Relaxation Of Arteries Research Articles

The effects of Xiaotan Huayu Liqiao formula on mesenteric artery function in rats exposed to chronic intermittent hypoxia

To investigate the effects of Xiaotan Huayu Liqiao formula (the Chinese Medicine) on mesenteric artery function in rats exposed to chronic intermittent hypoxia (CIH), and to explore the related mechanism. Forty-eight male SD rats were randomly divided into four groups as Normoxia, CIH, Formula+CIH and formula group. Rats were exposed to normoxia in the Normoxia and Formula group, or intermittent hypoxia in CIH or Formula+CIH group. Xiaotan Huayu Liqiao formula was given at 24g/kg by intragastric administration before intermittent hypoxia exposure. The pathological changes of mesenteric artery were determined by HE staining, and the relaxation of mesenteric artery (induced by acetylcholine(ACh) and L-arginine(L-Arg)) was recorded by microvascular ring technique. Serums of all rats were collected (0 d and 21 d) and the content of NO was detected by ELISA. The levels of endothelial nitric oxide synthase (eNOS) and p-eNOS were measured by Western blot. Compared with Normoxia group, the mesenteric arterial endothelial injury and media thickening were observed and the relaxation of mesenteric artery was significantly reduced in rats exposed to CIH. The level of NO in serum and the ratio of p-eNOS/eNOS were also decreased in the CIH group. Xiaotan Huayu Liqiao formula administration improved the pathologic changes and dilatation function of mesenteric artery, increased the levels of NO and p-eNOS. Compared with Normoxia group,all the results were not observed significant difference in Formula group. Xiaotan Huayu Liqiao formula increased the bioavailability of NO, and ameliorated the CIH induced mesenteric artery function injury.

Analisis Senyawa L-arginine pada Bawang Putih Tunggal dan Black Garlic Tunggal Dengan Menggunakan Metode HPLC (High Performance Liguid Chromatography)

L-Arginine is a semi essential amino acid and has a function in arterial vascular relaxation and improves blood flow. Single-bulb black garlic was produced from ordinary single-bulb garlic clove by process from maillard browning at temperature at 700C and 95Y of humidity controlled room for a month. It is known that single-bulb black garlic has benefits as antidiabetes, uric acid, cholesterol, hypertension, endurance and others. This study aims to determine the content of L-Arginine compound on single-bulb garlic and single-bulb black garlic by using HPLC (High Performance Liguid Chromatography)method.The mobile phase used consists of buffer A and buffer B. Buffer A consists of Na-Acetate (pH 6.5), Na-EDTA, Methanol, and Tetrahidrofuran. While B buffer consists of Methanol and Water Pro Injection. Thermo Scientific Column ODS-2 Hyersil C18, flow rate 1 ml / min, using Flouresensi detector with 350 nm wavelength. Based on the results obtained L-Arginine compound contained in a single-bulb garlic of 2.504 while for single-bulb black garlic that is egual to 06704. The content of L-Arginine compounds in single garlic is greater than that of a single black garlic. Normal 0 false false false IN X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Table Normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0cm; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Cambria,serif; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Times New Roman; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin;} L-Arginine is a semi essential amino acid and has a function in arterial vascular relaxation and improves blood flow. Single-bulb black garlic was produced from ordinary single-bulb garlic clove by process from maillard browning at temperature at 700C and 95Y of humidity controlled room for a month. It is known that single-bulb black garlic has benefits as antidiabetes, uric acid, cholesterol, hypertension, endurance and others. This study aims to determine the content of L-Arginine compound on single-bulb garlic and single-bulb black garlic by using HPLC (High Performance Liguid Chromatography)method.The mobile phase used consists of buffer A and buffer B. Buffer A consists of Na-Acetate (pH 6.5), Na-EDTA, Methanol, and Tetrahidrofuran. While B buffer consists of Methanol and Water Pro Injection. Thermo Scientific Column ODS-2 Hyersil C18, flow rate 1 ml / min, using Flouresensi detector with 350 nm wavelength. Based on the results obtained L-Arginine compound contained in a single-bulb garlic of 2.504 while for single-bulb black garlic that is egual to 06704. The content of L-Arginine compounds in single garlic is greater than that of a single black garlic. Normal 0 false false false IN X-NONE X-NONE

Endothelial PPARγ (Peroxisome Proliferator-Activated Receptor-γ) Protects From Angiotensin II-Induced Endothelial Dysfunction in Adult Offspring Born From Pregnancies Complicated by Hypertension.

Preeclampsia is a hypertensive disorder of pregnancy associated with vascular dysfunction and cardiovascular risk to offspring. We hypothesize that endothelial PPARγ (peroxisome proliferator-activated receptor-γ) provides cardiovascular protection in offspring from pregnancies complicated by hypertension. C57BL/6J dams were bred with E-V290M sires, which express a dominant-negative allele of PPARγ selectively in the endothelium. Arginine vasopressin was infused throughout gestation. Vasopressin elevated maternal blood pressure at gestational day 14 to 15 and urinary protein at day 17 consistent. Systolic blood pressure and vasodilation responses to acetylcholine were similar in vasopressin-exposed offspring compared to offspring from control pregnancies. We treated offspring with a subpressor dose of angiotensin II to test if hypertension during pregnancy predisposes offspring to hypertension. Male and female angiotensin II-treated E-V290M offspring from vasopressin-exposed but not control pregnancy exhibited significant impairment in acetylcholine-induced relaxation in carotid artery. Endothelial dysfunction in angiotensin II-treated E-V290M vasopressin-exposed offspring was attenuated by tempol, an effect which was more prominent in male offspring. Nrf2 (nuclear factor-E2-related factor) protein levels were significantly elevated in aorta from male E-V290M offspring, but not female offspring compared to controls. Blockade of ROCK (Rho-kinase) signaling and incubation with a ROCK2-specific inhibitor improved endothelial function in both male and female E-V290M offspring from vasopressin-exposed pregnancy. Our data suggest that interference with endothelial PPARγ in offspring from vasopressin-exposed pregnancies increases the risk for endothelial dysfunction on exposure to a cardiovascular stressor in adulthood. This implies that endothelial PPARγ provides protection to cardiovascular stressors in offspring of a pregnancy complicated by hypertension and perhaps in preeclampsia.

