tissue biopsies were incubated in a basal medium for 24 hours. Conditioned medium was collected and used for wire myography assays to examine vascular reactivity. Participants/materials, setting, methods Second order mesenteric arteries from female Wistar rats were mounted on a wire myograph system and toned with uridine triphosphate (UTP) before being exposed to control (non-conditioned), normal myometrium (n = 4) or fibroid (n = 9) conditioned medium. Vascular activity was measured relative to UTP induced vasoconstriction. To determine the mediatory factors of observed vasoreactivity, inhibitors of well characterised pathways were systematically tested in the presence of conditioned medium. Main results and the role of chance Fibroid conditioned medium caused a marked dilation of UTP induced vasoconstriction in rat mesenteric arteries, which was significantly greater than that induced by non-conditioned, or control myometrium conditioned medium. Blockage of ATP-sensitive potassium (KATP) channels with PNU37883A completely inhibited fibroid induced vasodilation. However, blockage of other potassium channels did not inhibit dilation, suggesting that a KATP-mediated PKA-dependent pathway is being stimulated. To test the potential involvement of endothelial-derived nitric oxide synthase in vasodilation, vessels were treated with the inhibitor L-NAME, which partially blocked fibroid induced vasodilation. The specificity of nitric oxide synthase inhibition was confirmed by treating vessels with acetylcholine or carbachol, which induced vasodilation via activation of nitric oxide synthase. In the presence of L-NAME, the vasodilatory effects of acetylcholine and carbachol were blocked, thus confirming inhibitor specificity. An adenosine receptor inhibitor, theophylline, had no effect on fibroid induced vasodilation, thus ruling out the adenosine pathway. Blockage of bradykinin and adrenergic receptors also failed to prevent fibroid induced dilation in mesenteric arteries. However, blocking opioid receptors with naloxone completely inhibited vasodilation in the presence of fibroid conditioned medium. These findings suggest that fibroid induced vasodilation is mediated via endothelial nitric oxide synthase and an unknown opioid compound. Limitations, reasons for caution This study was performed using excised tissue in an ex vivo environment, which may not fully simulate the fibroid secretome in vivo. Although rat mesenteric arteries are a well-established model, human uterine arteries may be more appropriate to study fibroid induced vasoreactivity. Wider implications of the findings Fibroids are responsible for up to half of all hysterectomies and healthcare costs run into the billions globally. This study demonstrates that fibroid secreted factors can impact vasoreactivity, which has important connotations for disease pathophysiology regarding HMB. Targeting vasoactive compounds produced by fibroids may be a future novel treatment option. Trial registration number NA

Vitamin E Attenuates Red-Light-Mediated Vasodilation: The Benefits of a Mild Oxidative Stress.

Red light (670 nm) energy controls vasodilation via the formation of a transferable endothelium-derived nitric oxide (NO)-precursor-containing substance, its intracellular traffic, and exocytosis. Here we investigated the underlying mechanistic effect of oxidative stress on light-mediated vasodilation by using pressure myography on dissected murine arteries and immunofluorescence on endothelial cells. Treatment with antioxidants Trolox and catalase decreased vessel dilation. In the presence of catalase, a lower number of exosomes were detected in the vessel bath. Light exposure resulted in increased cellular free radical levels. Mitochondrial reactive oxygen species were also more abundant but did not alter cellular ATP production. Red light enhanced the co-localization of late exosome marker CD63 and cellular S-nitrosoprotein to a greater extent than high glucose, suggesting that a mild oxidative stress favors the localization of NO precursor in late exosomes. Exocytosis regulating protein Rab11 was more abundant after irradiation. Our findings conclude that red-light-induced gentle oxidative stress facilitates the dilation of blood vessels, most likely through empowering the traffic of vasodilatory substances. Application of antioxidants disfavors this mechanism.

Risk association of the nitric oxide synthase VNTR intron 4 a/b variant with diabetic nephropathy – a pilot study

Diabetic nephropathy (DN) is known to be a leading complication of type 2 diabetes mellitus (T2D). This study evaluated whether the VNTR intron 4 a/b and rs1799983 polymorphisms of endothelial-derived nitric oxide synthase (eNOS) gene modulated the risk of developing DN in Asian Indian patients. The eNOS variants were genotyped in 200 patients, 100 with DN and 100 without DN. A significant risk association was observed for the VNTR intron 4 a/b (p < 0.05). Haplotype analysis revealed that the allele combination of rs1799983894 G/Intron 4b and rs1799983894 T/Intron 4b had a statistically significant inverse association with DN.

