Response Of Vascular Smooth Muscle Research Articles

Role of NaV1.7 in postganglionic sympathetic nerve function in human and guinea-pig arteries.

NaV1.7 plays a crucial role in inducing and conducting action potentials in pain-transducing sensory nociceptor fibres, suggesting that NaV1.7 blockers could be effective non-opioid analgesics. While SCN9A is expressed in both sensory and autonomic neurons, its functional role in the autonomic system remains less established. Our single neuron rt-PCR analysis revealed that 82% of sympathetic neurons isolated from guinea-pig stellate ganglia expressed NaV1.7 mRNA, with NaV1.3 being the only other tetrodotoxin-sensitive channel expressed in approximately 50% of neurons. We investigated the role of NaV1.7 in conducting action potentials in postganglionic sympathetic nerves and in the sympathetic adrenergic contractions of blood vessels using selective NaV1.7 inhibitors. Two highly selective NaV1.7 blockers, GNE8493 and PF05089771, significantly inhibited postganglionic compound action potentials by approximately 70% (P<0.01), with residual activity being blocked by the NaV1.3 inhibitor, ICA121431. Electrical field stimulation (EFS) induced rapid contractions in guinea-pig isolated aorta, pulmonary arteries, and human isolated pulmonary arteries via stimulation of intrinsic nerves, which were inhibited by prazosin or the NaV1 blocker tetrodotoxin. Our results demonstrated that blocking NaV1.7 with GNE8493, PF05089771, or ST2262 abolished or strongly inhibited sympathetic adrenergic responses in guinea-pigs and human vascular smooth muscle. These findings support the hypothesis that pharmacologically inhibiting NaV1.7 could potentially reduce sympathetic and parasympathetic function in specific vascular beds and airways. KEY POINTS: 82% of sympathetic neurons isolated from the stellate ganglion predominantly express NaV1.7 mRNA. NaV1.7 blockers inhibit action potential conduction in postganglionic sympathetic nerves. NaV1.7 blockade substantially inhibits sympathetic nerve-mediated adrenergic contractions in human and guinea-pig blood vessels. Pharmacologically blocking NaV1.7 profoundly affects sympathetic and parasympathetic responses in addition to sensory fibres, prompting exploration into the broader physiological consequences of NaV1.7 mutations on autonomic nerve activity.

Asymmetric Dimethylarginine Enables Depolarizing Spikes and Vasospasm in Mesenteric and Coronary Resistance Arteries.

Increased vasoreactivity due to reduced endothelial NO bioavailability is an underlying feature of cardiovascular disease, including hypertension. In small resistance arteries, declining NO enhances vascular smooth muscle (VSM) reactivity partly by enabling rapid depolarizing Ca2+-based spikes that underlie vasospasm. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) is metabolized by DDAH1 (dimethylarginine dimethylaminohydrolase 1) and elevated in cardiovascular disease. We hypothesized ADMA might enable VSM spikes and vasospasm by reducing NO bioavailability, which is opposed by DDAH1 activity and L-arginine. Rat isolated small mesenteric arteries and myogenic rat-isolated intraseptal coronary arteries (RCA) were studied using myography, VSM intracellular recording, Ca2+ imaging, and DDAH1 immunolabeling. Exogenous ADMA was used to inhibit NO synthase and a selective DDAH1 inhibitor, NG-(2-methoxyethyl) arginine, to assess the functional impact of ADMA metabolism. ADMA enhanced rat-isolated small mesenteric arteries vasoreactivity to the α1-adrenoceptor agonist, phenylephrine by enabling T-type voltage-gated calcium channel-dependent depolarizing spikes. However, some endothelium-dependent NO-vasorelaxation remained, which was sensitive to DDAH1-inhibition with NG-(2-methoxyethyl) arginine. In myogenically active RCA, ADMA alone stimulated depolarizing Ca2+ spikes and marked vasoconstriction, while NO vasorelaxation was abolished. DDAH1 expression was greater in rat-isolated small mesenteric arteries endothelium compared with RCA, but low in VSM of both arteries. L-arginine prevented depolarizing spikes and protected NO-vasorelaxation in rat-isolated small mesenteric artery and RCA. ADMA increases VSM electrical excitability enhancing vasoreactivity. Endothelial DDAH1 reduces this effect, and low levels of DDAH1 in RCAs may render them susceptible to endothelial dysfunction contributing to vasospasm, changes opposed by L-arginine.

