Loss Of Nitric Oxide Bioavailability Research Articles

Eicosapentaenoic Acid Improves Endothelial Nitric Oxide Bioavailability Via Changes in Protein Expression During Inflammation.

Endothelial cell (EC) dysfunction involves reduced nitric oxide (NO) bioavailability due to NO synthase uncoupling linked to increased oxidation and reduced cofactor availability. Loss of endothelial function and NO bioavailability are associated with inflammation, including leukocyte activation. Eicosapentaenoic acid (EPA) administered as icosapent ethyl reduced cardiovascular events in REDUCE-IT (Reduction of Cardiovascular Events With Icosapent Ethyl-Intervention Trial) in relation to on-treatment EPA blood levels. The mechanisms of cardiovascular protection for EPA remain incompletely elucidated but likely involve direct effects on the endothelium. In this study, human ECs were treated with EPA and challenged with the cytokine IL-6 (interleukin-6). Proinflammatory responses in the ECs were confirmed by ELISA capture of sICAM-1 (soluble intercellular adhesion molecule-1) and TNF-α (tumor necrosis factor-α). Global protein expression was determined using liquid chromatography-mass spectrometry tandem mass tag. Release kinetics of NO and peroxynitrite were monitored using porphyrinic nanosensors. IL-6 challenge induced proinflammatory responses from the ECs as evidenced by increased release of sICAM-1 and TNF-α, which correlated with a loss of NO bioavailability. ECs pretreated with EPA modulated expression of 327 proteins by >1-fold (P<0.05), compared with IL-6 alone. EPA augmented expression of proteins involved in NO production, including heme oxygenase-1 and dimethylarginine dimethylaminohydrolase-1, and 34 proteins annotated as associated with neutrophil degranulation. EPA reversed the endothelial NO synthase uncoupling induced by IL-6 as evidenced by an increased [NO]/[peroxynitrite] release ratio (P<0.05). These direct actions of EPA on EC functions during inflammation may contribute to its distinct cardiovascular benefits.

Increased oxidative stress alters coronary microvascular tone in exercising swine with multiple comorbidities

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Cardiovascular Alliance Background/Objectives We have previously shown, in female swine that the combination of three cardiovascular risk factors (diabetes mellitus (DM), hypercholestolemia (HC) and chronic kidney disease (CKD)) induces systemic inflammation, oxidative stress, coronary endothelial dysfunction and impaired myocardial perfusion due to impaired nitric oxide (NO) bioavailability. In the present study we hypothesized that the reduced bioavailability of nitric oxide (NO) is the result of increased production of reactive oxygen species (ROS) and ROS scavenging will restore myocardial oxygen balance thereby improving myocardial perfusion. Methods 12 female swine, with DM (streptozotocin 3x50mg/kg iv), CKD (renal embolization), and HC (high fat, high sugar, high salt diet) and 9 female healthy swine on normal pig chow (Normal) were included. The effect of ROS scavenging on the coronary microvascular tone control was studied in vivo, at rest and during graded treadmill exercise before and after infusion of the ROS scavengers (MPG+Tempol). Additionally, vasodilation to bradykinin was studied in the presence of MPG and TEMPOL and after subsequent addition to catalase, in isolated left ventricle small coronary arteries, in vitro using wire-myography. Results 6 months of DM+HC+CKD resulted in hyperglycemia (18.0±1.3 vs 8.4±0.9mmol/L), hypercholesterolemia (13.4±2.1 vs 1.7±0.1mmol/l), renal dysfunction (plasma creatinine: 166±9 vs 119±3µmol/l), and systemic inflammation (TNFα: 63±8 vs 41±4pg/ml, all P&amp;lt;0.05) associated with impaired endothelium-dependent vasodilator response to bradykinin in vitro. Furthermore, myocardial oxidative stress was increased in DM+HC+CKD animals (8-isoprostane 13±1 vs 10±1pg/ml) and total antioxidant capacity was reduced (0.67±0.02 vs 0.72±0.01nmol Trolox eq./mg protein, both P&amp;lt;0.05). Surprisingly, in vivo ROS scavenging resulted in an even further increase in myocardial oxygen extraction in DM+HC+CKD while it had no effect in Normal, indicating the involvement of a vasodilator ROS (most likely H2O2) in the control of coronary microvascular tone. This was supported by increased activity of catalase in the myocardium of DM+HC+CKD animals (44±3 vs 31±4nmol/min/ml, both P&amp;lt;0.05) and the vascular relaxation response to bradykinin when catalase was added on top of the ROS scavenging in vitro. Conclusion In swine exposed for 6 months to multiple comorbidities, impaired myocardial perfusion is mediated by a loss of NO bioavailability due to increased oxidative stress which was partially compensated by increased H2O2-mediated coronary vasodilation.

2-(4-Methylthiazol-5-yl) Ethyl Nitrate Hydrochloride Ameliorates Cognitive Impairment via Modulation of Oxidative Stress and Nuclear Factor Kappa B (NF-κB) Signaling Pathway in Chronic Cerebral Hypoperfusion-Associated Spontaneously Hypertensive Rats.

