Endothelium-independent Responses Research Articles

Dehydroepiandrosterone (DHEA), a circulating steroid hormone precursor produced potent vasorelaxation in rat aorta and mesenteric arteries through blockade of L-type voltage-dependent calcium channels

Dehydroepiandrosterone (DHEA) is known for potent cardioprotective properties and diminished DHEA level in plasma is often associated with hypertension and age-related anomalies. However, putative ex-vivo vasorelaxation potential of DHEA in systemic resistance vessels like mesenteric arteries and conduit arteries like aorta are still to be worked out. The study aimed to explore vasorelaxation potential of DHEA in superior and resistance mesenteric arteries and aorta in rats and to determine the contribution L-type Voltage dependent calcium channel (L-VDCC) in the relaxation response in these arterial tissues. Ex-vivo vasorelaxation potential of DHEA in isolated arterial tissues were evaluated and the mechanism of vasorelaxation induced by DHEA was characterized by contraction experiment in isolated arterial tissue and in-vitro calcium imaging assay using Fluo-4 in primary vascular smooth muscle cells derived from aorta. In the current study, DHEA was found to exhibit potent concentration dependent, endothelium and potassium channel independent vasorelaxation response in conduit and resistance arteries. The block of L-type VDCCs was evident from the findings that DHEA in a concentration-dependent manner inhibited both BAY K-8644 and CaCl2-induced contractions. The results of the contraction experiment were further substantiated by Fluo-4 mediated calcium imaging assay in primary rat vascular smooth muscle wherein DHEA concentration dependently blocked noradrenaline and BAY K-8644-induced rise in intracellular calcium fluorescence. The present study showed potent endothelium and potassium channel independent vasorelaxation properties of DHEA in aorta, superior and resistance mesenteric artery mediated predominantly through blockade of L-VDCC.

Coronary microvascular dysfunction in heart failure: observations from the MICRO registry

Abstract Background Heart failure (HF) is one of the leading causes of hospitalization in the elderly. Coronary microvascular dysfunction (CMD), among other risk factors, is linked to the pathogenesis and clinical manifestations of HF. Purpose This abstract aims to summarize key findings from a comprehensive analysis of CMD in subgroups of HF, shedding light on its association with hemodynamic parameters. Methods Patients experiencing recurrent angina (CCS II-IV) and no relevant epicardial stenosis were enrolled in a prospective registry (MICRO). Epicardial and microvascular responses (pressure, resistance and flow) were assessed at rest and during adenosine and acetylcholine testing. The impact of an elevated left ventricular end-diastolic pressure (LVEDP) in the absence of clinical signs of HF was also investigated. Results Overall, 188 patients (63% (118) women, mean age 65±11 years) with angina and no epicardial stenosis were enrolled. Patients with HF (n=26) displayed lower hyperemic flow in response to endothelium-independent adenosine (5.2±3.1 vs. 7.3±4.4; P=0.004), lower coronary flow reserve (2.9±1.5 vs. 4.4±1.1; P<0.001) and microvascular resistance reserve (3.3±2.1 vs. 5.2±2.7; P<0.001), and increased microvascular resistance during adenosine-induced hyperemia (25±21 vs. 17±11; P=0.045). An increased LVEDP without clinical signs of HF was not associated with a lower coronary flow reserve (4.3±1.9 vs. 2.6±1.2; P=0.006) or microvascular resistance reserve (4.9±2.1 vs. 2.8±1.7; P=0.004). Microvascular resistance during acetylcholine (endothelium-dependent)-induced hyperemia was lower in HF patients (36.2±23.5 vs. 57.4±37.8; P=0.018). Conclusion(s) Patients with heart failure displayed signs of impaired microvascular endothelium-independent responses while endothelium-dependent reactivity appears to be maintained. An elevated LVEDP without clinical HF was not associated with impaired coronary microvascular function.

Coronary microvascular dysfunction in postmenopausal minipigs with multiple risk factors

