NO-dependent Vasodilation Research Articles

Collateral Arteriogenesis Involves a Sympathetic Denervation That Is Associated With Abnormal α-Adrenergic Signaling and a Transient Loss of Vascular Tone.

Stimulating collateral arteriogenesis is an attractive therapeutic target for peripheral artery disease (PAD). However, the potency of arteriogenesis-stimulation in animal models has not been matched with efficacy in clinical trials. This may be because the presence of enlarged collaterals is not sufficient to relieve symptoms of PAD, suggesting that collateral function is also important. Specifically, collaterals are the primary site of vascular resistance following arterial occlusion, and impaired collateral vasodilation could impact downstream tissue perfusion and limb function. Therefore, we evaluated the effects of arteriogenesis on collateral vascular reactivity. Following femoral artery ligation in the mouse hindlimb, collateral functional vasodilation was impaired at day 7 (17 ± 3 vs. 60 ± 8%) but restored by day 28. This impairment was due to a high resting diameter (73 ± 4 μm at rest vs. 84 ± 3 μm dilated), which does not appear to be a beneficial effect of arteriogenesis because increasing tissue metabolic demand through voluntary exercise decreased resting diameter and restored vascular reactivity at day 7. The high diameter in sedentary animals was not due to sustained NO-dependent vasodilation or defective myogenic constriction, as there were no differences between the enlarged and native collaterals in response to eNOS inhibition with L-NAME or L-type calcium channel inhibition with nifedipine, respectively. Surprisingly, in the context of reduced vascular tone, vasoconstriction in response to the α-adrenergic agonist norepinephrine was enhanced in the enlarged collateral (−62 ± 2 vs. −37 ± 2%) while vasodilation in response to the α-adrenergic antagonist prazosin was reduced (6 ± 4% vs. 22 ± 16%), indicating a lack of α-adrenergic receptor activation by endogenous norepinephrine and suggesting a denervation of the neuroeffector junction. Staining for tyrosine hydroxylase demonstrated sympathetic denervation, with neurons occupying less area and located further from the enlarged collateral at day 7. Inversely, MMP2 presence surrounding the enlarged collateral was greater at day 7, suggesting that denervation may be related to extracellular matrix degradation during arteriogenesis. Further investigation on vascular wall maturation and the functionality of enlarged collaterals holds promise for identifying novel therapeutic targets to enhance arteriogenesis in patients with PAD.

Women with a history of preeclampsia have preserved sensory nerve-mediated dilatation in the cutaneous microvasculature.

What is the central question of this study? Are sensory nerve-mediated vasodilatation and the NO-dependent contribution to that response attenuated in the cutaneous microvasculature of women who have had preeclampsia? What is the main finding and its importance? Women who have had preeclampsia demonstrate attenuated microvascular endothelium-dependent dilatation compared to women with a history of uncomplicated pregnancy. However, there are no differences in sensory nerve-mediated vasodilatation between groups. This suggests that the neurogenic response is not altered following preeclampsia, and that the NO-dependent vasodilatation of the neurogenic response is not related to endothelium-dependent NO-mediated dilatation in these women. Women who have had preeclampsia (PE) demonstrate microvascular endothelial dysfunction, mediated in part by reduced nitric oxide (NO)-dependent mechanisms. Localized heating of the skin induces a biphasic vasodilatation response: a sensory nerve-mediated initial peak, followed by a sustained endothelium-dependent plateau. We have previously shown that the endothelium-dependent plateau is attenuated in PE. However, it is unknown if the sensory nerve-mediated initial peak is similarly attenuated. Therefore, the purpose of this study was to examine the effect of PE history on sensory nerve-mediated vasodilatation and the NO-dependent contribution to that response. We hypothesized that PE would have an attenuated initial peak and a reduced NO-dependent contribution to that response compared to women with a history of normotensive pregnancy (healthy controls, HC). Nine HC (31±4years) and nine PE (28±6years) underwent a standard local heating protocol (42°C; 0.1°Cs-1 ). Two intradermal microdialysis fibres were placed in the skin of the ventral forearm for the continuous local delivery of lactated Ringer solution alone (control) or 15-mM NG -nitro-l-arginine methyl ester for nitric oxide synthase (NOS) inhibition. Red blood cell flux was measured at each site by laser Doppler flowmetry (LDF). Cutaneous vascular conductance was calculated (CVC=LDF/mean arterial pressure) and normalized to maximum (%CVCmax ; 28-mM SNP + local heat 43°C). There were no differences in the initial peak between groups (HC: 79±8 vs. PE: 80±10%CVCmax ; P=0.936). NOS inhibition attenuated the initial peak in both HC (57±18% CVCmax ; P=0.003) and PE (54±10%CVCmax ; P=0.002). However, there were no differences in the NO-dependent portion of the initial peak (HC: 23±16 vs. PE: 24±9%; P=0.777). The local heating plateau (HC: 99±4 vs. PE: 88±7%CVCmax ; P=0.001) and NO contribution to the plateau (HC: 31±9 vs. PE: 17±14%; P=0.02) were attenuated in PE. There was no relation between NO-dependent dilatation in the initial peak and NO-dependent dilatation in the plateau across groups (R2 =0.005; P=0.943). Women who have had PE demonstrate attenuated microvascular endothelium-dependent dilatation. However, there are no differences in sensory nerve-mediated vasodilatation following PE, suggesting that the NO-dependent vasodilatation of the neurogenic response is not related to endothelium-dependent NO-mediated dilatation in these women.