Hypertension reduces the bioavailability of vascular nitric oxide (NO) and contributes to the onset of vascular dementia (VaD). A loss of NO bioavailability increases inflammation and oxidative stress. 2-(4-Methylthiazol-5-yl) ethyl nitrate hydrochloride (W1302) is a novel nitric oxide donor (NOD) which is undergoing phase I clinical trials in China for the treatment of VaD. In this study, we investigated the protective effects of W1302 in VaD rats induced by the permanent occlusion of a bilateral common carotid arteries model related to spontaneous hypertension (SHR-2VO), and we further explored the underlying mechanisms. Nimodipine was used as a positive control. Our results showed that W1302 treatment for 4 weeks (10 mg/Kg/day) exhibited stronger improvement in the spatial learning and memory deficits in SHR-2VO rats compared with nimodipine with slightly lower systolic blood pressure (SBP). Meanwhile, W1302 treatment significantly increased NO and cGMP production, restored mitochondrial membrane potential and attenuated oxidative stress as evidenced by increasing ATP production and reducing malondialdehyde (MDA) levels in the brain. Furthermore, W1302 treatment markedly inhibited the iNOS activity and decreased TNF-α expression via inhibiting the nuclear factor kappa B (NF-κB) signaling pathway. Nimodipine treatment also restored these aberrant changes, but its ATP production was weaker than that of W1302, and there was no significant effect on NO release. Taken together, W1302 exhibited beneficial effects on complications in VaD with hypertension, which is involved in suppressing oxidative damage, and the inflammatory reaction might be mediated by an increase in NO release. Therefore, W1302 has therapeutic potential for the treatment of VaD caused by chronic cerebral hypoperfusion-associated spontaneous hypertension.

Loss of nitric oxide bioavailability promotes arterial stiffening in a tissue transglutaminase-dependent manner

Although arterial stiffening undeniably contributes to cardiovascular disease, the cellular and molecular mechanisms leading to increased arterial stiffness are not well understood. Increased arterial stiffness in disease states such as obesity and type 2 diabetes typically coexists with indices of endothelial dysfunction including diminished nitric oxide (NO) bioavailability. In fact, it is commonly assumed that endothelial dysfunction precedes, and causally contributes to, arterial stiffening; yet the molecular underpinnings for this causal relationship remain elusive. We and others previously showed that activation of tissue transglutaminase (TG2), a crosslinking enzyme that also works as a G-protein and cellular scaffold, promotes cytoskeletal actin polymerization and stiffening of vascular smooth muscle cells (VSMC), thus contributing to whole-artery stiffening. It is also reported that NO is an endogenous inhibitor of TG2. Accordingly, herein, we tested the hypothesis that increased exposure of VSMC to NO suppresses TG2 activity and actin polymerization. Conversely, we hypothesized that loss of NO bioavailability promotes arterial stiffening, and that this effect is mediated by TG2 activity and downstream activation of LIM kinase (LIMK), an enzyme that modulates fibrillar (F)-actin severing via cofilin phosphorylation. All differences reported herein are significant at P&lt;0.05. In support of our hypotheses, we show that treatment of cultured human aortic VSMC with the NO donor S-nitrosoglutathione (200μM for 2 hours) reduces TG2 activity (N-epsilon gamma glutamyl Lysine presence) and F-actin formation (phalloidin staining) induced by TG2 activation (dithiothreitol, 200μM). Furthermore, we show that exposure of mouse isolated mesenteric arteries to the NO synthase inhibitor L-NAME (300μM for 18 hours) causes an increase in arterial stiffness ( i.e., increased incremental modulus of elasticity) as determined using pressure myography and videomicroscopy. Of note, L-NAME-induced arterial stiffening is prevented when arteries are co-treated with a TG2 inhibitor (cystamine, 1mM) or LIMK inhibitor (LIMKi, 1μM). Taken together, these findings support the notion that NO is a key downregulator of TG2 activity and demonstrate that reduced NO bioavailability promotes arterial stiffening in a VSMC TG2-LIMK-F-actin dependent manner. As such, this work provides insight into plausible molecular mechanisms by which endothelial dysfunction leads to arterial stiffening. R01HL153264 (to LAML and JP), and MU Research Excellence Program (REP) postdoctoral fellowship (to FIRP). 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.