Proteomic approach to explore the role of Rho-related BTB Domain Containing 1 (RhoBTB1)-Cullin-3 (CUL-3) pathway in identifying potential therapeutic candidates in cardioprotection

Protein-protein interaction studies have contributed enormously to understand the fundamentals of human impairments including cardiovascular diseases. We previously identified Phosphodiesterase 5 (PDE5) as an interacting partner of RhoBTB1. The latter can function as an adapter for CUL-3 E3 ring ubiquitin ligase complex delivering PDE5 for ubiquitination and proteasomal degradation. This in turn regulates the nitric oxide response of vascular smooth muscle (VSM) and consequently blood pressure. Ubiquitous expression of RhoBTB1, its link with CUL-3, and PDE5 suggests that RhoBTB1 may have other binding partners in VSM and other cells. The binding sites of these partners on RhoBTB1 might be similar to PDE5. With the notion that PDE5 binding region on RhoBTB1 could be responsible for binding of unknown partners in RhoBTB1-CUL-3 pathway, we truncated full-length RhoBTB1 into its component domains, individually (GTPase, BTB1, BTB2, and C-terminal (CT)) and in combination (e.g. B1B2C, B2C, and PB1B2). HEK293 cells were used to express epitope-tagged RhoBTB1 constructs and PDE5. The interaction between different constructs of RhoBTB1 and PDE5 was determined by co-immunoprecipitation (Co-IP). We showed that RhoBTB1 could bind to PDE5 utilizing the combination of its B1B2C domains. Our data also suggest that the CT domain itself was essential for the interaction between RhoBTB1 and PDE5 but was not sufficient on its own to bind PDE5. The Co-IP data was further corroborated with the findings of immunofluorescence and proximity labeling assay (PLA). Subsequent protein stability and ubiquitination studies revealed that B1B2C is catalytically sufficient for CUL3-dependent proteasomal degradation like full-length RhoBTB1. Furthermore, macromolecular protein-protein docking revealed the binding interface between RhoBTB1 and CUL-3 comprising of 24 amino acids. Additional Co-IP data suggested that Ser363 and Pro353 on RhoBTB1 domain are required for binding to CUL-3 as mutants of RhoBTB1 (Ser363Ala and Pro353Ala) could not deliver PDE5 for proteasomal degradation. To further identify unknown interacting partners in RhoBTB1-CUL-3 axis, we utilized the ascorbate peroxidase (APEX2) proximity labelling system involving B1B2C as “bait.” Proteomic profiling of the biotinylated proteome through mass spectrometry has identified several unknown interacting partners involved in RhoBTB1-CUL-3 pathway. Some of these have demonstrated upregulation on pharmacological inhibition of proteasome and CUL-3 which establishes their potential involvement in RhoBTB1-CUL-3 pathway. Exploring the involvement of such interacting partners in RhoBTB1-CUL-3 axis may open novel therapeutic avenues in cardioprotection. NIH R35 HL144807 (to Dr. Curt D. Sigmund) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Resveratrol supplementation improves endothelial function in chronic kidney disease

Introduction: Chronic kidney disease (CKD) associates with increased risk of cardiovascular disease (CVD), in part, due to lower endothelial function. Thus, interventions that increase endothelial function could mitigate CVD in CKD. Resveratrol is a polyphenol that improves endothelial function in obese and aging adults. Whether resveratrol supplementation increases endothelial function in patients with CKD is unknown. Our objective was to test the effects of resveratrol supplementation on endothelial function in CKD. We hypothesized that resveratrol supplementation would increase endothelial function in patients with CKD. Methods: 25 adults with stage 3 CKD and type 2 diabetes were enrolled in a randomized, double-blind, placebo-controlled, crossover study to investigate the effects of 6-week resveratrol supplementation (400 mg/day) on endothelial function. Endothelial function was assessed via brachial artery flow-mediated dilation (BA-FMD). Vascular smooth muscle reactivity was determined from nitroglycerin-mediated dilation. Estimated glomerular filtration rate (eGFR) was calculated from serum cystatin C using the CKD-EPI formula. A mixed effects generalized linear model was used to evaluate differences in treatment arms over time. Results: The median age of the subjects was 70 (67, 74) years and 86% of the patients were male. The mean (SD) GFR and hemoglobin A1c were 42(9) ml/min/1.73m2 and 7.36(0.77)%, respectively. Within-group comparisons revealed BA-FMD increased by 0.91% after resveratrol supplementation (p=0.002), but not placebo (p=0.44), and between-group comparisons indicate resveratrol increased BA-FMD greater than placebo (p=0.001). Nitroglycerin mediated dilation was unchanged after both resveratrol supplementation and placebo (p&gt;0.05). GFR, hemoglobin A1c and blood pressure were unchanged following resveratrol supplementation suggesting that other mechanisms may underlie resveratrol-mediated benefits on endothelial function in this study. Conclusion: Resveratrol supplementation increases endothelial function in patients with CKD. Larger clinical trials for longer duration are needed to confirm our findings and to evaluate the mechanisms by which resveratrol improves endothelial function in CKD. Funding: This study was supported by the Renal Hypertension Training Grant (NIH NRSA T32 award) and Dr. Jalal's grant R01HL134738-01A1. Colin Gimblet is supported by the American Heart Association predoctoral fellowship grant (23PRE1012593). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Reduction of vascular reactivity in rat aortas following pilocarpine-induced status epilepticus