Moringa oleifera Seeds Improve Aging-Related Endothelial Dysfunction in Wistar Rats

Vascular aging is characterized by functional and structural changes of the vessel wall, including endothelial dysfunction, with decreased endothelial NO· bioavailability and elevated vasoconstrictor and inflammatory mediator production, vascular rigidity, and tone impairment. Moringa oleifera (MOI) is a little tree, and different parts of which are used in traditional medicine in tropical Africa, America, and Asia for therapeutic applications in several disorders including cardiovascular disease. The present study is aimed at assessing the effect of MOI on aging-associated alteration of the endothelial function in Wistar rats. Middle-aged Wistar rats (46-week-old males) have been fed with food containing or not 750 mg/kg/day of MOI seed powder for 4 weeks. A group of young Wistar rats (16-week-old) was used as control. Measurement of isometric contraction, western blot analysis, and immunostaining has then been performed in the aortas and mesenteric arteries to assess the endothelium function. MOI treatment improved carbachol-induced relaxation in both aortas and mesenteric arteries of middle-aged rats. In the aortas, this was associated with an increased Akt signalling and endothelial NO synthase activation and a downregulation of arginase-1. In the mesenteric arteries, the improvement of the endothelial-dependent relaxation was related to an EDHF-dependent mechanism. These results suggest a vascular protective effect of MOI seeds against the vascular dysfunction that develops during aging through different mechanisms in conductance and resistance arteries.

Heterocellular Contact Can Dictate Arterial Function.

Resistance arteries and conduit arteries rely on different relative contributions of endothelial-derived hyperpolarization versus nitric oxide to achieve dilatory heterocellular signaling. Anatomically, resistance arteries use myoendothelial junctions (MEJs), endothelial cell projections that make contact with smooth muscle cells. Conduit arteries have very few to no MEJs. Determine if the presence of MEJs in conduit arteries can alter heterocellular signaling. We previously demonstrated that PAI-1 (plasminogen activator inhibitor-1) can regulate formation of MEJs. Thus, we applied pluronic gel containing PAI-1 directly to conduit arteries (carotid arteries) to determine if this could induce formation of MEJs. We found a significant increase in endothelial cell projections resembling MEJs that correlated with increased biocytin dye transfer from endothelial cells to smooth muscle cells. Next, we used pressure myography to investigate whether these structural changes were accompanied by a functional change in vasodilatory signaling. Interestingly, PAI-1-treated carotids underwent a switch from a conduit to resistance artery vasodilatory profile via diminished nitric oxide signaling and increased endothelial-derived hyperpolarization signaling in response to the endothelium-dependent agonists acetylcholine and NS309. After PAI-1 application, we also found a significant increase in carotid expression of endothelial alpha globin, a protein predominantly expressed in resistance arteries. Carotids from mice with PAI-1, but lacking alpha globin (Hba1-/-), demonstrated that l-nitro-arginine methyl ester, an inhibitor of nitric oxide signaling, was able to prevent arterial relaxation. The presence or absence of MEJs is an important determinant for influencing heterocellular communication in the arterial wall. In particular, alpha globin expression, induced within newly formed endothelial cell projections, may influence the balance between endothelial-derived hyperpolarization and nitric oxide-mediated vasodilation.

Downregulation of endothelial transient receptor potential vanilloid type 4 channel underlines impaired endothelial nitric oxide-mediated relaxation in the mesenteric arteries of hypertensive rats.

The endothelium contributes to the maintenance of vasodilator tone by releasing endothelium-derived relaxing factors, including nitric oxide (NO). In hypertension, endothelial nitric oxide synthase (eNOS) produces less NO and could be one of the contributing factors to the increased peripheral vascular resistance. Agonist-induced Ca(2+) entry is essential for the activation of eNOS. The transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca(2+)-permeant cation channel, is expressed in the endothelial cells and involved in the regulation of vascular tone. The present study aimed to investigate the role of TRPV4 channel in endothelium-dependent NO-mediated relaxation of the resistance artery in hypertensive rats. Using a wire myograph, relaxation response to the TRPV4 activator, 4alpha-phorbol-12,13-didecanoate (4alphaPDD) was assessed in mesenteric arteries obtained from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs). Compared to WKY, SHR demonstrated a significantly attenuated 4alphaPDD-induced endothelium-dependent NO-mediated relaxation. Immunohistochemical analysis revealed positive staining for TRPV4 in the endothelium of mesenteric artery sections in both WKY and SHR. Furthermore, TRPV4 mRNA and protein expressions in SHR were significantly lower than their expression levels in WKY rats. We conclude that 4alphaPDD-induced endothelium-dependent NO-mediated vasorelaxation is reduced in SHR and downregulation of TRPV4 could be one of the contributing mechanisms.