Maladaptive Pulmonary Vascular Responses to Chronic Sustained and Chronic Intermittent Hypoxia in Rat.

Chronic sustained hypoxia (CSH), as found in individuals living at a high altitude or in patients suffering respiratory disorders, initiates physiological adaptations such as carotid body stimulation to maintain oxygen levels, but has deleterious effects such as pulmonary hypertension (PH). Obstructive sleep apnea (OSA), a respiratory disorder of increasing prevalence, is characterized by a situation of chronic intermittent hypoxia (CIH). OSA is associated with the development of systemic hypertension and cardiovascular pathologies, due to carotid body and sympathetic overactivation. There is growing evidence that CIH can also compromise the pulmonary circulation, causing pulmonary hypertension in OSA patients and animal models. The aim of this work was to compare hemodynamics, vascular contractility, and L-arginine-NO metabolism in two models of PH in rats, associated with CSH and CIH exposure. We demonstrate that whereas CSH and CIH cause several common effects such as an increased hematocrit, weight loss, and an increase in pulmonary artery pressure (PAP), compared to CIH, CSH seems to have more of an effect on the pulmonary circulation, whereas the effects of CIH are apparently more targeted on the systemic circulation. The results suggest that the endothelial dysfunction evident in pulmonary arteries with both hypoxia protocols are not due to an increase in methylated arginines in these arteries, although an increase in plasma SDMA could contribute to the apparent loss of basal NO-dependent vasodilation and, therefore, the increase in PAP that results from CIH.

Comodulation of NO-Dependent Vasodilation by Erythroid Band 3 and Hemoglobin: A GP.Mur Athlete Study.

GP.Mur, a red blood cell (RBC) hybrid protein encoded by glycophorin B-A-B, increases expression of erythroid band 3 (Anion Exchanger-1, SLC4A1). GP.Mur is extremely rare but has a prevalence of 1–10% in regions of Southeast Asia. We unexpectedly found slightly higher blood pressure (BP) among healthy Taiwanese adults with GP.Mur. Since band 3 has been suggested to interact with hemoglobin (Hb) to modulate nitric oxide (NO)-dependent hypoxic vasodilation during the respiratory cycle, we hypothesized that GP.Mur red cells could exert differentiable effects on vascular tone. Here we recruited GP.Mur-positive and GP.Mur-negative elite male college athletes, as well as age-matched, GP.Mur-negative non-athletes, for NO-dependent flow-mediated dilation (FMD) and NO-independent dilation (NID). The subjects were also tested for plasma nitrite and nitrate before and after arterial occlusion in FMD. GP.Mur+ and non-GP.Mur athletes exhibited similar heart rates and blood pressure, but GP.Mur+ athletes showed significantly lower FMD (4.8 ± 2.4%) than non-GP.Mur athletes (6.5 ± 2.1%). NO-independent vasodilation was not affected by GP.Mur. As Hb controls intravascular NO bioavailability, we examined the effect of Hb on limiting FMD and found it to be significantly stronger in GP.Mur+ subjects. Biochemically, plasma nitrite levels were directly proportional to individual band 3 expression on the red cell membrane. The increase of plasma nitrite triggered by arterial occlusion also showed small dependency on band 3 levels in non-GP.Mur subjects. By the GP.Mur comparative study, we unveiled comodulation of NO-dependent vasodilation by band 3 and Hb, and verified the long-pending role of erythroid band 3 in this process.

The associations of serum homoarginine to long-term prognosis in patients with chronic coronary syndrome and non-obstructive coronary artery disease