Abstract Background Coronary microvascular angina occurs in both men and women, however, the majority of patients are postmenopausal women with multiple cardiovascular risk factors, such as diabetes mellitus (DM), chronic kidney disease (CKD) and dyslipidemia. No treatment is available for coronary microvascular dysfunction, requiring more in depth knowledge on the mechanisms underlying this syndrome in this expanding group of patients. Purpose Here we studied microvascular function in vivo and and in vitro in isolated small coronary arteries, in post-menopausal miniswine with multiple cardiovascular risk factors. Methods DM (streptozotocin), hypercholesterolemia (HC, high fat, high sugar diet), CKD (renal artery embolization) and menopause (ovariectomy, OVX) were induced in 5 adult female minipigs (1.5-2 years old, ~40kg, DM+HC+CKD+OVX) for 6 months, while 11 healthy age- and weight-matched female minipigs on normal pig chow served as controls (Normal). At sacrifice, coronary flow reserve (CFR) in response to intracoronary infusion of adenosine was measured under anesthesia. Isolated small coronary arteries were used to study the endothelium-dependent response to bradykinin in the presence and absence of eNOS blockade with LNAME, and cyclooxygenase inhibition by indomethacin, and endothelium-independent response to the nitric oxide (NO) donor, SNAP. Results 6 months of sustained hyperglycemia (17.5±1.0 in DM+HC+CKD+OVX vs 8.2±0.9 mmol/l in Normal, P<0.05 by t-test), hypercholesterolemia (15.4±2.0 vs 1.7±0.1 mmol/l, P<0.05), renal dysfunction (NGAL: 205±72 vs 67±5 ng/ml, P<0.05) and low progesterone levels (13.1±5.7 in DM+HC+CKD+OVX vs 92.0±30.0 nmol/l in Normal, P=0.1) were accompanied by systemic inflammation (TNFα: 62±7 vs 47±1 pg/ml, P<0.05). CFR was significantly reduced in DM+HC+CKD+OVX as compared to the healthy animals (panel A). Coronary small arteries from DM+HC+CKD+OVX animals showed impaired endothelium-dependent vasodilation to bradykinin (panel B), which was mediated by a loss of NO (panels C & D). The loss of NO was partly compensated for by activation of the cyclooxygenase pathway, as indomethacin blunted the dilation to bradykinin in the DM+HC+CKD+OVX animals, but had no effect on the dilation to bradykinin in Normals (panels C & D). Endothelium-independent dilation to SNAP was also impaired in DM+HC+CKD+OVX animals, indicating blunted vascular smooth muscle responsiveness to nitric oxide (panel E). Conclusion Postmenopausal minipigs with multiple risk factors, displayed severe coronary microvascular dysfunction as evidenced by a significantly reduced coronary flow reserve and both reduced NO sensitivity/ production, indicating endothelial and smooth muscle cell dysfunction. Loss of NO-mediated vasodilation was partially compensated by activation of cyclooxygenase pathway. Such perturbations may contribute to reduced myocardial perfusion that is often observed in post-menopausal women with multiple cardiovascular risk factors.

Role of endothelial cell-selective adhesion molecule in the regulation of pulmonary vascular resistance

Endothelial cell-selective adhesion molecule (ESAM) is a member of the endothelial cell junction adhesion molecules family, which plays an important role in the maintenance of endothelial barrier function in the pulmonary circulation. The role of ESAM in affecting pulmonary vascular resistance remains unknown. To examine the role of ESAM in pulmonary vascular resistance, whole lungs were isolated from 16-18 weeks old ESAM knock out (KO) mice and wild type (WT) littermates, ventilated and perfused. We found that pulmonary vascular resistance is significantly increased in the lung of ESAM KO mice compared to WT mice. Bradykinin-induced, endothelium-dependent reduction in the pulmonary vascular resistance was impaired in ESAM KO mice, whereas the direct NO donor, sodium nitroprusside-induced endothelium independent responses remained similar in WT and ESAM KO mice. U46619 mediated increases in pulmonary vascular resistance was significantly higher in ESAM KO mice. Inhibition of the NO synthesis with the L-NAME (0.2 mM for 10 minutes) augmented the U46619-mediated increase in pulmonary vascular resistance in WT mice, but it did not affect the U46619 response in the ESAM KO mice. The mRNA expression of pro-inflammatory cytokines, including interleukin-6 and tumor necrosis factor, was increased in the lungs of ESAM KO mice, whereas expression of collagen isoforms (type I and type III) and fibronectin were similar in WT and ESAM KO mice. Collectively, we propose that ESAM plays an important role in the maintenance of pulmonary vascular resistance, likely via preserving endothelial nitric oxide production in pulmonary arteries. AHA predoctoral award ID 917057. 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.

Sodium valproate treatment reverses endothelial dysfunction in aorta from rabbits with acute myocardial infarction

Sodium valproate (VPA), a histone deacetylase (HDAC) inhibitor, could be a promising candidate to treat acute myocardial infarction (AMI). In this study, AMI was induced in New Zealand White rabbits by occluding the left circumflex coronary artery for 1 h, followed by reperfusion. The animals were distributed into three experimental groups: the sham-operated group (SHAM), the AMI group and the AMI + VPA group (AMI treated with VPA 500 mg/kg/day). After 5 weeks, abdominal aorta was removed and used for isometric recording of tension in organ baths or protein expression by Western blot, and plasma for the determination of nitrate/nitrite (NOx) levels by colorimetric assay. Our results indicated that AMI induced a reduction of the endothelium-dependent response to acetylcholine without modifying the endothelium-independent response to sodium nitroprusside, leading to endothelial dysfunction. VPA treatment reversed AMI-induced endothelial dysfunction and even increased NO sensitivity in vascular smooth muscle. This response was consistent with an antioxidant effect of VPA, as it was able to reverse the superoxide dismutase 1 (SOD 1) down-regulation induced by AMI. Our experiments also ruled out that the VPA mechanism was related to eNOS, iNOS, sGC and arginase expression or changes in NOx plasma levels. Therefore, we conclude that VPA improves vasodilation by increasing NO bioavailability, likely due to its antioxidant effect. Since endothelial dysfunction was closely related to AMI, VPA treatment could increase aortic blood flow, making it a potential agent in reperfusion therapy that can prevent the vascular damage.