Sulodexide promotes arterial relaxation via endothelium-dependent nitric oxide-mediated pathway

Sulodexide (SDX) is a highly purified glycosaminoglycan with antithrombotic and profibrinolytic properties and reported benefits in thrombotic and atherosclerotic vascular disorders. However, the effects of SDX on vascular function are unclear. We tested whether SDX affects vascular relaxation and examined the potential underlying mechanisms. Isolated segments of male rat abdominal aorta and mesenteric artery were suspended in a tissue bath, and the changes in arterial contraction/relaxation were measured. The α-adrenergic receptor agonist phenylephrine (Phe) (10−9–10−5 M) caused concentration-dependent aortic and mesenteric artery contraction that was reduced in tissues pretreated with SDX (1 mg/ml). In aortic and mesenteric arterial segments precontracted with submaximal concentration of Phe (3 × 10−7–6 × 10−7 M), SDX (0.001–1 mg/ml) caused concentration-dependent relaxation. To test the role of endothelium, SDX-induced relaxation was compared with that of acetylcholine (ACh), a known activator of endothelium-dependent relaxation. In Phe precontracted aorta, ACh relaxation was abolished and SDX relaxation was significantly inhibited by endothelium removal or the nitric oxide synthase (NOS) inhibitor Nω-nitro-l-arginine methyl ester (L-NAME), suggesting a role of NO. In mesenteric artery, ACh relaxation was abolished by endothelium removal, partially blocked by L-NAME, and completely blocked by a mixture of indomethacin, a cyclooxygenase inhibitor and blocker of the PGI2-cAMP pathway, and tetraethylammonium, a blocker of K+ channels and EDHF-dependent hyperpolarization pathway. In comparison, SDX relaxation of mesenteric artery was almost completely inhibited by endothelium removal or NOS inhibitor L-NAME. SDX enhanced vascular relaxation and increased nitrate/nitrite production in response to all ACh concentrations in the aorta, but only to low ACh concentrations (<10−7 M) in mesenteric artery. SDX did not affect aortic or mesenteric artery endothelium-independent relaxation to the NO donor sodium nitroprusside. Thus, SDX promotes arterial relaxation via a mechanism involving endothelium-dependent NO production; an effect that could enhance vasodilation and decrease vasoconstriction in vascular disorders.

Responsiveness of rat aorta and pulmonary artery to cGMP generators in the presence of thiol or heme oxidant

This study investigated the effects of thiol and heme oxidants on responsiveness to cGMP generators in isolated rat aorta and pulmonary artery using an organ chamber. The nitric oxide (NO) donor sodium nitroprusside (SNP)-induced relaxation was impaired by exposure to the thiol oxidant diamide in both the aorta and the pulmonary artery, whereas the soluble guanylate cyclase (sGC) stimulator BAY 41-2272- or the sGC activator BAY 60-2770-induced relaxation was not affected. The impairment by diamide of SNP-induced aortic and pulmonary arterial relaxation was completely restored by post-treatment with the thiol reductant dithiothreitol. However, regardless of the vessel type, the relaxant response to SNP or BAY 41-2272 was impaired by exposure to the heme oxidant ODQ, whereas the response to BAY 60-2770 was enhanced. The ODQ-induced effects were reversed partially by post-treatment with the heme reductant dithionite. These findings indicate that thiol oxidation attenuates only the vascular responsiveness to NO donors and that heme oxidation attenuates the responsiveness to NO donors and sGC stimulators but augments that to sGC activators. Therefore, under oxidative stress, the order of usability of the vasodilators is suggested to be: NO donors < sGC stimulators < sGC activators.

Endothelial‐Specific Interference with PPARγ Causes Endothelial Dysfunction with Sex‐ Specific Mechanisms in Offspring Born from AVP‐infused Pregnancies

Mutations in the ligand‐activated transcription factor PPARγ result in hypertension, and synthetic agonists of PPARγ reduce blood pressure. Previously we found that mice expressing dominant‐negative (DN) PPARγ driven by an endothelium‐specific promoter (E‐DN) exhibit vascular dysfunction. Preeclampsia (PE) is a hypertensive disorder of pregnancy and carries cardiovascular risk to offspring. PE is also associated with vascular dysfunction. Therefore, we hypothesized a role for endothelial PPARγ in the pathogenesis of PE and its sequelae. C57BL/6J dams were bred with E‐DN sires, and symptoms of PE were induced by infusion of vasopressin (AVP, 24 ng/hr sc) throughout gestation. Phenotypes of PE were first assessed in pregnant dams and then in adult offspring. Compared to saline infusion (SAL), AVP elevated maternal blood pressure (SBP: 116±3 vs 107±3, p&lt;0.05) at gestational day (GD) 14–15 and urine protein (70±6 vs 27±4 mg/mL, p&lt;0.05) at GD17. Offspring were phenotyped in adulthood. Data were stratified to sex, genotype, and exposure to AVP or SAL. Adult male and female offspring were exposed to a sub‐pressor dose of Angiotensin II (ANG; 120ng/kg/hr) for 14 days as a cardiovascular stressor. Male and female ANG‐treated E‐DN born to PE pregnancies, but not control pregnancies, exhibited significant impairment in ACh‐induced relaxation in carotid artery (% relaxation: Males − 56±9 vs 81±11, p&lt;0.05; Females − 68±10 vs 84±11, p&lt;0.05). Endothelial dysfunction in male ANG‐treated E‐DN born to PE pregnancies was attenuated by tempol (relaxation improved from 53±9% vs 84±12%, p&lt;0.05), indicative of a role for oxidative stress. Interestingly, tempol did not rescue endothelial dysfunction in female E‐DN exposed to PE (% relaxation: 69±10% vs 81±11%, ns). In keeping with this, Nrf2 protein levels was significantly elevated in aorta from male E‐DN (1.9±0.2 vs 0.2±0.1, p&lt;0.05), but not females (1.2±0.3 vs 0.6±0.2, ns) compared to genotype‐ and sex‐matched controls, suggesting that the mechanism of endothelial dysfunction might differ between sexes. Blockade of Rho‐kinase (ROCK) signaling with Y‐27632 improved endothelium‐dependent relaxation in male and female E‐DN offspring born from PE pregnancies (% relaxation: Males − 60±10 vs 87±12, p&lt;0.05; Females − 69±10 vs 98±13, p&lt;0.05). We observed a significant 4‐fold increase in ROCK2 protein levels in aorta from both male and female E‐DN mice exposed to PE (Males – 2.3±0.5 vs 0.6±0.4, p&lt;0.05; Females − 1.9±0.5 vs 0.5±0.2, p&lt;0.05). No changes were observed in ROCK1 expression. Further, incubation with a ROCK2 specific inhibitor, SLX‐2119, significantly improved relaxation in male and female E‐DN (Males – relaxation improved from 72±11 vs 91±10, p&lt;0.05; Females – relaxation improved from 68±10 vs 94±12, p&lt;0.05). Our data suggests that interference with endothelial PPARγ in pups born from PE pregnancies increases the risk for endothelial dysfunction upon exposure to a cardiovascular stressor in adulthood. Moreover, potential mechanisms accounting for endothelial dysfunction in offspring born from complicated pregnancies might be sex‐dependent.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Estrogen Replacement on ACh‐Induced Relaxation in Mesenteric Arteries of Prediabetic Ovariectomized Rats