Abstract Background Although generally at lower risk than those with obstructive CAD, patients with non-obstructive CAD (NOCAD) are heterogeneous concerning long-term prognosis. Notably, reversible ischemia due to vascular dysfunction is a frequent finding in NOCAD patients (1). The endogenous, non-proteinogenic amino acid homoarginine (hArg) increases the availability of nitric oxide (NO) and thereby the NO-dependent vasodilatation (2). Several studies have reported a strong inverse relation between serum hArg levels and cardiovascular disease (CVD) risk among patients with obstructive CAD. However, the prognostic implications of serum hArg levels in NOCAD patients have not been explored previously. Purpose We evaluated serum hArg as a predictor of long-term risk of CVD mortality among patients with NOCAD. Methods 1046 patients with chronic coronary syndrome (CCS) underwent elective coronary angiography during 2000–2004, with the findings of NOCAD. Serum hArg was measured by liquid chromatography-tandem mass spectrometry in samples that had been frozen and stored at −80°C. The association of serum hArg to CVD mortality risk was visualized in a generalized additive regression plot and further explored using Cox regression. The models included age, sex, body mass index, hypertension, diabetes, smoking status, serum LDL cholesterol and estimated glomerular filtration rate as independent covariables. We evaluated model discrimination and risk classification by calculating C-statistics and net reclassification improvement (NRI >0), respectively. Results Median (25th-75th percentiles) age at inclusion was 57 (51–65) years, 48.5% were women and median (25th-75th percentiles) level of serum hArg was 1.87 (1.47–2.38) μmol/L. During median (25th- 75th percentiles) 14.1 (13.2–15.4) years of follow-up 5.7% of the patients died from CVD. The multivariable adjusted hazard ratio (95% confidence interval) per SD increment of (log transformed) hArg was 0.53 (0.40–0.70) in relation to CVD mortality. The multivariable model without biomarker provided a C- statistics for CVD mortality of 0.79 which increased to 0.82 by the addition of serum-hArg to the model (Δ area =0.03, P=0.01). Further, serum hArg provided a high NRI (95% CI) of 0.53 (0.40–0.70), P<0.001. Conclusion We demonstrated a strong inverse relationship between serum hArg and long-term risk of CVD mortality among patients with NOCAD. Our study adds to previous literature linking low hArg with vascular dysfunction and adverse CVD prognosis. The potential clinical usefulness of serum hArg measurements for the identification of a high-risk phenotype in NOCAD warrants further evaluation. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): The Western Norway Regional Health Authority

Smooth muscle cell CYB5R3 preserves cardiac and vascular function under chronic hypoxic stress

Chronic hypoxia is a major driver of cardiovascular complications, including heart failure. The nitric oxide (NO) – soluble guanylyl cyclase (sGC) – cyclic guanosine monophosphate (cGMP) pathway is integral to vascular tone maintenance. Specifically, NO binds its receptor sGC within vascular smooth muscle cells (SMC) in its reduced heme (Fe2+) form to increase intracellular cGMP production, activate protein kinase G (PKG) signaling, and induce vessel relaxation. Under chronic hypoxia, oxidative stress drives oxidation of sGC heme (Fe2+→Fe3+), rendering it NO-insensitive. We previously showed that cytochrome b5 reductase 3 (CYB5R3) in SMC is a sGC reductase important for maintaining NO-dependent vasodilation and conferring resilience to systemic hypertension and sickle cell disease-associated pulmonary hypertension. To test whether CYB5R3 may be protective in the context of chronic hypoxia, we subjected SMC-specific CYB5R3 knockout mice (SMC CYB5R3 KO) to 3 weeks hypoxia and assessed vascular and cardiac function using echocardiography, pressure volume loops and wire myography. Hypoxic stress caused 1) biventricular hypertrophy in both WT and SMC CYB5R3 KO, but to a larger degree in KO mice, 2) blunted vasodilation to NO-dependent activation of sGC in coronary and pulmonary arteries of KO mice, and 3) decreased, albeit still normal, cardiac function in KO mice. Overall, these data indicate that SMC CYB5R3 deficiency potentiates bilateral ventricular hypertrophy and blunts NO-dependent vasodilation under chronic hypoxia conditions. This implicates that SMC CYB5R3 KO mice post 3-week hypoxia have early stages of cardiac remodeling and functional changes that could foretell significantly impaired cardiac function with longer exposure to hypoxia.

Thromboxane-prostanoid receptor activation blocks ATP-sensitive potassium channels in rat aortas.

K+ channel activation is one of the major mechanisms involved in vasodilation. Vasoconstrictor agonists such as angiotensin II promote ATP-dependent potassium channels (KATP ) dysfunction. This study evaluates whether thromboxane-prostanoid (TP receptor) activation by the agonist U46619 increases reactive oxygen species (ROS) production in rat aortas, which could contribute to KATP channel dysfunction and impaired NO-dependent vasodilation. TP receptor activation with the selective agonist U46619 increased ROS in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), but the TP receptor antagonist SQ29548 abolished this effect. ECs and VSMCs incubation with ROS scavengers like Tiron or PEG-Catalase impaired U46619-induced ROS production. U46619 at the concentrations of 0.1 and 1µmol/L induced contractions with similar amplitude. KATP channel activation with pinacidil-induced relaxation was lower for the contractions induced with 0.1 or 1µmol/L U46619 than with 10nmol/L U46619. Acetylcholine-induced relaxation provided similar results. In aortas pre-contracted with 10nmol/L U46619, neither Tiron (100µmol/L) nor catalase (300U/mL) affected pinacidil-induced relaxation. However, in aortas pre-contracted with 0.1µmol/L U46619, catalase potentiated pinacidil-induced relaxation. Pinacidil potentiated acetylcholine-induced relaxation in aortas pre-contracted with 0.1 and 1µmol/L U46619. Incubation with 10nmol/L U46619 increased NO concentration in ECs. Taken together, these results show that high concentrations of the TP receptor agonist U46619 impair KATP channels, which is probably due to ROS production. It is likely that hydrogen peroxide is the ROS.

Microelectrode Sensor for Real-Time Measurements of Nitrite in the Living Brain, in the Presence of Ascorbate.