Optimization of resting tension for wire myography in male rat pulmonary arteries.

Wire myography to test vasomotor functions of blood vessels ex-vivo are well-established for the systemic circulation, however, there is no consensus on protocols for pulmonary arteries. We created a standardized wire myography protocol for healthy rat PAs and validated this in a pulmonary hypertension (PH) model. Vessels stretched to higher initial tensions (5.0, 7.5 and 10.0 mN) exhibited a uniform response to phenylephrine, a larger dynamic range, and lower EC50 values. The endothelium-mediated relaxation showed that moderate tensions (7.5 and 10.0 mN) produced robust responses with higher maximum relaxation and lower EC50 values. For endothelium independent responses, the higher initial tension groups had lower and more consistent EC50 values than the lower initial tension groups. Pulmonary arteries from rats with PH were more responsive to vasoactive drugs when subjected to a higher initial tension. Notably, vessels in the PH group subjected to 15.0 mN exhibited high dynamic ranges in contractile and relaxation responses without tearing. Lastly, we observed attenuated cholinergic responses in these vessels-consistent with endothelial dysfunction in PH. Therefore, a moderate initial tension of 7.5-10.0 mN is optimal for healthy rat pulmonary arteries and a higher initial tension of 15.0 mN is optimal for pulmonary arteries from animals with PH.

High-fat diet causes endothelial dysfunction in the mouse ophthalmic artery

Obesity is a significant health concern that leads to impaired vascular function and subsequent abnormalities in various organs. The impact of obesity on ocular blood vessels, however, remains largely unclear. In this study, we examined the hypothesis that obesity induced by high-fat diet produces vascular endothelial dysfunction in the ophthalmic artery. Mice were subjected to a high-fat diet for 20 weeks, while age-matched controls were maintained on a standard diet. Reactivity of isolated ophthalmic artery segments was assessed in vitro. Reactive oxygen species (ROS) were quantified in cryosections by dihydroethidium (DHE) staining. Redox gene expression was determined in ophthalmic artery explants by real-time PCR. Furthermore, the expression of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2), the receptor for advanced glycation end products (RAGE), and of the lectin-like oxidized low-density-lipoprotein receptor-1 (LOX-1) was determined in cryosections using immunofluorescence microscopy. Ophthalmic artery segments from mice on a high-fat diet exhibited impaired vasodilation responses to the endothelium-dependent vasodilator acetylcholine, while endothelium-independent responses to nitroprusside remained preserved. DHE staining intensity in the vascular wall was notably stronger in mice on a high-fat diet. Messenger RNA expression for NOX2 was elevated in the ophthalmic artery of mice subjected to high fat diet. Likewise, immunostainings revealed increased expression of NOX2 and of RAGE, but not of LOX-1. These findings suggest that a high-fat diet triggers endothelial dysfunction by inducing oxidative stress in the ophthalmic artery via involvement of RAGE and NOX2.

Unexplained recurrent pregnancy loss and markers of arterial vascular health: An experimental case-control study.

To investigate whether women with unexplained recurrent pregnancy loss have impaired arterial vascular health compared with controls, and to evaluate whether this is modifiable by exercise. Experimental case-control pilot study. University medical centre in the Netherlands. Twelve women with unexplained recurrent pregnancy loss, 11 nulliparous women and 19 primiparous women with a history of uncomplicated pregnancies. In all three groups we measured baseline characteristics, metabolic components and arterial vascular health, and repeated this in women with unexplained recurrent pregnancy loss after 1 month of protocolled and supervised cycle training. Differences in arterial vascular health between women with unexplained recurrent pregnancy loss and controls, and the effect of exercise on arterial vascular health in women with unexplained recurrent pregnancy loss. Women with unexplained recurrent pregnancy loss have a significantly increased carotid intima media thickness in comparison with both controls (both P < 0.01), a significantly decreased brachial endothelial dependent flow-mediated vasodilation in comparison with both controls (nulliparous: P < 0.01; primiparous: P = 0.05) and a significantly decreased femoral endothelial dependent flow-mediated vasodilation in comparison with primiparous women (P = 0.01). The endothelium independent glyceryl trinitrate response was similar in all groups. With 1 month of exercise, the carotid intima media thickness decreased significantly by 7% (P = 0.05) and the femoral FMD increased significantly by 10% (P = 0.01) in women with unexplained recurrent pregnancy loss. Women with unexplained recurrent pregnancy loss have an impaired vascular health in comparison with controls. This impaired arterial vascular health can be improved by exercise.