Evaluation of the component’s contribution in endothelium-dependent acetylcholine-induced relaxation of the rat aorta

The regulation of rat aorta vascular tone involves various factors, including endothelium-derived hyperpolarization factor (EDHF), nitric oxide (NO), prostaglandins, and sensory nerves. While these elements can function independently, their pathways intersect at various points, complicating the assessment of their individual contributions. The aim of this study was to establish the numerical contributions of EDHF, NO, prostaglandins, and also the effect of the sensory nerve on acetylcholine-induced relaxation on the background of phenylephrine preconstriction using contraction and relaxation measurements in Wistar rat thoracic aorta. EDHF, whose action is mediated through potassium channels, emerges as a crucial regulator. Blockage of inward rectifier potassium (KIR) channels integral to EDHF significantly abolishes 50% of the relaxation amplitude in comparison to control conditions. Endothelial TRPV4 channel, exhibiting a fine-tuning role, contributes to a 25% reduction in the amplitude of acetylcholine-induced relaxation in comparison to control relaxation. NO demonstrates its vasodilatory prowess, with NO blockage eliminating 77% of the residual relaxation effect after KIR blockage. Blockage of prostaglandin functions, modulated by cyclooxygenase 1, reduces relaxation by 44% in comparison to control relaxation. Desensitization of sensory nerves with capsaicin, shows a minor yet significant role, in the reduction of acetylcholine-induced relaxation amplitude by 10%. In conclusion, we established that the main element of acetylcholine-induced relaxation is EDHF with approximately 50% of relaxation amplitude depending on it.

The short-chain fatty acid propionate prevents ox-LDL-induced coronary microvascular dysfunction by alleviating endoplasmic reticulum stress in HCMECs.

Coronary microvascular dysfunction (CMD) is a critical pathogenesis of cardiovascular diseases. Lower endothelial nitric oxide synthase (eNOS) phosphorylation leads to reduced endothelium-derived relaxing factor nitric oxide (NO) generation, causing and accelerating CMD. Endoplasmic reticulum stress (ER stress) has been shown to reduce NO production in umbilical vein endothelial cells. Oxidized low-density lipoprotein (ox-LDL) damages endothelial cell function. However, the relationship between ox-LDL and coronary microcirculation has yet to be assessed. Short-chain fatty acid (SCFA), a fermentation product of the gut microbiome, could improve endothelial-dependent vasodilation in human adipose arterioles, but the effect of SCFA on coronary microcirculation is unclear. In this study, we found ox-LDL stimulated expression of ER chaperone GRP78. Further, we activated downstream PERK/eIF2a, IRE1/JNK, and ATF6 signaling pathways, decreasing eNOS phosphorylation and NO production in human cardiac microvascular endothelial. Furthermore, SCFA-propionate can inhibit ox-LDL-induced eNOS phosphorylation reduction and raise NO production; the mechanism is related to the inhibition of ER stress and downstream signaling pathways PERK/eIF2a, IRE1/JNK, and ATF6. In summary, we demonstrate that ox-LDL induced CMD by activating ER stress, propionate can effectively counteract the adverse effects of ox-LDL and protect coronary microcirculation function via inhibiting ER stress.

The 2023 Walter B. Cannon Award Lecture: Mechanisms Regulating Vascular Function and Blood Pressure by the PPARγ-RhoBTB1-CUL3 Pathway.

Human genetic and clinical trial data suggest that peroxisome proliferator activated receptor γ (PPARγ), a nuclear receptor transcription factor plays an important role in the regulation of arterial blood pressure. The examination of a series of novel animal models, coupled with transcriptomic and proteomic analysis, has revealed that PPARγ and its target genes employ diverse pathways to regulate vascular function and blood pressure. In endothelium, PPARγ target genes promote an antioxidant state, stimulating both nitric oxide (NO) synthesis and bioavailability, essential components of endothelial-smooth muscle communication. In vascular smooth muscle, PPARγ induces the expression of a number of genes that promote an antiinflammatory state and tightly control the level of cGMP, thus promoting responsiveness to endothelial-derived NO. One of the PPARγ targets in smooth muscle, Rho related BTB domain containing 1 (RhoBTB1) acts as a substrate adaptor for proteins to be ubiquitinated by the E3 ubiquitin ligase Cullin-3 and targeted for proteasomal degradation. One of these proteins, phosphodiesterase 5 (PDE5) is a target of the Cullin-3/RhoBTB1 pathway. Phosphodiesterase 5 degrades cGMP to GMP and thus regulates the smooth muscle response to NO. Moreover, expression of RhoBTB1 under condition of RhoBTB1 deficiency reverses established arterial stiffness. In conclusion, the coordinated action of PPARγ in endothelium and smooth muscle is needed to maintain NO bioavailability and activity, is an essential regulator of vasodilator/vasoconstrictor balance, and regulates blood vessel structure and stiffness.