The risk of cardiovascular diseases (CVD) is lower in premenopausal women than in men, and this lower risk is related to the presumptive cardioprotective effects of female sex hormones, estrogen in particular. However, premenopausal women with diabetes not only lose this protection, they also experience a higher relative risk of CVD compared to diabetic men. Several lines of evidence suggest that endothelial dysfunction represents early steps in the development of vascular complications in diabetes, and hyperglycemia is the central initiating factor for those complications. Nonetheless, there is insufficient evidence to establish the timeline of the loss of this female‐specific protection in premenopausal patients with diabetes. The objective of this study was to investigate the effects of 17β‐estradiol (E2) on mesenteric arterial function in UC Davis type 2 diabetes mellitus (UCD‐T2DM) rats at the prediabetic stage.Mesenteric arteries (MA) were obtained from four groups of ovariectomized (ovex) female rats implanted with a subcutaneous pellet for 45 days: (1) Control, ovex + placebo, (2) Control, ovex + E2 (1.5mg/pellet), (3) prediabetic, ovex + placebo, (4) prediabetic, ovex + E2 (1.5mg/pellet). Endothelium‐dependent vasodilation (EDV) to acetylcholine (ACh, 10−8 to 10−5 M) in phenylephrine pre‐contracted mesenteric arterial rings were measured before and after indomethacin (Indo, 10μm), a cyclooxygenase (COX) inhibitor, followed by addition of L‐NAME (200μM), a nitric oxide synthase (NOS) inhibitor. When Indo and L‐NAME are administrated, the remaining EDV to ACh is referred to as the endothelium‐dependent hyperpolarization factor (EDHF)‐type relaxation.There were no significant differences in ACh‐ induced relaxation between placebo and E2 treated controls. However, the ACh responses in MA was markedly reduced in prediabetic treated with placebo. Treatment of prediabetic with E2 enhanced the ACh‐induced relaxation compared with its respective placebo group. Blocking of both COX and NOS completely abolished the relaxation responses to ACh in prediabetic, regardless of E2 replacement, suggesting no contribution of EDHF‐type relaxation in this group. The E2 replacement, however, increased the contribution of NO‐mediated vasodilation in MA of prediabetic rats. When compared with prediabetic groups, the effect of L‐NAME was much smaller in controls, specifically after E2 replacement, indicating an elevated contribution of EDHF‐type relaxation in E2‐treated control.These data show that EDV was impaired in ovex rats at prediabetic stage, possibly due to loss of EDHF. However, an intriguing observation of this study was that, at the pre‐diabetic stage, estrogen treated ovex rats exhibited intact EDV, regardless of loss of EDHF. This may be due to elevated contribution of NO to EDV in this group. Additional studies are required to establish the role of estrogen replacement in diabetic stages.Support or Funding InformationSupported by NHLBIThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sodium nitroprusside encapsulated in Silica nanoparticles as a strategy for safer nitric oxide delivery

Sodium nitroprusside (SNP) has been used as a nitric oxide (NO) donor in medicine for several decades, even before any knowledge of the biological role of NO. This medicine is used in cases of emergency of hypertension due to its fast release of NO causing fast drop in blood pressure. Unfortunately, this compound also releases cyanide (CN-) as a side product. Nanoencapsulated nitroprusside was synthesized by using silica nanoparticles functionalized with primary amine (MPSi-NH2) (the final compound was named APTES). The compound was bioassayed using vascular tissues from Wistar rats, albine guinea-pigs and New Zealand rabbits. All the protocols were approved by the Ethic committee of the Ceará State University under the protocol # 2897836/15. The thoracic aorta, pulmonary artery or corpora cavernosa were carefully removed and mounted in a 5 mL organ bath containing Krebs–Henseleit solution at 37°C, gassed with 95% O2: 5% CO2, attached to isometric transducers coupled to a powerlab data acquisition system. After equilibration, the tissues were precontracted with phenylephrine (PE, 10−6–10−5 M) and after a plateau was achieved increasing concentrations of SNP or APTES (10−10 to 10−5 M) were cumulatively added in intervals of 5 minutes. The relaxant effect was measured as percentage of the maximal contraction induced by phenylephrine. In addition, we also measured the level of phosphorylation of Ser239 of VASP, a useful marker for monitoring cGMP-dependent protein kinase activation and signaling. SNP APTEs relaxed rat aortic rings with similar potency than non-modified SNP with a concentration necessary to induce 50% vasodilation (CE50) of 26 nM [95% confidence interval=20–33 nM] compared to 14 nM [95% confidence interval=7,9–21 nM] for non-modified SNP. However, the maximal vasodilation induced by SNP APTEs (177.4±7.1%) was higher than the maximal relaxation induced by nonmodified SNP (143.8±9.6%). When comparing different vascular beds, i.e., guinea pig thoracic aorta vs. pulmonary arteries vs. rabbit corpora cavernosa, SNP APTEs was shown to have similar potency and efficacy (119.6±5.7% and 130nM vs. 108±6.3% and 275nM vs. 116±4.5% and 98 nM, respectively). Interestingly, SNP increase VASP phosphorylation by 3.8-fold and APTES increased by 4.13-fold (P<0.05 vs. control). Our synthetic strategy to make functionalized silica nanoparticles showed quite efficient to incorporate sodium nitroprusside, where we succeeded to have up to 320 umol g−1. Remarkably, we noticed a minor release of cyanide or even NP, despite full effect of NO was observed during a series of biological assays. Indeed, EC50 and maximal response for relaxation in aorta, pulmonary arteries and Rabbit corpora cavernosa were quite similar. Support or Funding Information CAPES, CNPq and FUNCAP Vasorelaxant Effect of Sodium nitroprusside encapsulated in Silica nanoparticles Original tracings depicting the strong and potent vascular relaxant effect of SNP APTES (panel A). Effect of SNP APTES on the phenylephrine precontracted aortic rings compared with non-modified sodium nitroprusside (Panel B). Panel C depicts the Effect of 1 μM SNP or SNP APTES compared with the addition of vehicle only in the level of phosphorylation of the residues Serine 239 of the protein Vasodilator-stimulated phosphoprotein (VASP) a substrate of activated cGMP-activated protein kinase (PKG) *p<0.05 vs SNP at the same concentration This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