MICROVASCULAR ENDOTHELIUM FUNCTION RESPONSE TO N-3 POLYUNSATURATED FATTY ACIDS, VITAMIN E, SELENIUM AND LUTEIN ENRICHED FUNCTIONAL FOOD IN PATIENTS WITH CHRONIC CORONARY SYNDROME

Objective: Ischemic heart disease is the leading cause of death at a global level. A healthy lifestyle, and especially a healthy diet, can have the effect of reducing the risk of subsequent cardiovascular events and mortality. The aim of this study was to examine the effect of hen eggs enriched with n-3 polyunsaturated fatty acids (n-3 PUFAs), vitamin E, selenium and lutein consumption on oxidative stress level and microvascular endothelium function in patients with existing chronic coronary syndrome (CCS). Design and method: A total of 30 patients (9 women and 21 men) with existing CCS participated in this study. The participants were divided in two groups; Control group (N = 15) who ate three regular hen eggs per day (n-3 PUFAs 146 mg, vitamin E 0.595 mg, selenium 0.0183 mg, lutein 0.11mg/per egg), and Nutri4 group (N = 15) who ate three enriched hen eggs per day (n-3 PUFAs 432 mg, vitamin E 1.098 mg, selenium 0.0191 mg, lutein 0.616 mg/per egg) for three weeks. Peripheral microvascular reactivity in response to vascular occlusion (PORH), and iontophoresis of acetylcholine (AChID) and sodium nitroprusside (SNPID), as well as serum lipid and fatty acids profile, pro- and anti-inflammatory cytokines level, as well as biomarkers of oxidative stress and antioxidant capacity were measured before and after the respective diet protocol. Results: Consumption of Nutri4 eggs significantly decreased serum triglycerides levels and n6/n3 PUFAs ratio, decreased levels of cytokines IL-17A and TGF-1beta, and improved endothelium-dependent microvascular PORH and AChID, but not endothelium-independent response in patients with CCS. Consumption of both Nutri4 and regular hen eggs did not affect biomarkers of oxidative stress and antioxidant capacity in CCS patients. Conclusions: Consumption of hen eggs enriched with n-3 PUFAs, vitamin E, selenium and lutein for three weeks induced an anti-inflammatory response, a favorable effect on lipid and fatty acids profile, especially on triglycerides levels and n6/n3 PUFAs ratio, as well as an improved endothelium-dependent peripheral microvascular reactivity in patients with CCS.

Influence of tobacco smoking on the functional state of the vascular endothelium in young people

The present article investigates the effect of tobacco smoking on the functional state of the vascular endothelium in healthy young people without other risk factors for coronary heart disease and arterial hypertension. Based on the analysis of the results obtained by rheoplethysmography, the endothelium-dependent and endothelium-independent responses according to Celermajer D.S.(1992) were studied. The increase in pulse blood flow to reactive hyperemia in students - smokers and non- smokers was analyzed. It was found that tobacco smoking has an adverse effect on the functional state of the vascular endothelium, namely endothelium-dependent vasodilation, which may further be a trigger for the development of a number of diseases of the cardiovascular system.

Sulodexide Prevents Hyperglycemia-Induced Endothelial Dysfunction and Oxidative Stress in Porcine Retinal Arterioles

Diabetes mellitus may cause severe damage to retinal blood vessels. The central aim of this study was to test the hypothesis that sulodexide, a mixture of glycosaminoglycans, has a protective effect against hyperglycemia-induced endothelial dysfunction in the retina. Functional studies were performed in isolated porcine retinal arterioles. Vessels were cannulated and incubated with highly concentrated glucose solution (HG, 25 mM D-glucose) +/- sulodexide (50/5/0.5 μg/mL) or normally concentrated glucose solution (NG, 5.5 mM D-glucose) +/- sulodexide for two hours. Endothelium-dependent and endothelium-independent vasodilatation were measured by videomicroscopy. Reactive oxygen species (ROS) were quantified by dihydroethidium (DHE) fluorescence. Using high-pressure liquid chromatography (HPLC), the intrinsic antioxidant properties of sulodexide were investigated. Quantitative PCR was used to determine mRNA expression of regulatory, inflammatory, and redox genes in retinal arterioles, some of which were subsequently quantified at the protein level by immunofluorescence microscopy. Incubation of retinal arterioles with HG caused significant impairment of endothelium-dependent vasodilation, whereas endothelium-independent responses were not affected. In the HG group, ROS formation was markedly increased in the vascular wall. Strikingly, sulodexide had a protective effect against hyperglycemia-induced ROS formation in the vascular wall and had a concentration-dependent protective effect against endothelial dysfunction. Although sulodexide itself had only negligible antioxidant properties, it prevented hyperglycemia-induced overexpression of the pro-oxidant redox enzymes, NOX4 and NOX5. The data of the present study provide evidence that sulodexide has a protective effect against hyperglycemia-induced oxidative stress and endothelial dysfunction in porcine retinal arterioles, possibly by modulation of redox enzyme expression.