Endothelial-dependent relaxation of α-pinene and two metabolites, myrtenol and verbenol, in isolated murine blood vessels.

Epidemiological evidence shows that residential proximity to greenspaces is associated with lower risk of all-cause and cardiovascular mortality; however, the mechanism(s) underlying this link remains unclear. Plants emit biogenic volatile organic compounds such as α-pinene that could elicit beneficial cardiovascular effects. To explore the role of α-pinene more directly, we studied the metabolism and the vascular effects of α-pinene. We found that exposure of mice to α-pinene (1 ppm, 6 h) generated two phase I oxidation metabolites, cis- and trans-verbenol [(1R,2R,5R)-verbenol and (1 R,2S,5R)-verbenol)] and myrtenol [(1S,5R)-(+)-myrtenol] that were identified in urine by GC-MS. Precontracted naïve murine male and female aorta and superior mesenteric artery (SMA) were relaxed robustly (60% tension reduction) by increasing concentrations of α-pinene, myrtenol, and verbenol to 0.3 mM, whereas 1 mM α-pinene was vasotoxic. The SMA was six times more sensitive than the aorta to α-pinene. Both myrtenol and verbenol were equally potent and efficacious as parent α-pinene in male and female SMA. The sensitive portion of the α-pinene-, myrtenol-, and verbenol-induced relaxations in male SMA was mediated by 1) endothelium, 2) eNOS-derived NO, and 3) guanylyl cyclase (GC) activity. Moreover, α-pinene activated the transient receptor potential ankyrin-1 (TRPA1) channel whereas the metabolites did not. Endothelial-derived NO regulates blood flow, blood pressure, and thrombosis, and it is plausible that inhaled (and ingested) α-pinene (or its metabolites) augments NO release to mediate the cardiovascular benefits of exposure to greenness.NEW & NOTEWORTHY A common plant-derived biogenic volatile organic compound, α-pinene, and two of its metabolites, myrtenol and verbenol, stimulate vasorelaxation in murine superior mesenteric artery. Both α-pinene- and its metabolites induce vasorelaxation by activation of the endothelium, nitric oxide, and guanylyl cyclase. α-Pinene also activates the transient receptor potential ankyrin-1. Positive associations between greenness exposure and human cardiovascular health may be a result of the vascular action of α-pinene and its metabolites, a novel consideration.

Vasodilator Responses of Perivascular Adipose Tissue-Derived Hydrogen Sulfide Stimulated with L-Cysteine in Pregnancy Hypertension-Induced Endothelial Dysfunction in Rats.

Endothelium-derived nitric oxide (NO)-induced vasodilation is impaired in pregnancy hypertension. However, the role of perivascular adipose tissue (PVAT)-derived hydrogen sulfide (H2S), as an alternative for counteracting vascular dysfunction, is incompletely clear in hypertensive disorders of pregnancy. Therefore, PVAT-derived H2S-induced vasodilation was investigated in pregnancy hypertension-induced endothelial dysfunction. Non-pregnant (Non-Preg) and pregnant (Preg) rats were submitted (or not) to the deoxycorticosterone (DOCA)-salt protocol and assigned as follows (n = 10/group): Non-Preg, Non-Preg+DOCA, Preg, and Preg+DOCA groups. Systolic blood pressure (SBP), angiogenesis-related factors, determinant levels of H2S (PbS), NO (NOx), and oxidative stress (MDA) were assessed. Vascular changes were recorded in thoracic aortas with PVAT and endothelium (intact and removed layers). Vasorelaxation responses to the substrate (L-cysteine) for the H2S-producing enzyme cystathionine-γ-lyase (CSE) were examined in the absence and presence of CSE-inhibitor DL-propargylglycine (PAG) in thoracic aorta rings pre-incubated with cofactor for CSE (pyridoxal-5 phosphate: PLP) and pre-contracted with phenylephrine. Hypertension was only found in the Preg+DOCA group. Preg+DOCA rats showed angiogenic imbalances and increased levels of MDA. PbS, but not NOx, showed increased levels in the Preg+DOCA group. Pre-incubation with PLP and L-cysteine elevated determinants of H2S in PVAT and placentas of Preg-DOCA rats, whereas no changes were found in the aortas without PVAT. Aortas of Preg-DOCA rats showed that PVAT-derived H2S-dependent vasodilation was greater compared to endothelium-derived H2S, whereas PAG blocked these responses. PVAT-derived H2S endogenously stimulated with the amino acid L-cysteine may be an alternative to induce vasorelaxation in endothelial dysfunction related to pregnancy hypertension.