The impaired blood flow to the brain causes a decrease in the supply of oxygen that can result in cerebral ischemia; if the blood flow is not restored quickly, neuronal injury or death will occur. Under hypoxic conditions, the production of nitric oxide (●NO), via the classical L-arginine–●NO synthase pathway, is reduced, which can compromise ●NO-dependent vasodilation. However, the alternative nitrite (NO2−) reduction to ●NO, under neuronal hypoxia and ischemia conditions, has been viewed as an in vivo storage pool of ●NO, complementing its enzymatic synthesis. Brain research is thus demanding suitable tools to probe nitrite’s temporal and spatial dynamics in vivo. In this work, we propose a new method for the real-time measurement of nitrite concentration in the brain extracellular space, using fast-scan cyclic voltammetry (FSCV) and carbon microfiber electrodes as sensing probes. In this way, nitrite was detected anodically and in vitro, in the 5–500 µM range, in the presence of increasing physiological concentrations of ascorbate (100–500 µM). These sensors were then tested for real-time and in vivo recordings in the anesthetized rat hippocampus; using fast electrochemical techniques, local and reproducible transients of nitrite oxidation signals were observed, upon pressure ejection of an exogenous nitrite solution into the brain tissue. Nitrite microsensors are thus a valuable tool for investigating the role of this inorganic anion in brain redox signaling.

Hyperuricemia-induced endothelial insulin resistance: the nitric oxide connection.

Hyperuricemia, defined as elevated serum concentrations of uric acid (UA) above 416µmol L-1, is related to the development of cardiometabolic disorders, probably via induction of endothelial dysfunction. Hyperuricemia causes endothelial dysfunction via induction of cell apoptosis, oxidative stress, and inflammation; however, it's interfering with insulin signaling and decreased endothelial nitric oxide (NO) availability, resulting in the development of endothelial insulin resistance, which seems to be a major underlying mechanism for hyperuricemia-induced endothelial dysfunction. Here, we elaborate on how hyperuricemia induces endothelial insulin resistance through the disruption of insulin-stimulated endothelial NO synthesis. High UA concentrations decrease insulin-induced NO synthesis within the endothelial cells by interfering with insulin signaling at either the receptor or post-receptor levels (i.e., proximal and distal steps). At the proximal post-receptor level, UA impairs the function of the insulin receptor substrate (IRS) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) in the insulin signaling pathway. At the distal level, high UA concentrations impair endothelial NO synthase (eNOS)-NO system by decreasing eNOS expression and activity as well as by direct inactivation of NO. Clinically, UA-induced endothelial insulin resistance is translated into impaired endothelial function, impaired NO-dependent vasodilation, and the development of systemic insulin resistance. UA-lowering drugs may improve endothelial function in subjects with hyperuricemia.

Women with a History of Preeclampsia Have Preserved Neurogenically‐Mediated Dilation in the Cutaneous Microvasculature

Women who have had preeclampsia (PrEC) demonstrate microvascular endothelial dysfunction, mediated in part by reduced nitric oxide (NO)-dependent mechanisms. Localized heating of the skin induces a biphasic vasodilation response: a neurogenically-mediated initial peak, followed by a sustained endothelium-dependent plateau. We have previously shown that the endothelium-dependent plateau is attenuated in PrEC, due to attenuated NO-dependent dilation. However, it is unknown if the same is true concerning the neurogenically-mediated initial peak. The purpose of this study was to (1) examine the effect of preeclampsia history on neurogenically-mediated vasodilation and the NO-dependent contribution to that response, and (2) examine the relation between NO-dependent neurogenic dilation and the NO-dependent portion of the endothelium-dependent plateau. We hypothesized that PrEC would have an attenuated initial peak response and a reduced NO-dependent contribution to that response compared to women with a history of normotensive pregnancy (HC). Six HC (31±2yrs, 7±2 months post-partum) and 7 PrEC (29±2yrs, 7±3months post-partum) underwent a standard local heating protocol (42°C; 0.1°C·s-1). Two intradermal microdialysis fibers were placed in the skin of the ventral forearm for the continuous local delivery of Lactated Ringer's alone (control) or 15mM NG-nitro-L-arginine methyl ester (L-NAME) for nitric oxide synthase (NOS)-inhibition. Red blood cell flux was measured at each site via laser-Doppler flowmetry (LDF). Cutaneous vascular conductance was calculated (CVC=LDF/mean arterial pressure) and normalized to maximum (%CVCmax; 28mM SNP + local heat 43°C). NO-dependent dilation was calculated within-subject as the difference between control and NOS-inhibited sites. There were no differences in the initial peak between groups (HC: 80±5 vs PrEC: 79±5 %CVCmax; p=.88). NOS-inhibition attenuated the initial peak in both HC (Control: 80±5 vs L-NAME: 57±9 %CVCmax; p=0.02) and PrEC (Control: 79±5 vs L-NAME: 53±6 %CVCmax; p=0.004). However, there were no group differences in the NO-dependent portion of initial peak (HC: 23±8 vs PrEC: 24±5 %CVCmax; p=0.97). The local heating plateau (HC: 99±2 vs PrEC: 85±1 %CVCmax; p<0.001) and NO-contribution to the plateau (HC: 47±5 vs PrEC: 14±5 %CVCmax; p<0.001) were attenuated in PrEC compared to HC. There was no relation between NO-dependent dilation in the initial peak and NO-dependent dilation in the plateau across groups (R2 = 0.05; p = 0.51). Women who have had preeclampsia demonstrate attenuated microvascular endothelium-dependent dilation compared to women with a history of uncomplicated pregnancy. However, there are no differences in neurogenically-mediated vasodilation between the groups, suggesting that the NO-dependent vasodilation of the neurogenic response is not related to endothelium-dependent NO-mediated dilation in these women.