Mesenteric Artery Reactivity in the Development of Metabolic Syndrome in Rats Fed on a High-Fat Diet

Higt fat diet can lead to the development of metabolic syndrome (MS). However, the question of the mechanisms of pathophysiological processes in MS has not been studied enough. The aim of the work was to study the effect of a high-fat diet (HFD) on the reactivity of the mesenteric arteries of Wistar rats in vivo, as well as to evaluate the change in the mechanisms of endothelium-dependent arterial dilatation in HFD. The HFD-group of rats (n = 25) received HFD containing 50% animal fat for 10 weeks, the control group (n = 25) received a standard diet. The effect of HFD on endothelium-dependent and endothelium-independent responses of the mesenteric arteries under the action of agonists in the absence and with the use of blockers of NO-synthase (L-NAME), cyclooxygenase (indomethacin), and K+-channels (tetraethylammonium) was assessed using photomicrography and video recording of mesenteric artery diameter in vivo. HFD in rats led to the development of MS, including dyslipidemia, hyperglycemia and insulin resistance, and an increase in blood pressure. MS was accompanied by impaired functional state of the mesenteric arteries. In rats of the HFD group, compared with the control group, there was an increase in the constrictor reaction to phenylephrine by 29%, as well as a decrease in the reactivity of vessels previously contracted by phenylephrine under the action of acetylcholine by 36%. Pre-incubation of vessels with blockers reduced the amplitude of relaxation under the action of acetylcholine, compared with the initial acetylcholine-induced vasorelaxation, in HFD-group rats: with L-NAME – by 47%, L-NAME and indomethacin – by 50%, L-NAME, indomethacin and tetraethylammonium – by 65%; in the control group – by 69, 72 and 83%, respectively. HFD had no significant effect on the amplitude of vasodilation under the action of sodium nitroprusside. Thus, endothelial dysfunction in HFD-treated rats was mediated both by impairment of NO-dependent mechanisms of vasodilation, in particular, by a decrease in NO production by the endothelium, and by a decrease in the effectiveness of ВКСа. The decrease in NO bioavailability in HFD was partially compensated by the activation of endothelial hyperpolarization mechanisms (mediated by IKCa and SKCa activities) in acetylcholine-induced vasodilation.

Treatment with Resveratrol Reverses Priapism in the Sickle Cell Mouse

Introduction: Sickle cell disease (SCD) men display priapism. Townes and Berkeley SCD mice models are widely studied in basic sciences because they express 100% HbS in their circulating erythrocytes and exhibit features of priapism, and, as in SCD humans, this appears to be due to decreased nitric oxide (NO) signaling and increased oxidative stress, which leads to lower PDE5 in the penis. Thus, when an erectile stimulus occurs in vivo, cGMP accumulates into the cavernosal smooth muscle cells, rendering the penile vasculature uncontrollably dilated, and penile erection persists (i.e., priapism) as cGMP is not degraded as a consequence of PDE5 downregulation. Moreover, oxidative/nitrosative stress has been associated with priapism in SCD mice.Resveratrol (trans-3,5,4′-trihydroxystilbene) is a natural phytoalexin product with potent antioxidant activity found in peanuts, berries, and grapes. In this study, since decreased NO bioavailability and increased oxidative stress leads to lower PDE5 in the penis, we aimed to evaluate the effects of resveratrol on functional and molecular alterations of erectile function in SCD mice. We have focused on the dysregulated NO-cGMP-PDE5 pathway and oxidative/nitrosative stress markers (4-HNE and 3-nitrotyrosine) in the erectile tissue of SCD. Methods: Wild type (WT, C57BL/6) and Townes SCD transgenic male mice (3-5 mo old) were treated with resveratrol (100 mg /kg/day) or its vehicle daily for two weeks via gavage. Corpus cavernosum (CC) strips were mounted in isolated organ baths, and the relaxing responses to acetylcholine (ACh; endothelium-dependent response) and sodium nitroprusside (SNP; endothelium-independent response), as well as electrical-field stimulation (EFS; nitrergic relaxation), were obtained in CC strips precontracted with the α1-adrenergic receptor agonist phenylephrine (3-10 µM). Results: The cumulative addition of ACh (0.001-10 µM) produced concentration-dependent CC relaxations in WT and SCD groups, but the ACh potency (pEC50) value was higher (P<0.05) in CC of SCD compared to WT mice, which was reversed by resveratrol. Likewise, the nitrergic relaxation induced by EFS was also higher (P<0.05) in SCD mice compared to control mice (4 Hz: 30 ± 8 and 80 ± 5 %, respectively; n=5), which was reversed by resveratrol treatment. Similarly, SNP (0.01- 10 µM) produced concentration-dependent CC relaxations in WT and SCD groups, but, again, the maximal relaxations elicited by this agent were higher (P<0.05) in SCD (90 ± 1%) group compared to the WT mice (65 ± 6%), which were fully restored to WT values with resveratrol treatment (n=6). The mRNA expression for the PDE5 was reduced (P<0.05) by approximately 57% in penile tissues from SCD compared to the WT group, respectively. Resveratrol treatment restored (P<0.05) the mRNA expression of PDE5 in the penises from the SCD group (n=5-6). The protein expressions for 4-HNE and 3-nitrotyrosine (3-NT) were significantly higher (P<0.05) in erectile tissues from SCD compared to the WT group, which were reduced (P<0.05) by resveratrol treatment (n=6). In WT mice, PDE5, 4-HNE, and 3-NT protein expressions were not affected by resveratrol, as well as CC relaxations induced by ACh, SNP, and EFS. Conclusion: Resveratrol treatment reduced enhanced NO-mediated CC relaxations along with normalization of PDE5 expression and reduced oxidative stress markers expressions (3-NT and 4-HNE) in the penises from SCD. Therefore, resveratrol may constitute an additional strategy to prevent priapism in SCD. Financial Support: FAPESP.