Roles of endothelial and smooth muscle cell dysfunction and vasa vasorum in vasomotor disorders in ischemia with no obstructive coronary artery disease

Recently, the importance has emerged of ischemia with no obstructive coronary artery disease (INOCA), for which endothelial and vascular smooth muscle cell (VSMC) dysfunctions and alterations in coronary vasa vasorum are involved. Regarding endothelial vasodilator functions, both endothelium-derived nitric oxide and endothelium-derived hyperpolarizing factor play important roles in modulating vascular tone, especially in the microcirculation. Recent studies have suggested systemic endothelial dysfunction in INOCA. Regarding VSMC dysfunction, Rho-kinase has been identified as a key molecular mechanism of VSMC hyperconstriction in INOCA. Finally, recent advances of coronary imaging have demonstrated the important role of altered adventitial vasa vasorum functions in INOCA.

Nitric oxide is required for lung alveolarization revealed by deficiency of argininosuccinate lyase.

Inhaled nitric oxide (NO) therapy has been reported to improve lung growth in premature newborns. However, the underlying mechanisms by which NO regulates lung development remain largely unclear. NO is enzymatically produced by three isoforms of nitric oxide synthase (NOS) enzymes. NOS knockout mice are useful tools to investigate NO function in the lung. Each single NOS knockout mouse does not show obvious lung alveolar phenotype, likely due to compensatory mechanisms. While mice lacking all three NOS isoforms display impaired lung alveolarization, implicating NO plays a pivotal role in lung alveolarization. Argininosuccinate lyase (ASL) is the only mammalian enzyme capable of synthesizing L-arginine, the sole precursor for NOS-dependent NO synthesis. ASL is also required for channeling extracellular L-arginine into a NO-synthetic complex. Thus, ASL deficiency (ASLD) is a non-redundant model for cell-autonomous, NOS-dependent NO deficiency. Here, we assessed lung alveolarization in ASL-deficient mice. Hypomorphic deletion of Asl (AslNeo/Neo) results in decreased lung alveolarization, accompanied with reduced level of S-nitrosylation in the lung. Genetic ablation of one copy of Caveolin-1, which is a negative regulator of NO production, restores total S-nitrosylation as well as lung alveolarization in AslNeo/Neo mice. Importantly, NO supplementation could partially rescue lung alveolarization in AslNeo/Neo mice. Furthermore, endothelial-specific knockout mice (VE-Cadherin Cre; Aslflox/flox) exhibit impaired lung alveolarization at 12weeks old, supporting an essential role of endothelial-derived NO in the enhancement of lung alveolarization. Thus, we propose that ASLD is a model to study NO-mediated lung alveolarization.

Pregnancy hypertension-associated endothelial dysfunction is attenuated by isoflurane anesthesia: Evidence of protective effect related to increases in nitric oxide

AimsPregnancy hypertension-induced endothelial dysfunction associated with impairment of nitric oxide (NO) bioavailability and hemodynamic derangements is a challenging for urgent procedures requiring maternal anesthesia. The volatile anesthetic isoflurane has demonstrated NO-associated protective effects. However, this isoflurane-induced effect is still unclear in pregnancy hypertension. Therefore, the present study examined the potential protective effects of isoflurane anesthesia on endothelial dysfunction and hemodynamic changes induced by hypertensive pregnancy associated with fetal and placental growth restrictions. Materials and methodsAnimals were distributed into four groups: normotensive pregnant rats (Preg), anesthetized pregnant rats (Preg+Iso), hypertensive pregnant rats (HTN-Preg), and anesthetized hypertensive pregnant rats (HTN-Preg+Iso). Systolic and diastolic pressures, mean arterial pressure (MAP), heart rate, fetal and placental weights, vascular contraction, endothelium-derived NO-dependent vasodilation, and NO levels were assessed. The vascular endothelial growth factor (VEGF) levels and endothelial NO synthase (eNOS) Serine (1177) phosphorylation (p-eNOS) expression were also examined. Key findingsIsoflurane produced more expressive hypotensive effects in the HTN-Preg+Iso versus Preg+Iso group, with respective reductions in MAP by 50 ± 13 versus 25 ± 4 mmHg (P < 0.05). Also, HTN-Preg+Iso compared to the HTN-Preg group showed (respectively) preventions against the weight loss of the fetuses (4.0 ± 0.6 versus 2.8 ± 0.6 g, P < 0.05) and placentas (0.37 ± 0.06 versus 0.30 ± 0.06 mg, P < 0.05), hyper-reactive vasocontraction response (1.8 ± 0.4 versus 2.8 ± 0.6 g, P < 0.05), impaired endothelium-derived NO-dependent vasodilation (84 ± 8 versus 50 ± 17 %, P < 0.05), reduced VEGF levels (147 ± 46 versus 25 ± 13 pg/mL, P < 0.05), and decreased p-eNOS expression (0.24 ± 0.07 versus 0.09 ± 0.05 arbitrary units, P < 0.05). SignificanceIsoflurane anesthesia protects maternal endothelial function in pregnancy hypertension, and possibly endothelium-derived NO is involved.