ObjectiveThe authors investigated changes in vascular reactivity in rats following pilocarpine-induced status epilepticus. MethodMale Wistar rats weighing between 250g and 300g were used. Status epilepticus was induced using 385 mg/kg i.p. pilocarpine. After 40 days the thoracic aorta was dissected and divided into 4 mm rings and the vascular smooth muscle reactivity to phenylephrine was evaluated. ResultsEpilepsy decreased the contractile responses of the aortic rings to phenylephrine (0.1 nM–300 mM). To investigate if this reduction was induced by increasing NO production with/or hydrogen peroxide L-NAME and Catalase were used. L-NAME (N-nitro-L arginine methyl ester) increased vascular reactivity but the contractile response to phenylephrine increased in the epileptic group. Catalase administration decreased the contractile responses only in the rings of rats with epilepsy. ConclusionsOur findings demonstrated for the first time that epilepsy is capable of causing a reduction of vascular reactivity in rat aortas. These results suggest that vascular reactivity reduction is associated with increased production of Nitric Oxide (NO) as an organic attempt to avoid hypertension produced by excessive sympathetic activation.

REATIVIDADE DA AORTA TORÁCICA EM PACIENTES OBESOS SUBMETIDOS AO EXERCÍCIO AERÓBICO

ABSTRACT Introduction: Aerobic exercise can improve the function of the cardiovascular circulatory system, reducing morbidity and mortality from cardiovascular disease by stimulating the production of endogenous self-protection. Activating potassium channels in vascular smooth muscle cells can cause vasodilation and increase blood flow, lowering blood pressure. There is a sensitivity to intracellular ATP and ADP concentration among the variety of potassium channels distributed in vascular smooth muscle cells, which vary mainly during aerobic physical activity. Objective: Explore the effect of aerobic exercise on the vascular reactivity of the thoracic aorta in patients with obesity and hyperlipidemia. Methods: Randomized controlled trial in twenty male Wistar rats weighing 250g and two months old. The control group remained at rest while the experimental group performed aerobic exercise on a treadmill at increasing speed for eight weeks. The rats were dissected, and dilatators and vasoconstrictors drugs stimulated their blood vessels in a tamponade solution. Observation of vascular changes was measured under controlled tensioning. Results: The blockade of KATP channels in vascular smooth muscle caused tonic contraction of vascular smooth muscle cells and increased blood pressure. Conclusion: Long-term regular aerobic exercise may induce changes in rats’ thoracic aortic vascular function and vascular smooth muscle reactivity. Aerobic exercise can also significantly improve the activity of KATP channels. Evidence Level II; Therapeutic Studies – Investigating the results.

Abstract P136: Exploring The Interactions Between Rho-related BTB Domain Containing 1 (RhoBTB1) And Phosphodiesterase 5 (PDE5) To Identify Novel Therapeutic Candidates To Counter Hypertension

Protein-protein interaction studies have provided valuable insights to understand fundamental mechanisms in cells. We previously identified that PDE5 is a binding partner of RhoBTB1 and stringently regulates PDE5 activity in vascular smooth muscle (VSM). This in turn regulates the nitric oxide response of VSM and consequently blood pressure. We showed that RhoBTB1 can function as an adapter for Cullin-3 (CUL3) E3 ring ubiquitin ligase complex delivering PDE5 for ubiquitination and proteasomal degradation. In order to identify other RhoBTB1 binding partners, we assessed the binding interface of RhoBTB1 which binds to PDE5 with the notion that the same region will bind to other RhoBTB1-CUL3 substrates. Full-length RhoBTB1 was truncated into its component domains, separately (GTPase, BTB1, BTB2, and C-terminal (CT)) and in combination (e.g. B1B2C) domains. HEK293 cells were co-transfected with epitope-tagged PDE5 and RhoBTB1 domain constructs, and their interaction was analyzed by co-immunoprecipitation (Co-IP). Interestingly, the CT domain itself was essential for the interaction between RhoBTB1 and PDE5 but was not sufficient on its own to bind to PDE5. This was supported by our finding that the domains lacking CT region could not bind with PDE5. The Co-IP data was further corroborated by immunofluorescence and proximity labeling assay (PLA). Our data suggest that RhoBTB1 could bind to PDE5 utilizing its BTB1-BTB2-CT domains. Moreover, protein stability and ubiquitination studies revealed that the BTB1-BTB2-CT domain is sufficient for CUL3-dependent proteasomal degradation. Molecular approaches including site-directed mutagenesis are being explored to investigate potential “hotspots” underlying the RhoBTB1-PDE5 interaction. We are currently utilizing ascorbate peroxidase (APEX2) fusion system involving BTB1-BTB2-CT domain as “bait” to identify unknown interacting partner in RhoBTB1-Cullin3 axis. Preliminary experiments have been performed to validate the system. For example, BTB1-BTB2-CT-APEX2 can bind to PDE5. These studies are designed to assess if there are other proteins which are involved in the regulation of blood pressure in VSM which may become novel therapeutic candidates against hypertension.