Breast cancer chemotherapy induces vascular dysfunction and hypertension through a NOX4-dependent mechanism.

Cardiovascular disease is the major cause of morbidity and mortality in breast cancer survivors. Chemotherapy contributes to this risk. We aimed to define the mechanisms of long-term vascular dysfunction caused by neoadjuvant chemotherapy (NACT) and identify novel therapeutic targets. We studied arteries from postmenopausal women who had undergone breast cancer treatment using docetaxel, doxorubicin, and cyclophosphamide (NACT) and from women with no history of such treatment matched for key clinical parameters. We explored mechanisms in WT and Nox4–/– mice and in human microvascular endothelial cells. Endothelium-dependent, NO-mediated vasodilatation was severely impaired in patients after NACT, while endothelium-independent responses remained normal. This was mimicked by a 24-hour exposure of arteries to NACT agents ex vivo. When applied individually, only docetaxel impaired endothelial function in human vessels. Mechanistic studies showed that NACT increased inhibitory eNOS phosphorylation of threonine 495 in a Rho-associated protein kinase–dependent (ROCK-dependent) manner and augmented vascular superoxide and hydrogen peroxide production and NADPH oxidase activity. Docetaxel increased expression of the NADPH oxidase NOX4 in endothelial and smooth muscle cells and NOX2 in the endothelium. A NOX4 increase in human arteries may be mediated epigenetically by diminished DNA methylation of the NOX4 promoter. Docetaxel induced endothelial dysfunction and hypertension in mice, and these were prevented in Nox4–/– mice and by pharmacological inhibition of Nox4 or Rock. Commonly used chemotherapeutic agents and, in particular, docetaxel alter vascular function by promoting the inhibitory phosphorylation of eNOS and enhancing ROS production by NADPH oxidases.

Does the Endothelium of Competitive Athletes Benefit from Consumption of n-3 Polyunsaturated Fatty Acid-Enriched Hen Eggs?

The present study aimed to determine the effect of n-3 polyunsaturated fatty acid (PUFA)-enriched hen eggs on microvascular vasodilation, microvascular responsiveness to a stress challenge and markers of oxidative stress in competitive athletes. Competitive athletes (n=23) were divided to a control group (n=9), who consumed three regular hens’ eggs daily (249 mg n-3 PUFAs/d), and n-3 PUFAs group (n=14), who consumed three n-3 PUFA-enriched hen eggs daily (1,053 g n-3 PUFAs/d) for 3 weeks. Endothelium-dependent responses [post-occlusive reactive hyperemia (PORH) and acetylcholine-induced dilation (AChID)] and endothelium-independent responses [sodium nitroprusside-induced dilation (SNPID)] of skin microvascular blood flow were assessed by laser Doppler flowmetry in pre- and post-acute exhausting exercise (AEE) sessions. Blood pressure, serum lipid, free fatty acids profiles, and biomarkers of oxidative stress were measured before and after each dietary protocol. Consumption of serum n-3 PUFAs significantly decreased the n-6/n-3 ratio and enhanced PORH and AChID, but did not affect SNPID at rest. Furthermore, serum glutathione peroxidase and superoxide dismutase activities were significantly decreased in the n-3 PUFAs group but remained unchanged in the control group. In both groups, PORH, AChID, and SNP were significantly reduced post-AEE compared with pre-AEE, both before and after consumption of each diet. Only AChID responsiveness to AEE (ΔAChID) significantly increased following consumption of n-3 PUFAs. Overall, n-3 PUFAs supplementation as n-3 PUFA-enriched hen eggs enhanced microvascular endothelial function at rest and may contribute to adaptation to AEE in competitive athletes.

Abstract 9356: The Effect of Intraoperative Oxygenation on Vascular Reactivity in Human Resistance Arterioles

Introduction: Hyperoxia during surgery is common and may promote oxidative stress. Oxidative stress uncouples endothelial nitric oxide synthase and may impair vascular reactivity, a key regulator of tissue perfusion. Hypothesis: Hyperoxia during cardiac surgery impairs vascular reactivity compared to normoxia. Methods: We isolated resistance-level arterioles from mediastinal fat collected from subjects in a clinical trial of hyperoxia versus normoxia during cardiac surgery. Tissue was obtained at the end of surgery, and arterioles from 24 subjects were mounted on a wire myograph. After norepinephrine contraction, we measured the endothelium-dependent response to acetylcholine and the endothelium-independent response to sodium nitroprusside. To further identify if hyperoxygenation impairs nitric oxide mediated vasodilation via oxidation of the soluble guanylyl cyclase heme moiety, we measured the response to the heme-independent soluble guanylyl cyclase activator cinaciguat. We compared relaxation to increasing doses of each vasodilator in normoxia vs. hyperoxia treated arterioles using repeated measures ANOVA and nonlinear mixed effects regression. Results: The acetylcholine response was uniformly impaired (P=0.98, Figure ). The sodium nitroprusside response was selectively impaired in hyperoxia treatment vs. normoxia as reflected by a higher EC50 (EC50: 50.1±6.3 x10-8 M for hyperoxia vs. 7.9 ± 0.8 x 10-8 M for normoxia, P=0.02) and decreased peak response. In striking contrast to the response to sodium nitroprusside, vasodilation evoked by cinaciguat was not impaired and was slightly enhanced for hyperoxia vs. normoxia (EC50: 7.9±0.8 x10-8 M vs. 15.8±6.2 x10-8 M; P=0.07, Figure). Conclusions: Intraoperative hyperoxia impaired human arteriole vasodilation likely via oxidation of the soluble guanylyl cyclase heme moiety. These data provide additional mechanistic insight into potential harmful effects of intraoperative hyperoxia.