Sodium Nitrite Attenuates Reduced Activity of Vascular Matrix Metalloproteinase-2 and Vascular Hyper-Reactivity and Increased Systolic Blood Pressure Induced by the Placental Ischemia Model of Preeclampsia in Anesthetized Rats.

Preeclampsia is a maternal hypertension disorder associated with vascular dysfunction and fetal and placental growth restrictions. Placental ischemia is suggested as the primary trigger of preeclampsia-associated impairments of both endothelium-derived nitric oxide (NO) and the vascular activity of extracellular matrix metalloproteinase-2 (MMP-2). Reduced uteroplacental perfusion pressure (RUPP) is a placental ischemia model of preeclampsia. Reduction of sodium nitrite to NO may occur during ischemic conditions. However, sodium nitrite effects in the RUPP model of preeclampsia have not yet been investigated. Pregnant rats were divided into four groups: normotensive pregnant rats (Norm-Preg), pregnant rats treated with sodium nitrite (Preg + Nitrite), preeclamptic rats (RUPP), and preeclamptic rats treated with sodium nitrite (RUPP + Nitrite). Maternal blood pressure and fetal and placental parameters were recorded. Vascular function, circulating NO metabolites, and the gelatinolytic activity of vascular MMP-2 were also examined. Sodium nitrite attenuates increased blood pressure, prevents fetal and placental weight loss, counteracts vascular hyper-reactivity, and partially restores NO metabolites and MMP-2 activity. In conclusion, sodium nitrite reduction to NO may occur during RUPP-induced placental ischemia, thereby attenuating increased blood pressure, fetal and placental growth restriction, and vascular hyper-reactivity associated with preeclampsia and possibly restoring NO and MMP-2 activity, which underlie the blood pressure-lowering effects.

A Redox Modulatory SOD Mimetic Nanozyme Prevents the Formation of Cytotoxic Peroxynitrite and Improves Nitric Oxide Bioavailability in Human Endothelial Cells.

The endothelium-derived signalling molecule nitric oxide (NO) in addition to controlling multifarious servo-regulatory functions, suppresses key processes in vascular lesion formation and prevents atherogenesis and other vascular abnormalities. The conversion of NO into cytotoxic and powerful oxidant peroxynitrite (ONOO- ) in a superoxide (O2 .- )-rich environment has emerged as a major reason for reduced NO levels in vascular walls, leading to endothelial dysfunction and cardiovascular complications. So, designing superoxide dismutase (SOD) mimetics that can selectively catalyze the dismutation of O2 .- in the presence of NO, considering their rapid reaction is challenging and is of therapeutic relevance. Herein, the authors report that SOD mimetic cerium vanadate (CeVO4 ) nanozymes effectively regulate the bioavailability of both NO and O2 .- , the two vital constitutive molecules of vascular endothelium, even in the absence of cellular SOD enzyme. The nanozymes optimally modulate the O2 .- level in endothelial cells under oxidative stress conditions and improve endogenously generated NO levels by preventing the formation of ONOO- . Furthermore, nanoparticles exhibit size- and morphology-dependent uptake into the cells and internalize via the clathrin-mediated endocytosis pathway. Intravenous administration of CeVO4 nanoparticles in mice caused no definite organ toxicity and unaltered haematological and biochemical parameters, indicating their biosafety and potential use in biological applications.