HIGH-INTENSITY STATINS COMBINED WITH EICOSAPENTAENOIC ACID (EPA) IMPROVES ENDOTHELIAL FUNCTION DURING EXPOSURE TO OXIDIZED LDL

BackgroundDuring atherosclerosis, endothelial cell (EC) dysfunction results in reduced nitric oxide (NO) bioavailability and increased cytotoxic peroxynitrite (ONOO−). This loss of NO bioavailability results in abnormal vasodilation and inflammatory changes. Eicosapentaenoic acid (EPA) administered as icosapent ethyl (IPE) reduced cardiovascular (CV) events in high-risk patients treated with statins (REDUCE-IT). We tested the effects of high-intensity statins and EPA in ECs exposed to oxidized LDL (oxLDL).Methods and ResultsHuman umbilical vein ECs (HUVECs) were pretreated with 20 mg/dL oxLDL for 20 min, then treated with atorvastatin (active metabolite, ATM) and rosuvastatin (rosuva) at 1.0 μM ± EPA (10μM) for 1 hr. Cells were stimulated with calcium and assayed for the NO/ONOO− release ratio using nanosensors. ECs exposed to oxLDL showed a 60% reduction in NO release compared with vehicle (386±29 to 156±18 nM, p< 0.001) concomitant with a pronounced increase in ONOO− release (205±31 to 283±16 nM, p< 0.001), resulting in a >70% decrease in the NO/ONOO− release ratio (p < 0.001). ECs treated with ATM had an improved NO/ONOO− release ratio (53%) that increased in combination with EPA by 216% (p < 0.01). Similar results were observed for EPA in combination with rosuvastatin. When either statin was combined with EPA, there was also decreased ONOO− release compared to statin alone (p < 0.01).ConclusionIn combination with high intensity statins, EPA enhanced NO bioavailability in dysfunctional human ECs. The ability of EPA to reverse vascular EC dysfunction may lead to reduced ischemic events in statin-treated patients, as evidenced in outcome trials. BackgroundDuring atherosclerosis, endothelial cell (EC) dysfunction results in reduced nitric oxide (NO) bioavailability and increased cytotoxic peroxynitrite (ONOO−). This loss of NO bioavailability results in abnormal vasodilation and inflammatory changes. Eicosapentaenoic acid (EPA) administered as icosapent ethyl (IPE) reduced cardiovascular (CV) events in high-risk patients treated with statins (REDUCE-IT). We tested the effects of high-intensity statins and EPA in ECs exposed to oxidized LDL (oxLDL). During atherosclerosis, endothelial cell (EC) dysfunction results in reduced nitric oxide (NO) bioavailability and increased cytotoxic peroxynitrite (ONOO−). This loss of NO bioavailability results in abnormal vasodilation and inflammatory changes. Eicosapentaenoic acid (EPA) administered as icosapent ethyl (IPE) reduced cardiovascular (CV) events in high-risk patients treated with statins (REDUCE-IT). We tested the effects of high-intensity statins and EPA in ECs exposed to oxidized LDL (oxLDL). Methods and ResultsHuman umbilical vein ECs (HUVECs) were pretreated with 20 mg/dL oxLDL for 20 min, then treated with atorvastatin (active metabolite, ATM) and rosuvastatin (rosuva) at 1.0 μM ± EPA (10μM) for 1 hr. Cells were stimulated with calcium and assayed for the NO/ONOO− release ratio using nanosensors. ECs exposed to oxLDL showed a 60% reduction in NO release compared with vehicle (386±29 to 156±18 nM, p< 0.001) concomitant with a pronounced increase in ONOO− release (205±31 to 283±16 nM, p< 0.001), resulting in a >70% decrease in the NO/ONOO− release ratio (p < 0.001). ECs treated with ATM had an improved NO/ONOO− release ratio (53%) that increased in combination with EPA by 216% (p < 0.01). Similar results were observed for EPA in combination with rosuvastatin. When either statin was combined with EPA, there was also decreased ONOO− release compared to statin alone (p < 0.01). Human umbilical vein ECs (HUVECs) were pretreated with 20 mg/dL oxLDL for 20 min, then treated with atorvastatin (active metabolite, ATM) and rosuvastatin (rosuva) at 1.0 μM ± EPA (10μM) for 1 hr. Cells were stimulated with calcium and assayed for the NO/ONOO− release ratio using nanosensors. ECs exposed to oxLDL showed a 60% reduction in NO release compared with vehicle (386±29 to 156±18 nM, p< 0.001) concomitant with a pronounced increase in ONOO− release (205±31 to 283±16 nM, p< 0.001), resulting in a >70% decrease in the NO/ONOO− release ratio (p < 0.001). ECs treated with ATM had an improved NO/ONOO− release ratio (53%) that increased in combination with EPA by 216% (p < 0.01). Similar results were observed for EPA in combination with rosuvastatin. When either statin was combined with EPA, there was also decreased ONOO− release compared to statin alone (p < 0.01). ConclusionIn combination with high intensity statins, EPA enhanced NO bioavailability in dysfunctional human ECs. The ability of EPA to reverse vascular EC dysfunction may lead to reduced ischemic events in statin-treated patients, as evidenced in outcome trials. In combination with high intensity statins, EPA enhanced NO bioavailability in dysfunctional human ECs. The ability of EPA to reverse vascular EC dysfunction may lead to reduced ischemic events in statin-treated patients, as evidenced in outcome trials.