EFFECTS OF RADIATION ON CUTANEOUS MICROVASCULAR FUNCTION IN THE INTACT HUMAN CIRCULATION

Radiation is a commonly used treatment for various types of cancer, however it is often associated with an increased risk of cardiovascular disease (CVD). Previous animal studies have shown a significant decreased relaxation in irradiated arteries in response to Acetylcholine. However, there is a paucity of knowledge on the effects of radiation within the intact human circulation. Given that changes vascular function occurs early in the progression of numerous cardiovascular and metabolic diseases characterization of the microvascular responses to radiation treatment is critical. Therefore, the primary was to determine the effects of cancer treatment radiation on cutaneous microvascular reactivity in breast cancer patients.METHODSThe present study utilized a longitudinal study design in breast cancer patients receiving radiation therapy. Cutaneous microvascular reactivity to Acetylcholine (ACh) was evaluated following radiation exposure in breast cancer patients at the site of radiation treatment and at a control non‐irradiated site. Briefly, red blood cell flux was measured as an index of cutaneous blood flow via laser Doppler flowmetry with ACh mediated vasodilation determined by iontophoresis drug delivery. Data are expressed as cutaneous vascular conductance.RESULTSTo date, 4 patients have completed the study (planned enrollment of 15 patients). Cutaneous vascular conductance to ACh was significantly attenuated following a total cumulative dose of 1950 ± 719 cGy. Similarly, the total ACh response was decreased in the radiated tissue. The forth coming patients will provide additional insight into the effects of radiation exposure on microvascular reactivity. Furthermore, we will be able to provide insight into the role coexisting risk factors of metabolic syndrome and preexisting atherosclerosis on radiation‐induced vascular dysfunction.CONCLUSIONThis study demonstrates that low doses of radiation for cancer treatment attenuate cutaneous microvascular function, which may have long‐term implications for cardiovascular health in these patients.Support or Funding InformationN/AThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

D. russelii Venom Mediates Vasodilatation of Resistance Like Arteries via Activation of Kv and KCa Channels.

Russell’s viper (Daboia russelii) venom causes a range of clinical effects in humans. Hypotension is an uncommon but severe complication of Russell’s viper envenoming. The mechanism(s) responsible for this effect are unclear. In this study, we examined the cardiovascular effects of Sri Lankan D. russelii venom in anaesthetised rats and in isolated mesenteric arteries. D. russelii venom (100 μg/kg, i.v.) caused a 45 ± 8% decrease in blood pressure within 10 min of administration in anaesthetised (100 μg/kg ketamine/xylazine 10:1 ratio, i.p.) rats. Venom (1 ng/mL–1 μg/mL) caused concentration-dependent relaxation (EC50 = 145.4 ± 63.6 ng/mL, Rmax = 92 ± 2%) in U46619 pre-contracted rat small mesenteric arteries mounted in a myograph. Vasorelaxant potency of venom was unchanged in the presence of the nitric oxide synthase inhibitor, L-NAME (100 µM), or removal of the endothelium. In the presence of high K+ (30 mM), the vasorelaxant response to venom was abolished. Similarly, blocking voltage-dependent (Kv: 4-aminopryidine; 1000 µM) and Ca2+-activated (KCa: tetraethylammonium (TEA; 1000 µM); SKCa: apamin (0.1 µM); IKCa: TRAM-34 (1 µM); BKCa; iberiotoxin (0.1 µM)) K+ channels markedly attenuated venom-induced relaxation. Responses were unchanged in the presence of the ATP-sensitive K+ channel blocker glibenclamide (10 µM), or H1 receptor antagonist, mepyramine (0.1 µM). Venom-induced vasorelaxtion was also markedly decreased in the presence of the transient receptor potential cation channel subfamily V member 4 (TRPV4) antagonist, RN-1734 (10 µM). In conclusion, D. russelii-venom-induced hypotension in rodents may be due to activation of Kv and KCa channels, leading to vasorelaxation predominantly via an endothelium-independent mechanism. Further investigation is required to identify the toxin(s) responsible for this effect.