Modulation of Vasomotor Function by Perivascular Adipose Tissue of Renal Artery Depends on Severity of Arterial Dysfunction to Nitric Oxide and Severity of Metabolic Parameters.

Perivascular adipose tissue (PVAT) enhances vascular relaxation of mesenteric arteries in SHRSP.Z-Leprfa/IzmDmcr rats (SPZF), a metabolic syndrome model. We investigated and compared the effects of PVAT on the renal artery in SPZF with those on SHR/NDmcr-cp rats (CP). Renal arteries with and without PVAT were isolated from 23-week-old SPZF and CP. The effects of PVAT on acetylcholine- and nitroprusside-induced relaxation were examined using bioassays with phenylephrine-contracted arterial rings. Acetylcholine-induced relaxations without PVAT in SPZF and CP were 0.7- and 0.5-times lower in females than in males, respectively. In the presence of PVAT, acetylcholine-induced relaxations increased 1.4- and 2-times in male and female CP, respectively, but did not differ in SPZF. Nitroprusside-induced relaxation with and without PVAT was 0.7-times lower in female than in male SPZF but did not differ in CP. Angiotensin-II type-1 receptor (AT1R)/AT1R-associated protein mRNA ratios were lower in CP than in the SPZF and negatively correlated with the difference in arterial relaxation with and without PVAT. The effects of renal artery PVAT differed between the SPZF and CP groups. Higher levels of enhanced AT1R activity in SPZF PVAT may drive these differences by impairing the vascular smooth muscle responses to nitric oxide.

AICAR promotes endothelium-independent vasorelaxation by activating AMP-activated protein kinase via increased ZMP and decreased ATP/ADP ratio in aortic smooth muscle.

AICAR, an adenosine analog, has been shown to exhibit vascular protective effects through activation of AMP-activated protein kinase (AMPK). However, it remains unclear as to whether adenosine kinase-mediated ZMP formation or adenosine receptor activation contributes to AICAR-mediated AMPK activation and/or vasorelaxant response in vascular smooth muscle. In the present study using endothelium-denuded rat aortic ring preparations, isometric tension measurements revealed that exposure to 1mM AICAR for 30min resulted in inhibition of phenylephrine (1μM)-induced smooth muscle contractility by ∼35%. Importantly, this vasorelaxant response by AICAR was prevented after pretreatment of aortic rings with an AMPK inhibitor (compound C, 40µM) and adenosine kinase inhibitor (5-iodotubercidin, 1µM), but not with an adenosine receptor blocker (8-sulfophenyltheophylline, 100µM). Immunoblot analysis of respective aortic tissues showed that AMPK activation seen during vasorelaxant response by AICAR was abolished by compound C and 5-iodotubercidin, but not by 8-sulfophenyltheophylline, suggesting ZMP involvement in AMPK activation. Furthermore, LC-MS/MS MRM analysis revealed that exposure of aortic smooth muscle cells to 1mM AICAR for 30min enhanced ZMP level to 2014.9±179.4picomoles/mg protein (vs. control value of 8.5±0.6; p<0.01), which was accompanied by a significant decrease in ATP/ADP ratio (1.08±0.02 vs. 2.08±0.06; p<0.01). Together, the present findings demonstrate that AICAR-mediated ZMP elevation and the resultant AMPK activation in vascular smooth muscle contribute to vasorelaxation.

Type 2 diabetes impairs vascular responsiveness to nitric oxide, but not the venoarteriolar reflex or post-occlusive reactive hyperaemia in forearm skin.

The venoarteriolar reflex (VAR) is a local mechanism by which vasoconstriction is mediated in response to venous congestion. This response may minimize tissue overperfusion, preventing capillary damage and oedema. Post-occlusive reactive hyperaemia (PORH) is used to assess microvascular function by performing a brief local arterial occlusion resulting in a subsequent rapid transient vasodilation. In the current study, we hypothesized that type 2 diabetes (T2D) attenuates VAR and PORH responses in forearm skin in vivo. In 11 healthy older adults (Control, 58±8years) and 13 older adults with controlled T2D (62±10years), cutaneous blood flow measured by laser-Doppler flowmetry was monitored following a 3-min venous occlusion of 45mmHg that elicited the VAR, followed by a 3-min recovery period and then a 5-min arterial occlusion of 240mmHg that induced PORH. Finally, sodium nitroprusside, a nitric oxide donor, was administered to induce maximum vasodilation. VAR and PORH variables were similar between groups. By contrast, maximal cutaneous blood flow induced by sodium nitroprusside was lower in the T2D group. Taken together, our observations indicate that T2D impairs vascular smooth muscle responsiveness to nitric oxide, but not VAR and PORH in forearm skin.