Role of cytoglobin in cigarette smoke constituent-induced loss of nitric oxide bioavailability in vascular smooth muscle cells

Cytoglobin (Cygb) has been identified as the major nitric oxide (NO) metabolizing protein in vascular smooth muscle cells (VSMCs) and is crucial for the regulation of vascular tone. In the presence of its requisite cytochrome B5a (B5)/B5 reductase-isoform-3 (B5R) reducing system, Cygb controls NO metabolism through the oxygen-dependent process of NO dioxygenation. Tobacco cigarette smoking (TCS) induces vascular dysfunction; however, the role of Cygb in the pathophysiology of TCS-induced cardiovascular disease has not been previously investigated. While TCS impairs NO biosynthesis, its effect on NO metabolism remains unclear. Therefore, we performed studies in aortic VSMCs with tobacco smoke extract (TSE) exposure to investigate the effects of cigarette smoke constituents on the rates of NO decay, with focus on the alterations that occur in the process of Cygb-mediated NO metabolism. TSE greatly enhanced the rates of NO metabolism by VSMCs. An initial increase in superoxide-mediated NO degradation was seen at 4 h of exposure. This was followed by much larger progressive increases at 24 and 48 h, accompanied by parallel increases in the expression of Cygb and B5/B5R. siRNA-mediated Cygb knockdown greatly decreased these TSE-induced elevations in NO decay rates. Therefore, upregulation of the levels of Cygb and its reducing system accounted for the large increase in NO metabolism rate seen after 24 h of TSE exposure. Thus, increased Cygb-mediated NO degradation would contribute to TCS-induced vascular dysfunction and partial inhibition of Cygb expression or its NO dioxygenase function could be a promising therapeutic target to prevent secondary cardiovascular disease.

Reduced nitric oxide bioavailability impairs myocardial oxygen balance during exercise in swine with multiple risk factors

In the present study, we tested the hypothesis that multiple risk factors, including diabetes mellitus (DM), dyslipidaemia and chronic kidney disease (CKD) result in a loss of nitric oxide (NO) signalling, thereby contributing to coronary microvascular dysfunction. Risk factors were induced in 12 female swine by intravenous streptozotocin injections (DM), a high fat diet (HFD) and renal artery embolization (CKD). Female healthy swine (n = 13) on normal diet served as controls (Normal). After 5 months, swine were chronically instrumented and studied at rest and during exercise. DM + HFD + CKD swine demonstrated significant hyperglycaemia, dyslipidaemia and impaired kidney function compared to Normal swine. These risk factors were accompanied by coronary microvascular endothelial dysfunction both in vivo and in isolated small arteries, due to a reduced NO bioavailability, associated with perturbations in myocardial oxygen balance at rest and during exercise. NO synthase inhibition caused coronary microvascular constriction in exercising Normal swine, but had no effect in DM + HFD + CKD animals, while inhibition of phosphodiesterase 5 produced similar vasodilator responses in both groups, indicating that loss of NO bioavailability was principally responsible for the observed coronary microvascular dysfunction. This was associated with an increase in myocardial 8-isoprostane levels and a decrease in antioxidant capacity, while antioxidants restored the vasodilation to bradykinin in isolated coronary small arteries, suggesting that oxidative stress was principally responsible for the reduced NO bioavailability. In conclusion, five months of combined exposure to DM + HFD + CKD produces coronary endothelial dysfunction due to impaired NO bioavailability, resulting in impaired myocardial perfusion at rest and during exercise.

Phenotypic characterization of circulating endothelial cells induced by inflammation and oxidative stress in ankylosing spondylitis

Ankylosing spondylitis (AS) is a chronic auto-immune disease, affecting the spine, sacroiliac, and sometimes peripheral joints. It is also involved with cardio-vascular risk factors due to accelerated atherosclerosis. Oxidative burst, systemic inflammation coupled with endothelial dysfunction (ED), resulting in reduced bioavailability of the vasodilator nitric oxide (NO) and an increased number of circulating endothelial cells (CECs) may correlate with disease activity and its sustenance. Hence, the study was aimed to detect and quantify CECs and assess the oxidative stress and inflammatory status in AS patients vis-à-vis healthy controls, as well as relate these parameters with AS disease activity and atherosclerotic markers in patients. Our study showed an increased frequency of endothelial cells in peripheral blood of AS patients in pro-inflammatory conditions. In AS patient population, they showed significant reduction of flow-mediated dilatation (%FMD) (p < 0.05), and increased soluble adhesion molecules such as sICAM-1 (p < 0.01) and sVCAM-1 (p < 0.05) compared to healthy controls. A marked increase in pro-inflammatory markers such as TNF-α (p < 0.01) and IL-1β (p < 0.001) and reactive free radicals (p < 0.05) along with reduced serum nitrite in AS, provided a strong pro-inflammatory milieu which positively correlated with Bath ankylosing spondylitis disease activity and functional indices (BASDAI and BASFI). The observed significant upregulation in CECs (CD45-/CD31+/CD105+/CD144+) in patients compared to healthy controls positively correlated with disease activity and duration as well as with markers of oxidative stress. Thus, chronic inflammation and oxidative burst induce loss of NO bioavailability, leading to ED. This may cause the derangement of CECs that may be considered as a prognostic biomarker for ED.