Testosterone Replacement Enhances Internal Pudendal Artery Relaxation to Reverse Erectile Dysfunction in a Rat Model of Androgen Deprivation Therapy

Introduction/ObjectivesAndrogen deprivation therapy (ADT) is frequently used to manage prostate cancer; however, erectile dysfunction (ED) and cardiovascular disease are common side effects. After cessation of ADT, testosterone (T) levels may take years to recover leaving men prone to cardiovascular disease and long‐term ED. The internal pudendal arteries (IPA) supply blood to the penis and vascular injury to these vessels can lead to ED. This study will determine if castration impairs vascular function in systemic (aorta and mesenteric arteries) and penile vasculature (IPA), and if T can restore erectile and vascular physiology. We hypothesize the IPA will develop impaired relaxation prior to systemic arteries following castration, and T therapy will recover both vascular and erectile function.MethodsMale Sprague Dawley rats (12 wks) were divided into 3 groups (n=8/grp): control (CON), CAST (6 wks castration), and CAST+T (6 wks castration+T (3mg/kg) in last 2 wks). Erections were assessed via cavernous nerve stimulation and measurement of intracavernosal to mean arterial pressures (ICP/MAP). Aortas, mesenteric arteries, and IPA were excised, cut (2 mm) and mounted into tissue baths. Contractility to high concentration potassium solution (KCl), electrical field stimulation (EFS), and phenylephrine (PE) were measured. Endothelial dependent acetylcholine (ACh) relaxation and endothelial independent relaxation to DEA NONOate were assessed. Non‐adrenergic non‐cholinergic (NANC) relaxation was evaluated by EFS in the presence of guanethidine and atropine. Serum testosterone levels were measured via ELISA. Androgen receptor protein expression was measured in IPA.ResultsCAST markedly impaired erectile function (ICP/MAP; CON: 0.83±0.03, CAST: 0.31±0.02; p&lt;0.05) and decreased IPA ACh relaxation (CON: 50.4±3.5%, CAST: 31.2±2.3%; p&lt;0.05). DEA NONOate relaxation was unchanged with CAST. CAST diminished IPA NANC relaxation (CON: 40.8±5.1%, CAST: 22.1±3.9%; p&lt;0.05). However, CAST did not impact IPA contraction. Interestingly, CAST did not change vascular contraction or relaxation in aortas or mesenteric arteries. IPA androgen receptor expression decreased after CAST (CON: 1±0.17, CAST: 0.39±0.03; p&lt;0.05). T supplementation restored serum T (T (ng/ml); CON: 4.2±0.8, CAST: 0.1±0.02, CAST+T: 32.9±3.2: p&lt;0.05), erectile function (ICP/MAP; CAST+T 0.74±0.01 p&lt;0.05) and improved ACh relaxation in IPA to values greater than CON (CAST+T: 63.9±2.9%; p&lt;0.05). Similarly, CAST+T enhanced IPA NANC relaxation to levels greater than CON (CAST+T: 69.2±1.4%; p&lt;0.05). T supplementation did not impact IPA contractions and had no effect on aortic or mesenteric vasoreactivity.ConclusionsCastration induces severe IPA vascular damage, yet systemic vasculature is unaffected. T therapy dramatically enhances IPA endothelial dependent and NANC‐mediated relaxation. Though T therapy safety in cancer survivors remains controversial, a growing body of literature is finding no correlation between T therapy and cancer progression. In certain populations of prostate cancer survivors, T replacement is warranted.Support or Funding InformationBSOM start‐up grant, SMSNA, AUAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TRPM8 channel activation triggers relaxation of pudendal artery and corpus cavernosum with increased vascular sensitivity in spontaneously hypertensive rats: Is it a new target for erectile dysfunction?

Background and PurposeErectile dysfunction (ED) is frequently encountered in patients with arterial hypertension and there is a recently uncovered functional correlation between the expression of thermoreceptor channels TRPM8 (melastatin 8) and alterations in blood pressure. The aim of this study was to investigate the function of cold‐sensing TRPM8 channel in internal pudendal artery (IPA) in normotensive and hypertensive rats.Experimental ApproachWe performed experiments integrating physiological, pharmacological, biochemical, and cellular techniques.Key ResultsTRPM8 channels are expressed in the IPA and in vascular smooth muscle cells from IPA and corpus cavernosum (CC). In addition, TRPM8 activation, by both a cooling compound (icilin) and cold (thermal stimulus), induced relaxation in IPA and CC. In IPA, the concentration‐response curve to icilin was desensitized in different conditions, such as: the absence of vascular endothelium, the presence of L‐NAME (10−4 M) or indomethacin (10−5 M), a combination of charybdotoxin (10−7 M) and apamin (5×10−6 M), and Y27632 (10−6 M). Interestingly, icilin‐induced vasodilation was significantly higher in IPA from spontaneously hypertensive (SHR) compared to Wistar rats, despite no differences in the TRPM8 expression in IPA between the strains, suggesting that the sensitivity of TRPM8 channels is higher in SHR. In CC, the concentration‐response curve to icilin was shifted to the right in the presence of ROCK inhibitor Y27632, suggesting that ROCK, at least in part, is important to relaxation induced by TRPM8 activation. Supporting these functional data, we observed that the expression of RhoA and ROCK was not changed in CC treated with either vehicle, Phe, or Phe+icilin. However, MYPT1 phosphorylation was increased by Phe compared to control, and Phe+icilin reverted this effect, suggesting that TRPM8 activation inhibits RhoA/ROCK pathway in CC.ConclusionsThese data demonstrate, for the first time, the expression and function of TRPM8 channels in the IPA and CC involving, at least in part, endothelium‐derived relaxing factors and ROCK inhibition. Overall, this channel could potentially be a new target for the treatment of hypertension associated‐ED.Support or Funding InformationNational Council for Scientific and Technological Development (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ Brasil (CAPES); NIDDK Diacomp Pilot &amp; Feasibly Program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