R-(+)-WIN55212-2 protects pericytes from ischemic damage and restores retinal microcirculatory patency after ischemia/reperfusion injury

Background and purposeCannabinoids are vasoactive substances that act as key regulators of arterial tone in the blood vessels supplying peripheral tissues and the central nervous system. This study aimed to investigate the potential of R-(+)-WIN55212-2 (WIN), a cannabinoid receptor 1 agonist (CB1), as a treatment for retinal ischemia/reperfusion (I/R) injury. Experimental approachMale Wistar rats were subjected to retinal I/R injury by increasing intraocular pressure in the anterior chamber. The rats were randomly divided into four groups: normal control, I/R, vehicle (pre-treated with dimethyl sulfoxide [DMSO] via intraperitoneal injection), and experimental (pre-treated with WIN at a dose of 1 ml/kg via intraperitoneal injection). The rats were sacrificed at different time points of reperfusion (1 hour, 3 hours, 6 hours, and 1 day) after inducing retinal I/R injury, and their retinas were collected for analysis. Oxygen-glucose deprived/reperfusion (OGD/R) was performed by initially perfusing the retinas with oxygenated artificial cerebrospinal fluid (ACSF), then switching to an OGD solution to simulate ischemia, followed by another perfusion with ACSF. Pericyte contraction and the "no-reflow" phenomenon were observed using infrared differential interference contrast (IR-DIC) microscopy and immunohistochemistry. Western blot, enzyme-linked immunosorbent assay (ELISA), and nitric oxide (NO) detection were used to explore the potential mechanism. Key resultsIn both the OGD/R and I/R models, retinal pericytes exhibited persistent contraction even after reperfusion. The ability of WIN to regulate the tone of retinal pericytes and capillaries was specifically blocked by the BKCa inhibitor iberiotoxin (100 nM). WIN demonstrated a protective effect against retinal I/R injury by preserving blood flow in vessels containing pericytes. Pretreatment with WIN alleviated the persistent contraction and apoptosis of retinal pericytes in I/R-induced rats, accompanied by a reduction in intracellular calcium ion (Ca2+) concentration. The expression of CB1 decreased in a time-dependent manner in the I/R group. After I/R injury, endothelium-derived nitric oxide (eNOS) levels were reduced at all time points, which was successfully reversed by WIN therapy except for the 1 day group. Additionally, the downregulation of cyclic guanosine monophosphate (cGMP) and BKCa expression at 3 hours, 6 hours, and 1 day after I/R injury was restored by pretreatment of WIN. Conclusions & implicationsWIN exerted its protective effects on retinal I/R injury by inhibiting the contraction and apoptosis of pericytes through the CB1-eNOS-cGMP-BKCa signaling pathway, thus ameliorated the occlusion of retinal capillaries.

Sex differences in the development of coronary microvascular diseases in type-2 diabetes: possible role of gap junction protein connexin-40

Type-2 diabetes (T2D) increases the risk of cardiovascular diseases 2-4 folds in both sexes compared to non-diabetic individuals. However, more than 50% of female patients who experience chest pain have coronary microvascular disease (CMD) instead of obstructive CAD. On the other hand, 20% of men with chest pain have CMD. CMD is characterized by reduced coronary flow reserve, and coronary flow is regulated by the change in capillary density and vessel diameter. Gap junction protein connexin-40 (Cx40) plays a significant role in coronary microvascular function by controlling endothelium-dependent relaxation (EDR), cardiac endothelial cell (CEC) migration and capillary density. We hypothesized that T2D differentially affects the CX-40 expression and function in male and female coronary artery (CA) and causes CMD. The objective of this study is to identify the molecular mechanisms underlining the development of T2D-induced CMD in male and female mice.T2D mice were generated by a combination of high-fat diet feeding and a low-dose of streptozotocin (75 mg/kg, i.p.). The oral glucose tolerance was conducted at 4-, 8-, 12-, and 16 weeks after diabetes induction. Coronary flow velocity reserve (CFVR) was used to assess coronary microvascular function in mice. Coronary endothelial function was determined by 1) capillary density measurement in the left ventricle and 2) isometric tension experiment in small CAs (3rd order). CECs from male and female mice were isolated to conduct molecular biological experiments.There was no significant difference in CFVR and capillary density between control male and female mice. However, T2D mice of both sexes showed significant differences in the properties of EDR, CFVR and capillary density. T2D male mice exhibited a significant attenuation of EDR accompanied by impaired endothelium-derived nitric oxide(EDNO)-dependent relaxation and endothelium-dependent hyperpolarization (EDH)-mediated relaxation. On the contrary, there is no difference in EDR between control and diabetic female mice. Capillary density was reduced in T2D mice of both sexes compared to the respective controls. Our RNA sequencing data in CECs revealed that Cx40 is one of the genes which contributes to diabetes-mediated CMD. Systemic Cx40-KO male mice showed reduced EDR, EDH-mediated relaxation and capillary density compared to wild type control. T2D reduced the expression of Cx40 and overexpression of Cx40 restored EDR in CAs and increased capillary density in T2D male mice.T2D impaired coronary microvascular function in both sexes. However, the cause of reduced CFVR was different between diabetic male and female mice. In T2D females, capillary rarefaction was a primary cause of reduced CFVR. In contrast, attenuated EDNO- and EDH-mediated relaxation, reduced capillary density and downregulation of Cx40 contributed to decreased CFVR in T2D male mice. Future studies are required to establish the role of Cx40 in the development of CMD in T2D females. Supported by NIH R01HL142214 and DOD W81XWH2110472 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