To show the effect of intermittent fasting during ramadan on endothelial dysfunction via TIMI frame count.

Fasting and coronary functions are prestige fields for the study. There are a limited number of studies on these topics. The effect of Ramadan fasting on endothelial dysfunction, which can be manifested by loss of nitric oxide bioavailability, has been demonstrated via flow-mediated vasomotion in patients with the slow coronary flow in a small number of studies. To our knowledge, there is no study showing the relationship between TIMI frame count and Ramadan fasting. We aimed to prove that Ramadan fasting can improve endothelial dysfunction which can be documented via the TIMI frame count method in angiography. This retrospective study included 67 patients diagnosed with the coronary slow flow by coronary angiographic before Ramadan. All of them were evaluated again via TIMI frame count within a period of 1 to 3 months after Ramadan. We tested our hypothesis that fasting may improve endothelial dysfunction and it was proved by the TIMI frame count method in our study. TIMI frame counts measured angiographically from LAD, Cx, and RCA and they were significantly lower than the counts before fasting. All coronary frame count parameters showed significant improvement after Ramadan compared with the baseline values before the Ramadan fasting period (P < 0.001). Our results revealed that fasting and lifestyle changes during Ramadan may be beneficial for the improvement of endothelial dysfunctions in patients with the slow coronary flow and this can be showed easily using TIMI frame count. This is a practical and easy method for showing coronary functions.

BRG1 Activates PR65A Transcription to Regulate NO Bioavailability in Vascular Endothelial Cells.

Vascular endothelial cells contribute to the pathogenesis of cardiovascular diseases by producing and disseminating angiocrine factors. Nitric oxide (NO), catalyzed by endothelial NO synthase (eNOS), is one of the prototypical angiocrine factors. eNOS activity is modulated by site-specific phosphorylation. We have previously shown that endothelial-specific knockdown of BRG1 in Apoe–/– mice attenuates the development of atherosclerosis, in which eNOS-dependent NO catalysis plays an antagonizing role. Here we report that attenuation of atherogenesis in mice by BRG1 knockdown was accompanied by partial restoration of NO biosynthesis by 44% in the arteries and a simultaneous up-regulation of eNOS serine 1177 phosphorylation by 59%. Indeed, BRG1 depletion or inhibition ameliorated oxLDL-induced loss of NO bioavailability and eNOS phosphorylation in cultured endothelial cells. Further analysis revealed that BRG1 regulated eNOS phosphorylation and NO synthesis by activating the transcription of protein phosphatase 2A (PP2A) structural subunit a (encoded by PR65A). BRG1 interacted with ETS1, was recruited by ETS1 to the PR65A promoter, and cooperated with ETS1 to activate PR65A transcription. Finally, depletion of ETS1, similar to BRG1, repressed PR65A induction, normalized eNOS phosphorylation, and rescued NO biosynthesis in endothelial cells treated with oxLDL. In conclusion, our data characterize a novel transcriptional cascade that regulates NO bioavailability in vascular endothelial cells.

Mechanisms of Medicinal Plant Activity on Nitric Oxide (NO) Bioavailability as Prospective Treatments for Atherosclerosis.

Atherosclerosis is one of the leading causes of human morbidity globally and reduced bioavailability of vascular nitric oxide (NO) has a critical role in the progression and development of the atherosclerotic disease. Loss of NO bioavailability, for example via a deficiency of the substrate (L-arginine) or cofactors for endothelial nitric oxide synthase (eNOS), invariably leads to detrimental vascular effects such as impaired endothelial function and increased smooth muscle cell proliferation, deficiency of the substrate (Larginine) or cofactors for eNOS. Various medicinal plants and their bioactive compounds or secondary metabolites with fewer side effects are potentially implicated in preventing cardiovascular disease by increasing NO bioavailability, thereby ameliorating endothelial dysfunction. In this review, we describe the most notable medicinal plants and their bioactive compounds that may be appropriate for enhancing NO bioavailability, and treatment of atherosclerosis. The material in this article was obtained from noteworthy scientific databases, including Web of Science, PubMed, Science Direct, Scopus and Google Scholar. Medicinal plants and their bioactive compounds influence NO production through diverse mechanisms including the activation of the nuclear factor kappa B (NF-κB) signaling pathway, activating protein kinase C (PKC)-α, stimulating protein tyrosine kinase (PTK), reducing the conversion of nitrite to NO via nitrate-nitrite reduction pathways, induction of eNOS, activating the phosphatidylinositol 3-kinase (PI3K)/serine threonine protein kinase B (AKT) (PI3K/AKT/eNOS/NO) pathway and decreasing oxidative stress. Medicinal plants and/or their constituent bioactive compounds may be considered as safe therapeutic options for enhancing NO bioavailability and prospective preventative therapy for atherosclerosis.

Altered post-capillary and collecting venular reactivity in skeletal muscle with metabolic syndrome.