β‐Hydroxybutyrate (βHOB) Increases Nitric Oxide Synthase Activity in Resistance Arteries from Dahl Salt‐sensitive Rats

The deleterious link between overconsumption of dietary salt and hypertension is unequivocal. This dogma has recently expanded with the report that a high salt diet lowers the circulating levels of the ketone metabolite, beta‐hydroxybutyrate (βHOB), in Dahl salt‐sensitive rats (Dahl S‐HS). While stimulators of ketogenesis are well known for improving cardiovascular health (e.g., exercise and caloric restriction), less is known about negative‐regulation of ketone body metabolism and how it may impact hypertension and vascular aging. Therefore, we hypothesized that raising the circulating levels of βHOB would improve endothelium‐dependent relaxation in mesenteric resistance arteries (MRA) of Dahl S‐HS. To test this hypothesis, 1,3‐butanediol (20% v/v), which is a precursor of βHOB, was supplemented in the drinking water of 5 week old, male Dahl S‐HS for 8 weeks. Subsequently MRA function was analyzed using wire myographs. Although relaxation to endothelium‐dependent vasodilator acetylcholine (ACh) was not different [%Relaxation, Dahl S‐low salt (LS): 40±11 vs. Dahl S‐HS: 52±11 vs. Dahl S‐HS+βHOB: 44±11, p&gt;0.05], the mechanism of relaxation was different between the groups. Specifically, while nitric oxide synthase inhibitor (L‐NAME; 100 μM) completely prevented relaxation in Dahl S‐LS and Dahl S‐HS+βHOB groups, MRA from Dahl S‐HS still partially relaxed in the presence of L‐NAME (%Relaxation, Dahl S‐LS: 1±4 vs. Dahl S‐HS: 33±5 vs. Dahl S‐HS+βHOB: 6±4, p&lt;0.05; Figure 1A–C). These data suggest that MRA from Dahl S‐HS relax through nitric oxide‐independent mechanisms and that βHOB treatment restores nitric oxide synthase activity, despite consumption of a high salt diet. To test whether 1,3‐butanediol treatment had direct effects on arterial function, concentrations‐response curves were performed to βHOB and 1,3‐butanediol in MRA from naïve 5 week old, male Dahl S‐LS rats. We observed that βHOB and 1,3‐butanediol caused triphasic‐quadriphasic responses after pre‐contraction to phenylephrine (PE; Figure 2A–B). Specifically, we observed that low concentrations (&lt;3 nM) of βHOB caused pronounced vasodilation and this was inhibited by both L‐NAME and tetraethylammonium (TEA; potassium channel inhibitor; both, p&lt;0.05; Figure 3A–B). Intermediate concentrations of βHOB (3 nM–100 nM) caused contraction and this tended to be inhibited by indomethacin (cyclooxygenase inhibitor; p=0.08; Figure 3C). Finally, high concentrations (100 nM–30 μM) of βHOB caused relaxation again and this was predominately mediated by nitric oxide (p&lt;0.05; Figure 3A). Overall, these data suggest that βHOB stimulates the production of endothelium‐derived factors and low concentrations of βHOB could be a novel therapy against hypertension‐associated vascular aging by increasing nitric oxide synthesis.Support or Funding InformationAmerican Heart Association (18POST34060003) and National Institutes of Health (K99GM118885 and R01HL143082).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PP242, mTOR inhibitor, decreases phenylephrine‐induced vascular contractility in hypertensive and normotensive arteries

Cardiovascular disease is the leading cause of death in cancer survivors. The short‐term and long‐term cardiotoxic effects of targeted chemotherapeutics is not well known. PP242, a dual inhibitor to mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and 2 (mTORC2), is a targeted therapy in clinical trials for various malignancies including bladder, ovarian and gastric cancer. mTORC1 inhibition has been associated with heart failure, while mTORC2 inhibition has been considered cardioprotective. We hypothesized that PP242 leads to enhancedcontractile response to phenylephrine (PE), and impaired Acetylcholine (ACh)‐induced relaxation. We studied the effects of PP242 on the contractility and relaxation of aortic rings and mesenteric resistance arteries (MRA) of male Wistar rats (12–15 weeks old, n=4) and spontaneously hypertensive rats (SHR) (15–18 weeks old, n=6) on a wire myograph. Vessel segments were exposed to PP242 (500nM) for one‐hour incubation. Normotensive aortic rings exposed to PP242 showed a decreased contractile response to PE (LogEC50±SEM, Ctrl:−7.617±0.178 vs Drug: −7.292±0.129) and increased relaxation to ACh (LogEC50±SEM, Ctrl:−6.917±0.164 vs Drug: −7.588±0.164). SHR aortic rings exposed to PP242 (LogEC50±SEM, Ctrl:−7.606±0.129 vs Drug: −7.124±0.118) showed decreased contractility but no difference in ACh‐induced relaxation. No differences were observed in normotensive MRA. SHR MRA showed decreased PE‐induced contraction (LogEC50±SEM, Ctrl:−5.402±0.060 vs Drug:‐−5.322±0.065) and increased ACh‐induced relaxation (LogEC50±SEM, Ctrl:−7.992±0.117 vs Drug:−8.139±0.092). Our data suggest that dual inhibition of mTORC1 and mTORC2 with PP242 may provide a cardioprotective effect by limiting vasoconstriction and may be a good therapeutic agent for hypertensive cancer patients. However, long term treatments with this targeted therapy need to be evaluated for vascular dysfunction induction, as the hypocontractile effect could contribute to failure of the cardiovascular system, which could be associated with cardiotoxicity.Support or Funding InformationNIH P01 HL134604 (RCW), k99GM11888 (CFW)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abnormal neurovascular signaling in mouse model for familial hemiplegic migraine type 2