Skin Pigmentation is Negatively Associated with Both Circulating Vitamin D Concentration and Cutaneous Microvascular Endothelial Function

Given population disparities in cardiovascular disease (CVD) prevalence, it is imperative to better understand individual characteristics that result in predisposition to the development of hypertension and overt CVD. Skin color is an important determinant of vitamin D status because melanin has an inhibitory effect on UV-B-induced cutaneous vitamin D synthesis. In turn, vitamin D deficiency may result in reduced vascular endothelial function, an antecedent to CVD, via reduced nitric oxide (NO) bioavailability and/or increased oxidative stress. Purpose To investigate the associations between skin pigmentation (M-index, assessed using skin reflectance spectrophotometry), serum vitamin D concentration [25(OH)D], and the NO contribution to local heating-induced cutaneous vasodilation (%NO). We hypothesized that across a wide range of skin pigmentation, M-index would be negatively related to serum [25(OH)D] and %NO, and that serum [25(OH)D] would be positively related to %NO. Methods An intradermal microdialysis fiber was placed in the forearms of 18 healthy adults (9M/9F; 21±2 yrs) with M-indices ranging from 30 to 68 a.u. for local delivery of pharmacological agents. Lactated Ringer's solution was perfused through the microdialysis fiber during local heating (39°C) to induce cutaneous NO-dependent vasodilation. After attaining a stable plateau blood flow, 15mM NG-nitro-L-arginine methyl ester (L-NAME; nitric oxide synthase inhibiter) was infused at all sites to directly quantify %NO. Red cell flux was measured at each site by laser-Doppler flowmetry (LDF) and cutaneous vascular conductance (CVC=LDF/MAP) was expressed as a percentage of maximum (%CVCmax; 28mM sodium nitroprusside+43°C). Serum [25(OH)D] was analyzed via enzyme-linked immunosorbent assay (coefficient of variation=5.5%). Results As hypothesized, M-index was negatively associated with both serum [25(OH)D] (R2=0.43, β=-0.40, p<0.01) and %NO (R2=0.32, β=-0.50, p=0.02). Serum [25(OH)D] was positively associated with %NO (R2=0.21, β=0.67, p=0.055). Conclusions These data suggest that vitamin D plays an important role in promoting healthy endothelial function, and that ensuring adequate vitamin D status may be an effective intervention to mitigate the development of endothelial dysfunction in darkly-pigmented individuals living in relatively low UVR environments.

Impaired HSP70 Expression in the Aorta of Female Rats: A Novel Insight Into Sex-Specific Differences in Vascular Function.

Heat-shock protein 70 (HSP70) contributes to cellular calcium (Ca2+) handling mechanisms during receptor-mediated vascular contraction. Interestingly, previous studies have independently reported sex-related differences in HSP70 expression and Ca2+ dynamics. Still, it is unknown if sex, as a variable, plays a role in the impact that HSP70 has upon vascular contraction. To narrow this gap, we investigated if differences exist in the expression levels of HSP70 in the aorta, and if targeting this protein contributes to sex disparity in vascular responses. We report that, compared with male animals, female rats present a reduction in the basal levels of HSP70. More compelling, we found that the blockade of HSP70 has a greater impact on phenylephrine-induced phasic and tonic vascular contraction in female animals. In fact, it seems that the inhibition of HSP70 significantly affects vascular Ca2+ handling mechanisms in females, which could be associated with the fact that these animals have impaired HSP70 expression. Corroborating this idea, we uncovered that the higher sensitivity of female rats to HSP70 inhibition does not involve an increase in NO-dependent vasodilation nor a decrease in vascular oxidative stress. In summary, our findings reveal a novel mechanism associated with sex-specific differences in vascular responses to α-1 adrenergic stimulation, which might contribute to unraveling the network of intertwined pathways conferring female protection to (cardio)vascular diseases.

Pirfenidone Is a Vasodilator: Involvement of KV7 Channels in the Effect on Endothelium-Dependent Vasodilatation in Type-2 Diabetic Mice.