With the development of the metabolic syndrome, both post-capillary and collecting venular dilator reactivity within the skeletal muscle of obese Zucker rats (OZR) is impaired. The impaired dilator reactivity in OZR reflects a loss in venular nitric oxide and PGI2 bioavailability, associated with the chronic elevation in oxidant stress. Additionally, with the impaired dilator responses, a modest increase in adrenergic constriction combined with an elevated thromboxane A2 production may contribute to impaired functional dilator and hyperaemic responses at the venular level. For the shift in skeletal muscle venular function with development of the metabolic syndrome, issues such as aggregate microvascular perfusion resistance, mass transport and exchange within with capillary networks, and fluid handling across the microcirculation are compelling avenues for future investigation. While research into vascular outcomes of the metabolic syndrome has focused on arterial/arteriolar and capillary levels, investigation into venular function and how this impacts responses has received little attention. Using the in situ cremaster muscle of obese Zucker rats (OZR; with lean Zucker rats (LZR) as controls), we determined indices of venular function. At ∼17weeks of age, skeletal muscle post-capillary venular density was reduced by ∼20% in LZR vs. OZR, although there was no evidence of remodelling of the venular wall. Venular tone at ∼25μm (post-capillary) and ∼75μm (collecting) diameter was elevated in OZR vs. LZR. Venular dilatation to acetylcholine was blunted in OZR vs. LZR due to increased oxidant stress-based loss of nitric oxide bioavailability (post-capillary) and increased α1 - (and α2 -) mediated constrictor tone (collecting). Venular constrictor responses in OZR were comparable to LZR for most stimuli, although constriction to α1 -adrenoreceptor stimulation was elevated. In response to field stimulation of the cremaster muscle (0.5, 1, 3Hz), venular dilator and hyperaemic responses to lower frequencies were blunted in OZR, but responses at 3Hz were similar between strains. Venous production of TxA2 was higher in OZR than LZR and significantly higher than PGI2 production in either following arachidonic acid challenge. These results suggest that multi-faceted alterations to skeletal muscle venular function in OZR may contribute to alterations in upstream capillary pressure profiles and the transcapillary exchange of solutes and water under conditions of metabolic syndrome.

Metoprolol Improves Endothelial Function in Patients with Cardiac Syndrome X.

Endothelial dysfunction which is manifested by the loss of nitric oxide bioavailability, is an increasingly recognized cause of cardiac syndrome X (CSX) and beta blockers are used for the treatment of this syndrome. Thus, the aim of this study was to investigate effects of metoprolol, as a beta blocker, on endothelial function in CSX patients. The study included 25 CSX patients (20 female/ 5 male, mean age: 55.36±10.31 years) who received metoprolol (50 mg BID) for one month. In addition, 25 healthy controls (20 female/ 5 male, mean age: 54.32 ±9.27 years) were enrolled. Levels of endothelin-1, E-selectin, and vascular cell adhesion molecule-1 (VCAM-1) in controls and CSX patients were measured, both at the baseline and after the treatment, by the enzyme-linked immunosorbent assay. In CSX patients, at the baseline, levels of E-selectin and VCAM-1 were significantly higher than those of the controls. In addition, levels of these biomarkers in CSX patients after the treatment significantly decreased compared to the baseline. In spite of similar tendency, these differences were not significant for endothelin-1. In conclusion, metoprolol therapy improves endothelial function. Thus, it may be a suggested choice for CSX treatment. However, further studies are needed to confirm the clinical significance of metoprolol therapy for CSX patients.

Cerebral Cortical Microvascular Rarefaction in Metabolic Syndrome is Dependent on Insulin Resistance and Loss of Nitric Oxide Bioavailability.

Chronic presentation of the MS is associated with an increased likelihood for stroke and poor stroke outcomes following occlusive cerebrovascular events. However, the physiological mechanisms contributing to compromised outcomes remain unclear, and the degree of cerebral cortical MVD may represent a central determinant of stroke outcomes. This study used the OZR model of MS and clinically relevant, chronic interventions to determine the impact on cerebral cortical microvascular rarefaction via immunohistochemistry with a parallel determination of cerebrovascular function to identify putative mechanistic contributors. OZR exhibited a progressive rarefaction (to ~80% control MVD) of the cortical microvascular networks vs. lean Zucker rats. Chronic treatment with antihypertensive agents (captopril/hydralazine) had limited effectiveness in blunting rarefaction, although treatments improving glycemic control (metformin/rosiglitazone) were superior, maintaining ~94% control MVD. Chronic treatment with the antioxidant TEMPOL severely blunted rarefaction in OZR, although this ameliorative effect was prevented by concurrent NOS inhibition. Further analyses revealed that the maintenance of glycemic control and vascular NO bioavailability were stronger predictors of cerebral cortical MVD in OZR than was prevention of hypertension, and this may have implications for chronic treatment of CVD risk under stroke-prone conditions.