20-Hydroxyecdysone inhibits inflammation via SIRT6-mediated NF-κB signaling in endothelial cells

20-Hydroxyecdysone (20E) is known to have numerous pharmacological activities and can be used to treat diabetes and cardiovascular diseases. However, the protective effects of 20E against endothelial dysfunction and its targets remain unclear. In the present study, we revealed that 20E treatment could modulate the release of the endothelium-derived vasomotor factors NO, PGI2 and ET-1 and suppress the expression of ACE in TNF-α-induced 3D-cultured HUVECs. In addition, 20E suppressed the expression of CD40 and promoted the expression of SIRT6 in TNF-α-induced 3D-cultured HUVECs. The cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) and molecular docking results demonstrated that 20E binding increased SIRT6 stability, indicating that 20E directly bound to SIRT6 in HUVECs. Further investigation of the underlying mechanism showed that 20E could upregulate SIRT6 levels and that SIRT6 knockdown abolished the regulatory effect of 20E on CD40 in TNF-α-induced HUVECs, while SIRT6 overexpression further improved the effect of 20E. Moreover, we found that 20E could reduce the acetylation of NF-κB p65 (K310) through SIRT6, but the catalytic inactive mutant SIRT6 (H133Y) did not promote the deacetylation of NF-κB p65, suggesting that the inhibitory effect of 20E on NF-κB p65 was dependent on SIRT6 deacetylase activity. Additionally, our results indicated that 20E inhibited NF-κB via SIRT6, and the expression of CD40 was increased in HUVECs treated with SIRT6 siRNA and NF-κB inhibitor. In conclusion, the present study demonstrates that 20E exerts its effect through SIRT6-mediated deacetylation of NF-κB p65 (K310) to inhibit CD40 expression in ECs, and 20E may have therapeutic potential for the treatment of cardiovascular diseases.

Endothelial IK and SK channel activation decreases pulmonary arterial pressure and vascular remodeling in pulmonary hypertension.

Endothelial cells (ECs) from small pulmonary arteries (PAs) release nitric oxide (NO) and prostacyclin, which lower pulmonary arterial pressure (PAP). In pulmonary hypertension (PH), the levels of endothelium-derived NO and prostacyclin are reduced, contributing to elevated PAP. Small-and intermediate-conductance Ca2+-activated K+ channels (IK and SK)-additional crucial endothelial mediators of vasodilation-are also present in small PAs, but their function has not been investigated in PH. We hypothesized that endothelial IK and SK channels can be targeted to lower PAP in PH. Whole-cell patch-clamp experiments showed functional IK and SK channels in ECs, but not smooth muscle cells, from small PAs. Using a SU5416 plus chronic hypoxia (Su + CH) mouse model of PH, we found that currents through EC IK and SK channels were unchanged compared with those from normal mice. Moreover, IK/SK channel-mediated dilation of small PAs was preserved in Su + CH mice. Consistent with previous reports, endothelial NO levels and NO-mediated dilation were reduced in small PAs from Su + CH mice. Notably, acute treatment with IK/SK channel activators decreased PAP in Su + CH mice but not in normal mice. Further, chronic activation of IK/SK channels decreased PA remodeling and right ventricular hypertrophy, which are pathological hallmarks of PH, in Su + CH mice. Collectively, our data provide the first evidence that, unlike endothelial NO release, IK/SK channel activity is not altered in PH. Our results also demonstrate proof of principle that IK/SK channel activation can be used as a strategy for lowering PAP in PH.