Familial hemiplegic migraine type 2 (FHM2) is migraine with aura that is associated with mutations in the Na, K‐ATPase α2 isoform including G301R mutation. These FHM2‐associated mutations result in reduced expression of the α2 Na, K‐ATPase. Migraine attack in FHM2 patients is characterized by sequential hypo‐ and hyperperfusion of the affected hemisphere. Heterozygote mice bearing G301R mutation (α2+/−G301R) phenocopy several FHM2‐relevant disease traits.We hypothesize that α2+/−G301R mice have abnormal neurovascular coupling.We tested this hypothesis by comparing α2+/−G301R and wild type (WT) mice. The sex difference was considered. Increase in sensory cortex perfusion in response to whiskers stimulation was compared in anaesthetized mice using Laser Speckle imaging. In brain slices loaded with Calcium Green‐1, we assessed parenchymal artery diameter and intracellular Ca2+ changes in astrocytic endfeet in response to neuronal excitation by electric field stimulation (EFS). Isolated middle cerebral arteries were studied in isometric myograph for wall tension changes in response to elevation of bath K+. Smooth muscle membrane potential was assessed with sharp electrode impalements. Quantitative PCR, Western blot and whole mount immunohistochemistry were used to study the molecular background for the functional changes.Whisker stimulation induced a larger increase in blood flow in the sensory cortex of α2+/−G301R mice than in WT mice. Moreover, the response was larger in male than female α2+/−G301R mice but no significant differences between male and female WT mice was seen. EFS was associated with larger parenchymal artery dilation in brain slices from α2+/−G301R mice than in WT mice. EFS‐induced vasodilation was inhibited by 100 μM BaCl2, suggesting involvement of inward‐rectifying K+ channels (Kir). The EFS induced increase in astrocytic intracellular Ca2+ was similar in α2+/−G301R and WT mice. No sex differences in brain slice responses were found. Isolated middle cerebral arteries from α2+/−G301R mice had larger relaxation to elevated bath K+ than similar arteries from WT mice. Removal of endothelium suppressed the relaxations of arteries from α2+/−G301R mice and eliminated the difference between groups. This relaxation was inhibited by BaCl2. Expression of Kir2.1 channels was higher in endothelium of arteries from α2+/−G301R mice than in WT while the expression in smooth muscle cells was not changed.These data suggest that abnormal neurovascular coupling in mice bearing the FHM2‐associated mutation is a result of increased endothelial Kir2.1 channel expression. Such changes may underlie the abnormal vascular hyperperfusion in FHM2 patients during migraine attack.Support or Funding InformationSupported by the Lundbeck Foundation [nr. R183‐2014‐3618]; the Novo Nordisk Foundation [nrs. NNF14OC0012731, NNF17OC0025224 and NNF17OC0026198]; the Independent Research Fund Denmark – Medical Sciences [nrs. 7025‐00015B and 8020‐00084A].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fibrocytes are Increased in the Medial Layer of the Aorta from Spontaneously Hypertensive Rats

BackgroundChronic hypertension causes a state of inflammation and leads to vascular remodeling. Vascular remodeling involves alterations in vascular smooth muscle cells, endothelium, and extracellular matrix. Collagen deposition is increased in conductance and resistance vessels of hypertensive animals.Fibrocytes are bone marrow‐derived cells found in the circulation that migrate toward injured tissue. At the injured site, fibrocytes contribute to healing by serving as antigen presenting cells and secrete cytokines such as IL‐6 and IL‐8 to recruit T cells. Fibrocytes also lay down extracellular matrix such as collagen, and its activation is implicated in many fibrotic diseases including cardiac fibrosis. Studies have shown that patients with hypertensive heart disease have increased number of circulating fibrocytes and that hearts of hypertensive patients have increased collagen levels. The role of fibrocytes in vascular remodeling in hypertension have not been investigated.HypothesisThis study tests the hypothesis that fibrocytes are increased in hypertension and play a role in vascular remodeling.MethodsMale spontaneously hypertensive rats (SHR) and Wistar rats 13–16 weeks old were used in this study. Flow cytometry was used to determine the percentage of fibrocytes (CD34+/CD45+/CollagenI+) in the peripheral blood, aorta, mesenteric resistance arteries (MRA), and pudendal arteries. Wire myograph was used to evaluate the differences in vascular function in response to phenylephrine (PE), norepinephrine (NE), acetylcholine (ACh), and sodium nitroprusside (SNP) between SHR and Wistar. Immunohistochemistry was used to detect fibrocytes (CD34+/CD45+/Collagen I+) within the vascular walls of aorta, mesenteric, and pudendal arteries. Statistical analysis performed using nonlinear regression, two‐tailed unpaired t‐test, and two‐way ANOVA.ResultsFlow cytometry did not show a difference between the percentage of fibrocytes in SHR compared to Wistar. Wire myography demonstrated differences between the two groups in aortic contraction to PE (p&lt;0.0001) and NE (p&lt;0.0001), aortic relaxation to ACh (p&lt;0.0001) and SNP (p&lt;0.0001), MRA relaxation to ACh (p&lt;0.0001), and pudendal artery relaxation to SNP (p&lt;0.0001). Immunohistochemistry showed increased number of fibrocytes in the media layer of the SHR aorta (Figure 1) compared to Wistar (Figure 2).ConclusionsThe number of fibrocytes are increased in the media layer in the aorta in SHR compared to Wistar. The increased number of fibrocytes may contribute to increased collagen deposition leading to decreased vascular compliance in hypertensive animals.Support or Funding InformationNIH P01 HL‐134604This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel vasodilatory factor identified.

Singlet molecular oxygen generated by endothelial indoleamine 2,3-dioxygenase 1 has an important pathophysiological role in mammals in the redox regulation of arterial relaxation and blood pressure during inflammatory conditions through the formation of a tryptophan-derived metabolite