Acute and Chronic Inhibition of NOS Causes a Switch in Vasodilator Mechanism from Nitric Oxide to Hydrogen Peroxide in the Human Microcirculation

This study tests the hypothesis that inhibition of NO-synthase (NOS) increases mitochondria-derived (mt) reactive oxygen species (ROS), resulting in changes in the mediator of flow-induced dilation (FID). In healthy individuals nitric oxide (NO) is the primary vasodilator responsible for FID, while in microvessels (MV) of patients with coronary artery disease (CAD) mtROS are responsible. NO inhibits mtROS and reduces smooth muscle proliferation to contribute to prevention of atherosclerosis. Hydrogen peroxide (H2O2)promotes cell proliferation and is pro-atherosclerotic. MV (~200µm) obtained from surgical discarded adipose were dissected and cannulated on glass pipettes in a heated organ chamber, perfused with Krebs and aerated with 74% N2, 21% O2, 5% CO2. After equilibration, MV were constricted with endothelin-1 and responses to graded increases in flow were recorded. Papaverine (100µM) was used to assess endothelial independent relaxation. Neither 1h nor 16h incubation with L-NAME (100µM before and during experiment) affected the FID compared to Vehicle (V) (% Max dilation: V 76.1 ±5.1; L-NAME: 1h 84.3 ±3; 16h: 87.8±2.7 P< 0.05 ANOVA). In V treated MV FID was L-NAME inhibitable (10.1±8.3) but not effected by PEG-cat while with L-NAME pretreatment PEG-cat inhibited dilation (L-NAME: 1h +Peg-Cat 18.4±12.9 16h + Peg-Cat 12.6 ±1.6). Our results suggest that inhibition of NOS in MV from healthy individuals causes a primary shift in the mediator of dilation from NO to H2O2 , similar to subjects with CAD. Our data are consistent with the concept that that threshold levels of NO are necessary to suppress mtROS in human MV. Loss of NO bioavailability causes a compensatory increase in mtROS, which mediates FID.

Distinct temporal phases of microvascular rarefaction in skeletal muscle of obese Zucker rats.

Evolution of metabolic syndrome is associated with a progressive reduction in skeletal muscle microvessel density, known as rarefaction. Although contributing to impairments to mass transport and exchange, the temporal development of rarefaction and the contributing mechanisms that lead to microvessel loss are both unclear and critical areas for investigation. Although previous work suggests that rarefaction severity in obese Zucker rats (OZR) is predicted by the chronic loss of vascular nitric oxide (NO) bioavailability, we have determined that this hides a biphasic development of rarefaction, with both early and late components. Although the total extent of rarefaction was well predicted by the loss in NO bioavailability, the early pulse of rarefaction developed before a loss of NO bioavailability and was associated with altered venular function (increased leukocyte adhesion/rolling), and early elevation in oxidant stress, TNF-α levels, and the vascular production of thromboxane A2 (TxA2). Chronic inhibition of TNF-α blunted the severity of rarefaction and also reduced vascular oxidant stress and TxA2 production. Chronic blockade of the actions of TxA2 also blunted rarefaction, but did not impact oxidant stress or inflammation, suggesting that TxA2 is a downstream outcome of elevated reactive oxygen species and inflammation. If chronic blockade of TxA2 is terminated, microvascular rarefaction in OZR skeletal muscle resumes, but at a reduced rate despite low NO bioavailability. These results suggest that therapeutic interventions against inflammation and TxA2 under conditions where metabolic syndrome severity is moderate or mild may prevent the development of a condition of accelerated microvessel loss with metabolic syndrome.

Protective effect of sex on chronic stress- and depressive behavior-induced vascular dysfunction in BALB/cJ mice.

The presence of chronic, unresolvable stresses leads to negative health outcomes, including development of clinical depression/depressive disorders, with outcome severity being correlated with depressive symptom severity. One of the major outcomes associated with chronic stress and depression is the development of cardiovascular disease (CVD) and an elevated CVD risk profile. However, in epidemiological research, sex disparities are evident, with premenopausal women suffering from depressive symptoms more acutely than men, but also demonstrating a relative protection from the onset of CVD. Given this, we investigated the differential effect of sex on conduit artery and resistance arteriolar function in male and female mice following 8 wk of an unpredictable chronic mild stress (UCMS) protocol. In males, plasma cortisol and depressive symptom severity (e.g., coat status, anhedonia, delayed grooming) were elevated by UCMS. Endothelium-dependent dilation to methacholine/acetylcholine was impaired in conduit arteries and skeletal muscle arterioles, suggesting a severe loss of nitric oxide bioavailability and increased production of thromboxane A2 vs. prostaglandin I2 associated with elevated reactive oxygen species (ROS) and an increased level of systemic inflammation. Endothelium-independent dilation was intact. In females, depressive symptoms and plasma cortisol increases were more severe than in males, although alterations to vascular reactivity were blunted, including the effects of elevated ROS and inflammation on dilator responses. These results suggest that compared with males, female rats are more susceptible to chronic stress in terms of the severity of depressive behaviors, but that the subsequent development of vasculopathy is blunted owing to an improved ability to tolerate elevated ROS and systemic inflammatory stress.