Endothelial cell dysfunction and fibrosis are associated with worsening of the prognosis in patients with cardiovascular disease. Pirfenidone has a direct antifibrotic effect, but vasodilatation may also contribute to the effects of pirfenidone. Therefore, in a first study we investigated the mechanisms involved in the relaxant effect of pirfenidone in rat intrapulmonary arteries and coronary arteries from normal mice. Then in a second study, we investigated whether pirfenidone restores endothelial function in the aorta and mesenteric arteries from diabetic animals. From 16–18-week old normal male C57BL/6 mice and normoglycemic (db/db+), and type 2 diabetic (db/db) male and female mice, arteries were mounted in microvascular isometric myographs for functional studies, and immunoblotting was performed. In rat pulmonary arteries and mouse coronary arteries, pirfenidone induced relaxations, which were inhibited in preparations without endothelium. In mouse coronary arteries, pirfenidone relaxation was inhibited in the presence of a nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine (L-NOARG), a blocker of large-conductance calcium-activated potassium channels (BKCa), iberiotoxin, and a blocker of KV7 channels, XE991. Patch clamp studies in vascular smooth muscle revealed pirfenidone increased iberiotoxin-sensitive current. In the aorta and mesenteric small arteries from diabetic db/db mice relaxations induced by the endothelium-dependent vasodilator, acetylcholine, were markedly reduced compared to db/db + mice. Pirfenidone enhanced the relaxations induced by acetylcholine in the aorta from diabetic male and female db/db mice. An opener of KV7 channels, flupirtine, had the same effect as pirfenidone. XE991 reduced the effect of pirfenidone and flupirtine and further reduced acetylcholine relaxations in the aorta. In the presence of iberiotoxin, pirfenidone still increased acetylcholine relaxation in aorta from db/db mice. Immunoblotting for KV7.4, KV7.5, and BKCa channel subunits were unaltered in aorta from db/db mice. Pirfenidone failed to improve acetylcholine relaxation in mesenteric arteries, and neither changed acetylcholine-induced transient decreases in blood pressure in db/db+ and db/db mice. In conclusion, pirfenidone vasodilates pulmonary and coronary arteries. In coronary arteries from normal mice, pirfenidone induces NO-dependent vasodilatation involving BKCa and KV7 channels. Pirfenidone improves endothelium-dependent vasodilatation in aorta from diabetic animals by a mechanism involving voltage-gated KV7 channels, a mechanism that may contribute to the antifibrotic effect of pirfenidone.

Protective effects of pravastatin on the embryonic cardiovascular system during hypoxic development.

The use of statins in complicated pregnancy is being considered, as they protect endothelial function in the mother and placenta. However, whether statins affect cardiovascular function in the fetus is completely unknown. Here, we have determined the effects of pravastatin and underlying mechanisms on the cardiovascular system of the hypoxic chicken embryo, a model system that permits the direct effects of pravastatin on the developing offspring to be isolated independently of additional effects on the mother and/or placenta. Chicken embryos were incubated under normoxia or hypoxia (14% O2 ) from day 1±pravastatin (1mg/kg/d) from day 13 of incubation (term is 21days). On day 19 of incubation, hearts and vessels were isolated to determine changes in the cardiovascular structure and function. The data show that pravastatin protected the hypoxic chicken embryo against impaired cardiovascular dysfunction. Mechanisms involved in this protection included reduced oxidative stress, enhanced NO bioavailability, restored antioxidant defenses and normalized protein expression of RhoA in the embryonic heart, and improved NO-dependent vasodilator mechanisms in the peripheral circulation. Therefore, we show that the treatment of the chronically hypoxic chicken embryo with pravastatin from day 13 of incubation, equivalent to ca. 25weeks of gestation in human pregnancy, has direct beneficial effects on the embryonic cardiovascular system. Therefore, pravastatin may be a candidate for human clinical translation to rescue fetal cardiovascular dysfunction in risky pregnancy.

Abstract MP19: Smooth Muscle Cell Cytochrome B5 Reductase 3 Causes Heart Failure With Reduced Ejection Fraction Under Hypoxic Stress

Pulmonary hypertension causes increased pulmonary vascular resistance and right heart failure. Nitric oxide (NO) binds to its receptor soluble guanylyl cyclase (sGC) within vascular smooth muscle cells in its reduced heme (Fe 2+ ) form to increase intracellular cGMP production, activate protein kinase G signaling, and induce vessel relaxation. In pulmonary hypertension, endothelial damage leading to decreased NO bioavailability combined with oxidation of the sGC heme (Fe 3+ ) in vascular smooth muscle cells rendering it NO-insensitive results in vasonstriction. Notably, we have previously shown that cytochrome b5 reductase 3 (CYB5R3) in vascular smooth muscle cells is an sGC reductase (Fe 3+ to Fe 2+ ) that maintains NO-dependent vasodilation in vascular disease. We therefore hypothesized that CYB5R3 confers protection in pulmonary hypertension. To test this, we subjected smooth muscle cell-specific CYB5R3 knockout mice (SMC CYB5R3 KO) to 21 days of continuous hypoxia (10% O 2 ) and assessed vascular and cardiac function. We found that SMC CYB5R3 KO led to enhanced cardiac hypertrophy when compared to wild-type (WT) controls (n=8/ group). Specifically, SMC CYB5R3 KO mice had a larger right ventricle per tibia size, left ventricle mass, and Fulton index compared to WT (n=8/ group). Moreover, SMC CYB5R3 KO mice had a significantly impaired ejection fraction and fractional shortening, and increased left ventricular posterior wall pressure (n=3-5/group). No differences in right heart function or overall cardiac fibrosis were observed between groups (n=3-5/group). With respect to vascular function, hypoxic pulmonary arteries from SMC CYB5R3 KO mice also had a blunted response to sodium nitroprusside induced NO-dependent vasodilation though no difference in sGC activator BAY 58-2667 induced NO-independent vasodilation was observed as compared to WT (n=8-11/ group). No differences in pulmonary arterial sGC levels or medial area were observed between groups (n=6-7). Combined, these data implicate that loss of SMC CYB5R3 exacerbates cardiomyocyte hypertrophy and reduces cardiac function independent of pulmonary pressure differences.