In Vitro Model for Ischemic Stroke: Functional Analysis of Vascular Smooth Muscle Cells.

The Neurovascular Unit (NVU) is formed by vascular and neural cells controlling the cerebral hyperaemia. All the components are anatomically and functionally linked to each other, resulting in a highly efficient regulation of the cerebral blood flow, which, when interrupted, can lead to stroke. An ischemic stroke (IS) is the most common type of stroke with high rates of morbidity, mortality and disability. Therefore, it is of extreme importance to protect the functional and structural integrity of the NVU in patients with IS, understanding the mechanisms involved and how it affects each component of the NVU. Thus, the aim of this work is to analyse how the vascular smooth muscle cells from the rat middle cerebral artery function/react after an ischemic event. To mimic this event, primary cortical cultures were challenged to oxygen and glucose deprivation (OGD) for 4h and 6h, and the smooth muscle cells (SMCs) contractility was analysed after exposure to different media previously conditioned by the cortical cultures upon reperfusion. The results show a dual effect on the SMCs response to the vasorelaxant agent, only for cells exposed to the reperfusion media conditioned by neuron-glia cultures challenged by OGD, leading to increased relaxation of the SMCs for OGD 4h, whereas for OGD 6h the effect is reversed leading to contraction of the SMCs. These differences demonstrate that the astrocytes mediate the vasoactive response of vascular smooth muscle by releasing factors into the reperfusion medium, and the hypoxia time is fundamental for a beneficial/harmful response by the vascular smooth muscle.

PDIA1 acts as master organizer of NOX1/NOX4 balance and phenotype response in vascular smooth muscle

Changes in vascular smooth muscle cell (VSMC) phenotype underlie disease pathophysiology and are strongly regulated by NOX NADPH oxidases, with NOX1 favoring synthetic proliferative phenotype and NOX4 supporting differentiation. Growth factor-triggered NOX1 expression/activity strictly depends on the chaperone oxidoreductase protein disulfide isomerase-A1 (PDIA1). Intracellular PDIA1 is required for VSMC migration and cytoskeleton organization, while extracellular PDIA1 fine-tunes cytoskeletal mechanoadaptation and vascular remodeling. We hypothesized that PDIA1 orchestrates NOX1/NOX4 balance and VSMC phenotype. Using an inducible PDIA1 overexpression model in VSMC, we showed that early PDIA1 overexpression (for 24–48 h) increased NOX1 expression, hydrogen peroxide steady-state levels and spontaneous VSMC migration distances. Sustained PDIA1 overexpression for 72 h and 96 h supported high NOX1 levels while also increasing NOX4 expression and, remarkably, switched VSMC phenotype to differentiation. Differentiation was preceded by increased nuclear myocardin and serum response factor-response element activation, with no change in cell viability. Both NOX1 and hydrogen peroxide were necessary for later PDIA1-induced VSMC differentiation. In primary VSMC, PDIA1 knockdown decreased nuclear myocardin and increased the proliferating cell nuclear antigen expression. Newly-developed PDIA1-overexpressing mice (TgPDIA1) exhibited normal general and cardiovascular baseline phenotypes. However, in TgPDIA1 carotids, NOX1 was decreased while NOX4 and calponin expressions were enhanced, indicating overdifferentiation vs. normal carotids. Moreover, in a rabbit overdistension injury model during late vascular repair, PDIA1 silencing impaired VSMC redifferentiation and NOX1/NOX4 balance. Our results suggest a model in which PDIA1 acts as an upstream organizer of NOX1/NOX4 balance and related VSMC phenotype, accounting for baseline differentiation setpoint.

Traumatic Brain Injury (tbi) Attenuates Arterial Smooth Muscle Vasorelaxation In Pressurized Cerebral Arteries

Maintenance of cerebral blood flow (CBF) is impaired following traumatic brain injury (TBI), increasing the incidence of both ischemic and hemorrhagic events. Vascular tone is controlled by neurogenic, endothelial, and myogenic responses. PURPOSE: This work was designed to specifically determine the extent to which the myogenic response of vascular smooth muscle in the middle cerebral artery (MCA) is altered following TBI. METHODS: TBI was induced by controlled cortical impact (CCI) in 2-month-old male Sprague-Dawley rats. Twenty-four hours following injury or sham surgery, pressurized arterial myography was performed on endothelium-denuded MCA to examine the effect of TBI on smooth muscle-specific vasorelaxation using pharmacologic activators of the adenylyl cyclase (AC)-cAMP-PKA/PKG pathway (10-9M to 10-5M, n=6 at each dose). RESULTS: Myogenic tone was attenuated following TBI (25±1.1% vs. 18±1.2%, n=12; p<0.001). The mean change in myogenic tone in response to activators (10-6M) was reduced to 35% (AC), 33% (PKA), and 30% (PKG) in TBI as compared to sham controls. CONCLUSION: Attenuation of smooth muscle myogenic tone and resulting vasorelaxation following TBI may serve as a compensatory mechanism to protect sensitive brain tissue from cerebral ischemia. Future work will be needed to elucidate the role of pathways that could serve as potential targets for therapeutic intervention to reduce damaging vascular events. Supported by The Florida State University Graduate School Dissertation Award Grant.