The effects of Ramadan fasting on endothelial function in patients with cardiovascular diseases.

Endothelial dysfunction, which can be manifested by loss of nitric oxide bioavailability, is an increasingly recognized cause of cardiovascular diseases. Previous studies showed that diets affectendothelial function and modifycardiovascular risks. This study aimed to assess the effects of Ramadan fasting, as a diet intervention, on endothelial function. The study population consisted of 21 male patients (mean age: 52±9 years) with cardiovascular risks (coronary artery disease, cerebrovascular or peripheral arterial diseases). The biochemical variables in serum of patients were measured 2 days before and after Ramadan fasting. The levels of asymmetric dimethylarginine (ADMA) and vascular endothelial growth factor (VEGF) were evaluated using the enzyme-linked immunosorbent assay. Nitric oxide (NO) and Malondialdehyde (MDA) levels were measured by the Griess and thiobarbituric acid reaction substances assay, respectively. NO levels in patients after Ramadan fasting were significantly higher compared with thebaseline value (85.1±11.54 vs 75.8±10.7 μmol/l) (P<0.05). Post-Ramadan levels of ADMA decreased significantly in comparison with pre-Ramadan levels (802.6±60.9 vs 837.6±51.0 nmol/l) (P<0.05). In addition, the levels of VEGF and MDA changed during Ramadan fasting, but these changes were not statistically significant (228.1±27.1 vs 222.7±22.9 pg/ml and 3.2±0.7 vs 3.6±1.1 μmol/l, respectively). Ramadan fasting may have beneficial effects on endothelial function and can modulate cardiovascular risks. Further studies are needed to confirm the clinical significance of Ramadan fasting on cardiovascular health.

Functionally Defective High-Density Lipoprotein and Paraoxonase: A Couple for Endothelial Dysfunction in Atherosclerosis

The endothelium is the primary target for biochemical or mechanical injuries caused by the putative risk factors of atherosclerosis. Endothelial dysfunction represents the ultimate link between atherosclerotic risk factors that promote atherosclerosis. HDL-C is thought to exert at least some parts of its antiatherogenic facilities via stimulating endothelial NO production, nearby inhibiting oxidative stress and inflammation. HDL-C is capable of opposing LDL's inductive effects and avoiding the ox-LDL's inhibition of eNOS. Paraoxonase 1 (PON1) is an HDL-associated enzyme esterase which appears to contribute to the antioxidant and antiatherosclerotic capabilities of HDL-C. “Healthy HDL,” namely the particle that contains the active Paraoxonase 1, has the power to suppress the formation of oxidized lipids. “Dysfunctional HDL,” on the contrary, has reduced Paraoxonase 1 enzyme activity and not only fails in its mission but also potentially leads to greater formation of oxidized lipids/lipoproteins to cause endothelial dysfunction. The association of HDL-C PON1 and endothelial dysfunction depends largely on the molecules with exact damaging effect on NO synthase coupling. Loss of nitric oxide bioavailability has a pivotal role in endothelial dysfunction preceding the appearance of atherosclerosis. Analyses of HDL-C and Paraoxonase1 would be more important in the diagnosis and treatment of atherosclerosis in the very near future.

Endothelial function assessment in atherosclerosis. Comparison of brachial artery flow‑mediated vasodilation and peripheral arterial tonometry

Endothelial dysfunction, characterized by the loss of nitric oxide bioavailability, is a key element in the pathogenesis of atherosclerosis and an important prognostic factor in cardiovascular diseases. Therefore, the development of reliable, safe, and noninvasive methods of endothelial function assessment is important for their use in cardiovascular risk stratification. Brachial artery flow‑mediated dilation (FMD) is widely used in research but technical difficulties and problems with calibration between laboratories limit its clinical use. Reactive hyperemia-peripheral artery tonometry (RH‑PAT, EndoPAT) has been developed as a simpler, cheaper, and potentially more reproducible method. We aimed to investigate associations between RH‑PAT and FMD in relation to atherosclerotic risk factor profile. The study involved 80 subjects (52 men, 28 women) aged 43.6 ±14.8 years, with moderate‑to‑low cardiovascular risk (mean SCORE, 2.2% ±2%), in whom FMD, RH‑PAT, and intima-media thickness (IMT) were determined. The reactive hyperemia index (RHI) measured by RH‑PAT correlated with FMD (r = 0.35, P <0.01). However, no significant correlation was observed between RHI and IMT, SCORE, or the number of classical atherosclerotic risk factors (hypertension, smoking, diabetes, hypercholesterolemia), while FMD was significantly correlated with IMT (r = -0.53, P <0.001), risk factors (r = -0.55, P <0.05), and SCORE (r = -0.4, P <0.05). Despite its technical requirements, FMD is a more sensitive method than RH‑PAT in evaluating the effect of classical atherosclerotic risk factors on vascular endothelial function. Microvasculature response during RH‑PAT needs to be further studied, including the assessment of nonendothelial factors that may affect the measurements, before RH‑PAT becomes the universal tool for the evaluation of the endothelial cells.