Abstract P093: Cytochrome B5 Reductase 3 And Xanthine Oxidase Coordinately Regulate Soluble Guanylyl Cyclase Physiological Redox State

In cardiovascular disease, oxidative stress can drive soluble guanylyl cyclase (sGC) heme oxidation resulting in the loss of the sGC heme (apo-sGC), the impairment of nitric oxide (NO) binding and cGMP production, and vasoconstriction. Consequently, a new class of therapeutic compounds sGC activators have been developed which target oxidized and apo-sGC to cause irreversible, NO-independent reactivation of cGMP production and vasodilation. While sGC activators have had varied clinical success, surprisingly few studies have defined the impact of NO-independent sGC activation on vascular physiology in healthy conditions. We found mesenteric and pulmonary arteries are two log orders more sensitive to NO-independent sGC activator BAY 58-2667 induced vasodilation than aorta; no difference in NO-dependent sGC vasodilation between vessels was observed. These data indicate the presence of an activatable physiological pool of oxidized and/or apo-sGC in pulmonary and mesenteric arteries. We recently published that smooth muscle cell cytochrome b5 reductase 3 (CYB5R3) acts to reduce oxidized heme sGC back to its NO-sensitive reduced heme state during vascular disease. We found transgenic CYB5R3 overexpression (CYB5R3 OE) mice were more resistant to BAY 58-2667 mesenteric artery vasodilation and blood pressure lowering compared to wild-type controls (n=5-9) under physiologic conditions. Also, healthy CYB5R3 OE pulmonary arteries had a near complete loss of BAY 58-2667 vasodilation suggesting both mesenteric and pulmonary arteries contain a pool of oxidized sGC. We next asked if physiological H 2 O 2 production accounts for changes in BAY 58-2667 responsiveness. We found using mitochondrial-specific catalase overexpression mice, that BAY 58-2667 vasodilation did not differ from controls in any vascular bed (n=4-6). We next tested whether xanthine oxidase (XO), which can produce H 2 O 2 at the endothelial cell surface of vessels, can impact physiological BAY 58-2667 vasodilation. We found that Febuxostat, a XO inhibitor, led to a significant decrease in mesenteric artery BAY 58-2667 induced vasodilation from ~70% to ~30% (n=6). Combined, these data provide evidence for CYB5R3 and XO as regulators of physiological sGC resistance artery vasodilation.

Counterpoint: Investigators should not control for menstrual cycle phase when performing studies of vascular control that include women.

Observed sex differences in the epidemiology of cardiovascular disease incidence and the efficacy of clinical intervention strategies, coupled with recent mandates from the National Institutes of Health (NIH), and its international counterparts to consider sex as a biological variable in all preclinical and clinical work (15), have led to increased enrollment of women in human physiology studies of vascular function with the goal of improving external validity and clinical applicability of the findings. However, standard practice in these studies is to test women during the early follicular phase (~days 1-5) of the menstrual cycle, during which estrogen and progesterone concentrations are lowest, and either exclude or limit testing of women who use hormonal contraceptives to the placebo phase. This approach seeks to control for, or limit, differences in circulating hormone concentrations between men and women and is based on the dogmatic theory that cyclic changes in estrogen and progesterone concentrations lead to greater variability in study outcomes and uninterpretable datasets. Contrary to this common approach, we propose that limiting testing of women to this single phase 1) decreases external validity of the findings, 2) obfuscates potential physiological differences contributable to differences in sex hormone concentrations between men and women, and 3) is, in many instances, contradicted by literature that demonstrates limited or no variability in vascular measurements across the menstrual cycle.

Contribution of selected vasoactive systems to blood pressure regulation in two models of chronic kidney disease.