GPAT4-Generated Saturated LPAs Induce Lipotoxicity through Inhibition of Autophagy by Abnormal Formation of Omegasomes.

SummaryExcessive levels of saturated fatty acids are toxic to vascular smooth muscle cells (VSMCs). We previously reported that mice lacking VSMC-stearoyl-CoA desaturase (SCD), a major enzyme catalyzing the detoxification of saturated fatty acids, develop severe vascular calcification from the massive accumulation of lipid metabolites containing saturated fatty acids. However, the mechanism by which SCD deficiency causes vascular calcification is not completely understood. Here, we demonstrate that saturated fatty acids significantly inhibit autophagic flux in VSMCs, contributing to vascular calcification and apoptosis. Mechanistically, saturated fatty acids are accumulated as saturated lysophosphatidic acids (LPAs) (i.e. 1-stearoyl-LPA) possibly synthesized through the reaction of GPAT4 at the contact site between omegasomes and the MAM. The accumulation of saturated LPAs at the contact site causes abnormal formation of omegasomes, resulting in accumulation of autophagosomal precursor isolation membranes, leading to inhibition of autophagic flux. Thus, saturated LPAs are major metabolites mediating autophagy inhibition and vascular calcification.

Protein Kinase C Activity is Increased in Human Coronary Smooth Muscle Cells following Exposure to 5‐FU Chemotherapy

Background5‐Fluorouracil (5‐FU) is the third most commonly used chemotherapeutic agent used to treat cancer but is strongly associated with a dose‐limiting cardiotoxicity. Cancer patients treated with 5‐FU chemotherapy are at risk of cardiotoxicity that manifests as alterations in the myocardial circulation such as resting or exertional angina, arrhythmias, myocardial infarction, and coronary vasospasm. However, little is known about the primary signaling mechanisms that contribute to cardiotoxicity with 5‐FU. Recent work has suggested that the Protein Kinase C (PKC) family of kinases plays a role in altered vasoconstrictive responses in vascular smooth muscle following exposure to 5‐FU. Further, PKC signaling in other pathologies is known to be influenced by reactive oxygen species (ROS) and 5‐FU chemotherapy is known to promote the production of ROS. Therefore, we hypothesized that 5‐FU administration would increase PKC activity in human coronary smooth muscle cells (HCSMCs).MethodsIn vitro, HCSMCs were incubated with 5‐FU (7×10−3M) for 3h, 1h, and 10 minutes. Following the respective incubation periods, media was aspirated in control and 5‐FU groups and the HCSMCs were scraped, homogenized in lysis buffer, and analyzed for protein content. The activity of PKC was assessed by a PKC activity assay kit.ResultsAnalysis revealed relative PKC activity was significantly increased in HCSMCs following 10 minutes of exposure to 5‐FU chemotherapy (6.52±0.36 versus 4.84±0.36; p=0.02) and was not different at 1 hour versus control and 3 hours versus control (1h 5.28±0.356 versus 4.84±0.36; p=0.82, 3h 5.67±0.36 versus 4.84±0.36; p=0.37).ConclusionsOur findings suggest 5‐FU exposure acutely upregulates PKC activity in coronary smooth muscle cells immediately following drug administration and the PKC pathway may have a mechanistic role in the altered vasoconstrictive response to 5‐FU as previously shown. These findings are imperative given the limited knowledge of mechanistic pathways that lead to vascular damage following 5‐FU exposure. Future studies will focus on reactive oxygen species as a regulator of PKC activity and specific isoforms involved in this pathway.

Is Intact Endothelial Cell Autophagy Required for Training‐Induced Vascular Adaptations?