Lower endothelium-dependent microvascular function in adult breast cancer patients receiving radiation therapy

PurposeCancer patients with a history of radiotherapy are at an increased risk of ischemic heart disease. Preclinical animal studies demonstrate markedly impaired acetylcholine (ACh)-mediated endothelium-dependent vasorelaxation within days to weeks post-irradiation, however, whether microvascular function is affected in the intact human circulation during cancer radiation therapy has yet to be determined.Materials and methodsUsing laser-Doppler flowmetry, microvascular endothelium-dependent and independent responses were evaluated through iontophoresis of acetylcholine (ACh) (part 1, n = 7) and sodium nitroprusside (SNP) (part 2, n = 8), respectively, in women currently receiving unilateral chest adjuvant radiation therapy for breast cancer. Measurements were performed at the site of radiation treatment and at a contralateral control, non-radiated site. Cutaneous vascular conductance (CVC) was calculated by normalizing for mean arterial pressure.Results and ConculsionsIn part 1, patients received an average radiation dose of 2104 ± 236 cGy. A significantly lower peak ACh-mediated endothelium-dependent vasodilation was observed within the radiated microvasculature when compared to non-radiated (radiated: 532 ± 167%, non-radiated 1029 ± 263%; P = 0.02). In part 2, the average radiation dose received was 2251 ± 196 cGy. Iontophoresis of SNP elicited a similar peak endothelium-independent vasodilator response in radiated and non-radiated tissue (radiated: 179 ± 58%, non-radiated: 310 ± 158; P = 0.2). The time to 50% of the peak response for ACh and SNP was similar between radiated and non-radiated microvasculature (P < 0.05). These data provide evidence of early endothelium-dependent microvascular dysfunction in cancer patients currently receiving chest radiation and provide the scientific premise for future work evaluating coronary endothelial function and vasomotor reactivity using more detailed and invasive procedures.

Standardization of Kaempferia galanga L. rhizome and vasorelaxation effect of its key metabolite ethyl p-methoxycinnamate

Ethnopharmacological relevanceKaempferia galanga L. rhizome (KGR) is part of more than sixty-one Ayurvedic formulations and commonly known as ‘Chandramula’. KGR is widely used in traditional Indian medicines to treat fever (jwar), rheumatism (Amavata), respiratory (Shwasa), hypertension (Vyanabala vaishamya) and cardiovascular disorders (Vyanavayu Dushtijanya Hrudrog). Although ethnomedicinal properties have extensively been demonstrated in traditional medicines of south-east countries i.e. China, India, Indonesia, and Malaysia, the chemico-biological validation are still lacking. Aim of the studyChemico-biological standardization with respect to its vasorelaxation potential is the main objective of the present study. To investigate the vasorelaxation potential of key phytochemical of KGR, i.e., ethyl-p-methoxycinnamate (EPMC) and to study it's the mechanism of action. Materials and methodsA HPLC method was developed and validated for the quality assessment of KGR using its two major phytochemicals i.e. ethyl-p-methoxycinnamate (EPMC) and ethyl cinnamate (EC) in KGR. The vasorelaxation effect of major phytochemicals of KGR was evaluated on the main mesenteric arteries isolated from male Wistar rats. Specific BKca channel blocker tetraethylammonium (TEA), receptor antagonist, nitric oxide scavenging capacity, and antioxidant potential were also evaluated for its plausible mechanism. ResultsPresent validated HPLC method facilitates simultaneous quantitation of EPMC and EC faster than classical GC techniques. EPMC has shown a dose-dependent relaxation in rat main mesenteric arteries (MMA) contracted by U46619 with an Emax of 58.68 ± 3.31%. Similarly, in endothelium-denuded MMA rings, relaxation was also observed (Emax of 61.83 ± 3.38%). Moreover, relaxation response to EPMC has strongly inhibited (Emax 14.76 ± 2.29%) when the tissue exposed to depolarizing high K+ containing buffer for the contraction. The point correlation dimension (pD2) values were also significantly decreased in high K+ treated arterial rings compared to control. Interestingly, when MMA rings incubated with a specific BKca channel blocker (TEA, 1 mM), the relaxation response to EPMC was also significantly blocked. ConclusionsThe first time this study demonstrated the chemical standardization of K. galanga rhizome and EPMC is responsible for its vasorelaxation potential as demonstrated by the endothelium-independent response mediated by Ca2+ dependent potassium channels.