Berry consumption mitigates the hypertensive effects of a high-fat, high-sucrose diet via attenuation of renal and aortic AT1R expression resulting in improved endothelium-derived NO bioavailability

Dysregulation of the renin-angiotensin system (RAS) is a contributor to high-fat diet-related blood pressure (BP) increases. Deleterious effects of dysregulated RAS result in an overproduction of reactive oxygen species and a decrease in endothelial nitric oxide (NO) bioavailability due to increased NADPH oxidase (NOX) expression. Dietary polyphenols have been shown to mitigate the imbalance in the redox state and protect against endothelial dysfunction induced by a high-fat diet. Thus, we aim to determine whether polyphenol-rich blackberry and raspberry, alone and in combination, attenuate the detrimental effects of a high-fat, high-sucrose (HFHS) diet on the vascular endothelium and kidneys of mice. We show that a HFHS diet increased the expression of renal and aortic angiotensin type 1 receptor (AT1R). Further, NOX1 and NOX4 expression were increased in the kidney contributing to fibrotic damage. In human aortic endothelial cells (HAECs), palmitic acid increased the expression of NOX4, potentially driving oxidative damage in the aorta, as evidenced by increased nitrotyrosine expression. Berries reduced the expression of renal and aortic AT1R, leading to a subsequent decrease in renal NOX expression and reduced aortic oxidative stress evidenced by reduced nitrotyrosine expression. Blackberry and raspberry in combination increased the expression of NRF2 and its downstream proteins in HAECs, thereby reducing the oxidative burden to the endothelium. In combination, blackberry and raspberry also increased serum levels of NO metabolites. These findings indicate that blackberry and raspberry unique polyphenols may act synergistically to favorably modulate the abovementioned pathways and attenuate HFHS diet-induced increases in BP.

Role of adropin in arterial stiffening associated with obesity and type 2 diabetes.

Adropin is a peptide largely secreted by the liver and known to regulate energy homeostasis; however, it also exerts cardiovascular effects. Herein, we tested the hypothesis that low circulating levels of adropin in obesity and type 2 diabetes (T2D) contribute to arterial stiffening. In support of this hypothesis, we report that obesity and T2D are associated with reduced levels of adropin (in liver and plasma) and increased arterial stiffness in mice and humans. Establishing causation, we show that mesenteric arteries from adropin knockout mice are also stiffer, relative to arteries from wild-type counterparts, thus recapitulating the stiffening phenotype observed in T2D db/db mice. Given the above, we performed a set of follow-up experiments, in which we found that 1) exposure of endothelial cells or isolated mesenteric arteries from db/db mice to adropin reduces filamentous actin (F-actin) stress fibers and stiffness, 2) adropin-induced reduction of F-actin and stiffness in endothelial cells and db/db mesenteric arteries is abrogated by inhibition of nitric oxide (NO) synthase, and 3) stimulation of smooth muscle cells or db/db mesenteric arteries with a NO mimetic reduces stiffness. Lastly, we demonstrated that in vivo treatment of db/db mice with adropin for 4 wk reduces stiffness in mesenteric arteries. Collectively, these findings indicate that adropin can regulate arterial stiffness, likely via endothelium-derived NO, and thus support the notion that "hypoadropinemia" should be considered as a putative target for the prevention and treatment of arterial stiffening in obesity and T2D.NEW & NOTEWORTHY Arterial stiffening, a characteristic feature of obesity and type 2 diabetes (T2D), contributes to the development and progression of cardiovascular diseases. Herein we establish that adropin is decreased in obese and T2D models and furthermore provide evidence that reduced adropin may directly contribute to arterial stiffening. Collectively, findings from this work support the notion that "hypoadropinemia" should be considered as a putative target for the prevention and treatment of arterial stiffening in obesity and T2D.

The role of p53 in the alternation of vascular functions.

Ageing is a risk factor for many degenerative diseases. Cardiovascular diseases (CVDs) are usually big burdens for elderly, caregivers and the health system. During the aging process, normal functions of vascular cells and tissue progressively lost and eventually develop vascular diseases. Endothelial dysfunction, reduced bioavailability of endothelium-derived nitric oxide are usual phenomena observed in patients with cardiovascular diseases. Myriad of studies have been done to investigate to delay the vascular dysfunction or improve the vascular function to prolong the aging process. Tumor suppressor gene p53, also a transcription factor, act as a gatekeeper to regulate a number of genes to maintain normal cell function including but not limited to cell proliferation, cell apoptosis. p53 also crosstalk with other key transcription factors like hypoxia-inducible factor 1 alpha that contribute to the progression of cardiovascular diseases. Therefore, in recent three decades, p53 has drawn scientists’ attention on its effects in vascular function. Though the role of tumor suppressor gene p53 is still not clear in vascular function, it is found to play regulatory roles and may involve in vascular remodeling, atherosclerosis or pulmonary hypertension. p53 may have a divergent role in endothelial and vascular muscle cells in those conditions. In this review, we describe the different effects of p53 in cardiovascular physiology. Further studies on the effects of endothelial cell-specific p53 deficiency on atherosclerotic plaque formation in common animal models are required before the therapeutic potential can be realized.