Macroautophagy is operational during basal conditions to maintain intracellular organelle and protein quality control, but is upregulated during cellular stress (e.g, dynamic exercise) to adapt to changing nutritional and energy demands. We tested the hypothesis that intact endothelial cell (EC) autophagy is required to observe training‐induced vascular adaptations. Rationale for this hypothesis was provided by an earlier report that obese mice with germline, whole body mutation of a protein requisite for autophagy i.e., Bcl2‐AAA mice were refractory to training‐induced improvements concerning glucose homeostasis (He et al., Nature, 2012). First, we demonstrated that : (i) workload achieved during a maximal treadmill test; (ii) soleus muscle citrate synthase activity; (iii) mRNA and protein expression of vascular autophagy; and (iv) intraluminal flow‐mediated vasodilatory responses (FMD) of femoral arteries examined ex vivo, were greater (all p&lt;0.05) in ~ 4‐month old male C57Bl/6 mice that completed 10‐weeks of treadmill‐training vs. age‐matched sedentary animals (n=10 mice per group). These findings indicate that our efficacious training protocol improves vascular autophagy and arterial function in adult mice. Next, ~ 4‐month old male mice on a C75Bl/6 background with inducible Cre/LoxP‐based impairment of autophagy‐related gene 3 (Atg3) specifically in ECs (iecAtg3KO mice) and their Cre negative littermates (WT) were treated with tamoxifen. Two weeks later, one cohort of iecAtg3KO mice initiated a treadmill training program (ETR; 10–60 min per day × 0–20% grade × 6 days per week × 10 weeks) or maintained familiarity with the treadmill by running 10 min per day × 5% grade × 1 day per week (SED). Two cohorts of WT littermates were treated identically. After 10‐weeks efficacy of the training protocol was established as described earlier and verification of our mutant was assessed. With regard to the latter, primary ECs obtained from the carotid artery and aorta indicated Atg3 mRNA and Atg3 protein, respectively, was minimal (p&lt;0.05) in iecAtg3KO vs. WT mice, whereas vascular smooth muscle cell Atg3 was similar between groups. As expected, intraluminal FMD responses were greater (p&lt;0.05) in WT‐ETR vs. WT‐SED mice, while vascular smooth muscle responses to sodium nitroprusside were not different between groups. Further, as anticipated, intraluminal FMD was blunted (p&lt;0.05) in iecAtg3KO‐SED vs. WT‐SED mice, indicating the importance of intact EC autophagy to FMD. Contrary to our hypothesis, however, training‐induced vascular adaptations indeed were observed (p&lt;0.05) in iecAtg3KO‐ETR vs. iecAtg3KO‐SED mice, while vascular smooth muscle responses were not different between groups. These findings indicate that intact EC Atg3 is not necessary for training‐induced vascular adaptations to occur.Support or Funding InformationCR [APS UGRF, University of Utah (UU) Undergraduate Research Opportunities Program (UROP)]; JMC (UU Graduate Research Fellowship); SKP (AHA 17POST33670663); KL (APS STRIDE, UU UROP), LT (UU UROP); SB (NIDDK R01‐DK‐098646‐01A1, R01‐DK‐099110, AHA 16GRNT30990018), JDS (AHA16GRNT31050004, NIH RO3AGO52848, NIHRO1HL141540).

Acute experimental venous thrombosis impairs venous relaxation but not contraction

ObjectiveVenous thrombosis (VT) damages the vein wall, both physically by prolonged distension and from inflammation. These factors contribute to post-thrombotic syndrome (PTS). Interleukin (IL)-6 might play a role in experimental PTS and vein wall responses. Previous assessments of post-thrombotic vein wall injury used static measures such as histologic examination and immunologic assays. The purpose of the present study was to use myography to quantify the changes in contraction and relaxation of murine vessels exposed to an acute VT. MethodsWild-type (WT) C57BL/6 mice were used to determine the baseline vein wall passive tension on a DMT 610m myograph (DMT-USA, Inc., Ann Arbor, Mich), including dosing concentrations of phenylephrine (Phe) and acetylcholine (Ach). WT and IL-6−/− mice underwent VT using inferior vena cava (IVC) ligation (complete stasis) and stenosis (partial stasis), with no-surgery mice used as controls. The mice were harvested at 2 days (2D) and analyzed using a myograph. The vessels were stimulated with Phe and Ach to stimulate a contraction and relaxation response. The endothelial responses to VT were quantified by CD31 immunohistochemistry, Greiss assay, polymerase chain reaction, and Evans blue assay. ResultsOptimal passive tension was determined to be 2 mN, with an optimal concentration of Phe and Ach of 7E-3M and 1E-5M, respectively. No significant differences were found in the contractions when exposed to Phe between the WT control, WT 2D ligation, and WT 2D stenosis IVC segments and the IL-6−/− mice with and without thrombus (P > .05 for all). When treated with Ach, significantly more relaxation was found in the nonthrombosed control IVC segments than in those IVC segments that had had a 2D thrombus from either ligation- or stenosis-derived thrombotic mechanisms in both WT and IL-6−/− mice. CD31 staining showed ∼20% less luminal endothelium after stasis thrombosis (P ≤ .01) but no loss in the controls (P > .05). Evans blue staining showed a trend toward increased leakiness in post-thrombotic vein walls. No significant difference in the endothelial gene markers or nitric oxide production was found. ConclusionsCompared with the controls, acute thrombosis in the total or partial stasis models did not impair IVC contractile responses, suggesting no effect on the medial vascular smooth muscle response. The relaxation response was significantly reduced in the post-thrombotic groups, likely from direct endothelial injury. These findings suggest, at acute points, that VT impairs the endothelial function of a vein wall while retaining the vascular smooth muscle cell function and might be a mechanism that promotes PTS.

A Review of the Actions of Endogenous and Exogenous Vasoactive Substances during the Estrous Cycle and Pregnancy in Rats.