Vascular Ageing Features Caused by Selective DNA Damage in Smooth Muscle Cell.

Persistently unrepaired DNA damage has been identified as a causative factor for vascular ageing. We have previously shown that a defect in the function or expression of the DNA repair endonuclease ERCC1 (excision repair cross complement 1) in mice leads to accelerated, nonatherosclerotic ageing of the vascular system from as early as 8 weeks after birth. Removal of ERCC1 from endothelial alone partly explains this ageing, as shown in endothelial-specific Ercc1 knockout mice. In this study, we determined vascular ageing due to DNA damage in vascular smooth muscle cells, as achieved by smooth muscle-selective genetic removal of ERCC1 DNA repair in mice (SMC-KO: SM22αCre+ Ercc1fl/-). Vascular ageing features in SMC-KO and their wild-type littermates (WT: SM22αCre+ Ercc1fl/+) were examined at the age of 14 weeks and 25 weeks. Both SMC-KO and WT mice were normotensive. Compared to WT, SMC-KO showed a reduced heart rate, fractional shortening, and cardiac output. SMC-KO showed progressive features of nonatherosclerotic vascular ageing as they aged from 14 to 25 weeks. Decreased subcutaneous microvascular dilatation and increased carotid artery stiffness were observed. Vasodilator responses measured in aortic rings in organ baths showed decreased endothelium-dependent and endothelium-independent responses, mostly due to decreased NO-cGMP signaling. NADPH oxidase 2 and phosphodiesterase 1 inhibition improved dilations. SMC-KO mice showed elevated levels of various cytokines that indicate a balance shift in pro- and anti-inflammatory pathways. In conclusion, SMC-KO mice showed a progressive vascular ageing phenotype in resistant and conduit arteries that is associated with cardiac remodeling and contractile dysfunction. The changes induced by DNA damage might be limited to VSMC but eventually affect EC-mediated responses. The fact that NADPH oxidase 2 as wells as phosphodiesterase 1 inhibition restores vasodilation suggests that both decreased NO bioavailability and cGMP degradation play a role in local vascular smooth muscle cell ageing induced by DNA damage.

Endothelium-independent vasorelaxant effect of Asphodelus tenuifolius Cav. via inhibition of myosin light chain kinase activity in the porcine coronary artery

Ethnopharmacological relevanceAsphodelus tenuifolius Cav. (Asphodelaceae), a wild, terrestrial, annual stemless herb, is widely used in traditional medicine for the treatment of hypertension, diabetes, atherosclerosis and circulatory problems. A previous research study from our laboratory revealed that A. tenuifolius has beneficial effects in reducing blood pressure and improves aortic endothelial dysfunction in chronically glucose fed rats. Despite the fact that A. tenuifolius reduces blood pressure and improves endothelial function in vivo, there are no detailed studies about its possible mechanism of action. Aim of the studyThis study was designed to provide pharmacological basis and mechanism of action for the traditional use of A. tenuifolius in hypertension and circulatory problems. We explored the vasorelaxant effect of A. tenuifolius and its underlying vasorelaxation mechanism in porcine coronary artery rings. Materials and methodsAqueous methanolic crude extract of A. tenuifolius was prepared by maceration process and then activity guided fractionation was carried out by using different polarity based solvents. Phytochemical studies were carried out using LC-DAD-MS. Segments of porcine distal coronary artery were set up in a wire myograph for isometric force measurements. Extract/fractions of A. tenuifolius seeds were tested for vasodilator activity by measurement of changes in tone after pre-contraction with the thromboxane mimetic U46619 in the presence or absence of inhibitors of intracellular signaling cascades. ResultsCrude extract/fractions of A. tenuifolius produced dose dependent endothelium independent vasorelaxant response in coronary rings, whereas, the butanol fraction of A. tenuifolius (BS-AT) produced the largest relaxation response with 100% relaxation at 1 mg/ml, therefore the mechanism of relaxation of this fraction was determined. The relaxation to BS-AT was unaffected by removal of the endothelium, pre-contraction with KCl, or the presence of the non-selective potassium channel blocker tetraethylammonium, indicating that the relaxation was endothelium-independent, and does not involve activation of potassium channels. BS-AT (1 mg/ml) inhibited the contractile response to calcium,the L-type calcium channel activator BAY K8664,and ionomycin, indicating that it inhibits calcium-induced contractions. The relaxation response to BS-AT was attenuated in the absence of extracellular calcium. However, relaxations to BS-AT were also reduced after deletion of calcium from intracellular stores with cyclopiazonic acid. Incubation with 1 mg/ml BS-AT also inhibited phosphorylation of myosin light chains in homogenates of coronary artery. ConclusionThe butanol extract of Asphodelus tenuifolius produces a large endothelium-independent relaxation of the porcine coronary artery through inhibition of calcium-induced contractions. The effect appears to be downstream of calcium influx, possibly through inhibition of myosin light chain kinase. This study supports previous studies demonstrating that A. tenuifolius reduces blood pressure. Future studies will aim to determine the active compounds underlying this response.