It is generally accepted that angiotensin II plays an important role in high blood pressure (BP) development in both 2-kidney-1-clip (2K1C) Goldblatt hypertension and in partial nephrectomy (NX) model of chronic kidney disease (CKD). The contribution of sympathetic nervous system and nitric oxide to BP control in these models is less clear. Partial nephrectomy or stenosis of the renal artery was performed in adult (10-week-old) male hypertensive heterozygous Ren-2 transgenic rats (TGR) and normotensive control Hannover Sprague Dawley (HanSD) rats and in Wistar rats. One and four weeks after the surgery, basal blood pressure (BP) and acute BP responses to the consecutive blockade of renin-angiotensin (RAS), sympathetic nervous (SNS), and nitric oxide (NO) systems were determined in conscious rats. Both surgical procedures increased plasma urea, a marker of renal damage; the effect being more pronounced following partial nephrectomy in hypertensive TGR than in normotensive HanSD rats with a substantially smaller effect in Wistar rats after renal artery stenosis. We demonstrated that the renin-angiotensin system does not play so fundamental role in blood pressure maintenance during hypertension development in either CKD model. By contrast, a more important role is exerted by the sympathetic nervous system, the activity of which is increased in hypertensive TGR-NX in the developmental phase of hypertension, while in HanSD-NX or Wistar-2K1C it is postponed to the established phase. The contribution of the vasoconstrictor systems (RAS and SNS) was increased following hypertension induction. The role of NO-dependent vasodilation was unchanged in 5/6 NX HanSD and in 2K1C Wistar rats, while it gradually decreased in 5/6 NX TGR rats.

Vasodilation of Tea Polyphenols Ex Vivo Is Mediated by Hydrogen Peroxide Under Rapid Compound Decay.

Improvement of endothelial function represents a major health effect of tea in humans. Ex vivo, tea and tea polyphenols stimulate nitric oxide (NO)-dependent vasodilation in isolated blood vessels. However, it was reported that polyphenols can generate reactive oxygen species (ROS) in vitro. We therefore aimed to elucidate the role of ROS production in tea polyphenol-induced vasodilation in explanted aortic rings. Vasorelaxation of rat aortic rings was assessed in an organ chamber model with low concentrations of epigallocatechin-3-gallate (EGCG), theaflavin-3,3’-digallate (TF3), and with green and black tea, with or without pretreatment with catalase or superoxide dismutase (SOD). The stability of EGCG and TF3 was measured by HPLC, and the levels of hydrogen peroxide (H2O2) were determined. EGCG and green tea-induced vasorelaxation was completely prevented by catalase and slightly increased by SOD. TF3 and black tea yielded similar results. Both EGCG and TF3 were rapidly degraded. This was associated with increasing H2O2 levels over time. Hydrogen peroxide concentrations produced in a time range compatible with tea polyphenol decay induced NO-dependent vasodilation in aortic rings. In conclusion, tea polyphenol-induced vasodilation in vitro is mediated by low levels of H2O2 generated during compound decay. The results could explain the apparent lack of vasodilatory effects of isolated tea polyphenols in humans.

Acute Systemic Inhibition of Inflammation Augments Endothelium‐dependent Dilation in Women with a History of Preeclamptic Pregnancy

Women who have had preeclampsia demonstrate microvascular endothelial‐dysfunction, mediated in part by reduced nitric oxide (NO)‐dependent dilation. Preeclamptic pregnancies are associated with elevated inflammation, and inhibition of inflammation attenuates endothelial damage in animal models of preeclampsia. However, it is unclear if systemic inhibition of inflammation improves endothelial function in women after a preeclamptic pregnancy. Using the cutaneous microcirculation as a model, we hypothesized that acute systemic inhibition of inflammation (oral salsalate; 1500mg/twice daily/4 days) would improve endothelium‐ and NO‐dependent vasodilation in women with a history of preeclampsia (PrEC) but not in women with a history of uncomplicated pregnancy (HC). Eleven HC (30±1yrs, 9±2 months post‐partum) and 8 PrEC (29±3yrs, 6±1 months post‐partum) participated in a double‐blind placebo‐controlled study. Following each treatment, 2 intradermal microdialysis fibers were placed in the skin of the ventral forearm for graded infusion of acetylcholine (ACh, 10−7–102 mM) or ACh+15mM L‐NAME (NO synthase antagonist). Red cell flux was measured over each site by laser‐Doppler flowmetry (LDF). Cutaneous vascular conductance was calculated (CVC=LDF/mean arterial pressure) and normalized to maximum (%CVCmax; 28mM SNP + local heat 43°C). ACh‐induced (75±3 vs. 92±4%CVCmax; p=0.002) and NO‐dependent (18±8 vs. 32±4%; p=0.04) vasodilation were attenuated in PrEC compared to HC following placebo. Salsalate augmented ACh‐induced (92±3%CVCmax; p=0.001) and NO‐dependent (38±4%; p=0.04) dilation in PrEC compared to placebo but had no effect in HC (all p&gt;0.05). Women who have had preeclampsia demonstrate attenuated microvascular endothelial function compared to women with a history of uncomplicated pregnancy. Salsalate treatment augmented endothelium‐dependent vasodilation via NO‐mediated pathways in these women, suggesting that inflammatory signaling plays a role in mediating persistent endothelial‐dysfunction in women with a history of preeclampsia. ClinicalTrials.gov registration # NCT03482440Support or Funding InformationNIH K99 HL138133 (Stanhewicz); NIH R01 HL093238 (Alexander); NIH T32 AG049676 (Serviente)