Simple SummaryThe vascular endothelium plays an essential role in regulating cardiovascular homeostasis by controlling the vascular tone. However, it is known that variations of sex hormones in the female during the reproductive cycle have a significant impact on the homeostasis of the female cardiovascular system. Therefore, this review shows an overview of how the vascular smooth muscle response is altered in the presence of some endogenous and exogenous substances with vasoactive actions during the estrous cycle and during pregnancy in rats. We performed a systematic review of the literature using the Web of Science, PubMed, and Scielo database through multiple combinations of terms. We selected only those terms that coincided with the objectives of this study. The result of the review shows that several studies have observed both vasoconstrictive and vasodilatory responses induced by vasoactive substances during the estrous cycle and in ovariectomized and pregnant rats. The understanding of these effects is essential to optimize and develop new treatments for some vascular pathologies.Vascular endothelium plays a key role in regulating cardiovascular homeostasis by controlling the vascular tone. Variations in sex hormones during the reproductive cycle of females affect the homeostasis of the cardiovascular system. Also, the evidence shows that estrogens show a cardioprotective effect. On this basis, this study describes some vascular responses induced by vasoactive substances during the estrous cycle in rats. We obtained the information available on this topic from the online databases that included scientific articles published in the Web of Science, PubMed, and Scielo. Many investigations have evaluated the vasoactive response of substances such as acetylcholine and norepinephrine during the estrous cycle. In this review, we specifically described the vascular response to vasoactive substances in rats during the estrous cycle, pregnancy, and in ovariectomized rats. In addition, we discussed the existence of different signaling pathways that modulate vascular function. The knowledge of these effects is relevant for the optimization and development of new treatments for some vascular pathologies.

Cryptostrobin and catechin isolated from Eugenia mattosii D. Legrand leaves induce endothelium-dependent and independent relaxation in spontaneously hypertensive rat aorta.

Considering the therapeutic potential of phenolic compounds, the purpose of the present study was to investigate the mechanisms involved in the relaxation induced by cryptostrobin and catechin, isolated from Eugenia mattosii D. Legrand leaves, in the aorta of spontaneously hypertensive rats (SHR). The thoracic aorta was isolated from SHR and kept in the organ bath system by recording contractile or relaxant responses. The addition of cumulative concentrations of cryptostrobin and catechin induced endothelium-dependent and-independent relaxation in aorta rings from SHR, as well as both compounds were effective in reducing phenylephrine-induced contraction. Pretreatment of aortic rings with Nω-nitro-l-arginine methylester (L-NAME, an inhibitor of nitric oxide synthase) or 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of soluble guanylate cyclase), resulted in a significant change of relaxant effect induced by catechin, and a slight influence on cryptostrobin-induced relaxation. Muscarinic receptor and potassium channels are involved in catechin-induced relaxation as assessed using atropine (a muscarinic receptor antagonist), tetraethylammonium (a non-selective K+ channel blocker) and glibenclamide (an ATP-sensitive K+ channel blocker). Conversely, cryptostrobin, but not catechin, blunted the contraction induced by the addition of phenylephrine in a calcium-free solution. Besides that, cryptostrobin attenuated the contraction of rat aorta rings induced by internal Ca2+ release and external Ca2+ influx. These findings indicated that cryptostrobin and catechin alter vascular smooth muscle reactivity, and this effect may be involved, at least in part, by enhancing the endothelium NO/cGMP pathway and potassium channels activation. In addition, cryptostrobin reduced the phenylephrine, KCl and CaCl2-induced contractions in a calcium-free solution.

Ventilasi dan Perfusi, serta Hubungan antara Ventilasi dan Perfusi

Lung is a place for gas exchange where ventilation and perfusion occurs. Ventilation is the first step where sequential process of inhalation and exhalation take place. Meanwhile perfusion as the other step facilitates the gas exchange and tissue supply need. Blood flows through the lungs are equals as the amount of cardiac output where the factors that control cardiac output are mainly peripheral factors, also control pulmonary blood flow. In general condition, pulmonary blood vessels act as a passive tube, which can be increased with the increasing pressure and narrowed the pressure drop. Oxygen absorption level from lungs into bloodstream is a critical determinant for functional capacity, and an important factor wheter in normal conditions (including exercise) or even in illness state. Lung diffusion capacity is influenced by several geometric and functional factors. Gravitation influence systematic gradient in ventilation and perfusion distribution. Ventilation and blood flow variations at horizontal level also occur due to intrinsic anatomic variations and vascular geometry, as well as the differences in airway and vascular smooth muscle response which modifies the distribution. The change of integrity intrapleural chamber, hydrostatic pressure and osmotic imbalance, malfunction of surfactants, other intrinsic weakness of the branching system in the form of a progressive airway, and all the things that could potentially damage the structure of the lung can cause ventilation and diffusion dysfunction.