Pressure alters endothelial effects upon vascular smooth muscle cells by decreasing smooth muscle cell proliferation and increasing smooth muscle cell apoptosis

BACKGROUND: Although de-endothelialization after vascular intervention is associated with intimal hyperplasia, endothelial cells (ECs) increase smooth muscle cell (SMC) numbers in conventional cocultures. In previously published work, SMCs cocultured with ECs in a chronic high-pressure environment exhibited significantly decreased cell counts compared to monocultured SMCs in the same high pressure. This finding contrasted with SMCs cocultured with ECs in ambient pressure, which exhibited significantly higher cell counts than the monocultured SMCs in ambient pressure. We now hypothesize that extracellular pressure decreases SMC number during coculture with ECs by decreasing SMC proliferation through nuclear protein regulation and by increasing SMC apoptosis. Furthermore, this effect depends on the EC response to pressure. METHODS: Rat aortic SMCs were cultured independently (SMC/0) or cocultured with EC (SMC/EC) under either atmospheric or increased pressure (130-135 mmHg over ambient, SMC/0-P and SMC/EC-P) for 5 days. We assessed SMC proliferative potential by determining c-myc expression (by protein analysis), apoptosis (by cell counting, staining with acridine orange or TUNEL technique), and topoisomerase IIalpha levels. Parallel studies measured the effects of conditioned media from monocultured EC and SMC exposed for 5 days to control or increased pressure on recipient SMC growing in conventional culture. RESULTS: In high-pressure conditions, SMC/EC-P exhibited 42% less c-myc expression than SMC/0s (P = .00028). Significantly increased apoptotic activity (22 +/- 1.8%) in SMC/EC-Ps compared to SMC/0s was coupled with significantly lower topoisomerase IIalpha levels. Interestingly, pressure (SMC/0-P) and EC coculture (SMC/EC) each separately raised myocyte apoptotic activity to 15 +/- 1.3% and 17 +/- 2.0%, respectively. Conditioned media from pressurized ECs caused a 20% decrease in cell counts in target SMC compared to conditioned media from ECs in atmospheric pressure. Media from pressurized SMCs did not affect target SMCs. CONCLUSIONS: In a model designed to study SMC/EC interactions in a dynamic environment, EC exposure to pressure alters the growth characteristics and apoptotic activity of SMCs via a secreted factor. Extracellular pressure may alter EC regulation of SMC behavior and regulate intimal hyperplasia.     

The mechanisms of propofol-mediated hyperpolarization of in situ rat mesenteric vascular smooth muscle

Previously, we reported that propofol hyperpolarizes vascular smooth muscle (VSM) cells of small arteries and veins. The current study was designed to determine whether propofol-mediated hyperpolarization is the result of specific effects on potassium channels known to exist in VSM and on steps in the intracellular nitric oxide (NO), cyclic guanosine monophosphate (cGMP), and cyclic adenosine monophosphate (cAMP) second messenger pathways. VSM transmembrane potentials (E(m)) were measured in situ in sympathetically denervated, small mesenteric arteries and veins of Sprague-Dawley rats. Effects of propofol on VSM E(m) were determined before and during superfusion with specific inhibitors of VSM calcium-activated (K(Ca)), adenosine triphosphate-sensitive (K(ATP)), voltage-dependent (K(v)), and inward rectifying (K(IR)) potassium channels and with endogenous mediators of vasodilation. Propofol significantly hyperpolarized VSM in small mesenteric vessels. This hyperpolarization was abolished on inhibition of K(Ca) and K(ATP) channel activity and on inhibition of NO and cGMP (but not cAMP). Assuming a close inverse correlation between the magnitude of VSM E(m) and contractile force, these results suggest that propofol induces hyperpolarization and relaxation in denervated, small mesenteric vessels by activation of K(Ca) and K(ATP) channels. Such channel activation may be mediated by activation of NO and cGMP, but not cAMP, second messenger pathways. IMPLICATIONS: The results of this study indicate that propofol-mediated hyperpolarization in vascular smooth muscle can be attributed to the activation of calcium-activated, adenosine triphosphate-sensitive potassium channels, the nitric oxide, and cyclic guanosine monophosphate pathways.     

General anesthetics and vascular smooth muscle: direct actions of general anesthetics on cellular mechanisms regulating vascular tone

General anesthetics threaten cardiovascular stability by causing changes in cardiac function, vascular reactivity, and cardiovascular reflexes and significantly alter distribution of cardiac output to various organs. Their overall impact is often systemic hypotension, which is attributable to myocardial depression, peripheral vasodilation, and attenuated sympathetic nervous system activity. However, one could be more causative than the others, depending on anesthetic agents and cardiovascular factors inherent in patients (e.g., coexisting heart disease). It is generally believed that most general anesthetics attenuate sympathetic nervous system outflow from the central nervous system, thereby decreasing vascular resistance in peripheral circulations. Indeed, in previous in vivo studies, during administration of various general anesthetics, vascular resistance was decreased in most peripheral circulations; however, it was unaffected or increased in some peripheral circulations. General anesthetics may act directly on vascular smooth muscle and/or endothelial cells in various vascular beds, influencing total peripheral and/or regional vascular resistance, and hence organ blood flow. This article reviews previously reported direct (i.e., nonneural) vascular actions of general anesthetics and discusses their underlying mechanisms, their in vivo relevance, and the future of research for general anesthetic vascular pharmacology.     

Autonomic neuroeffector mechanisms in smooth muscle

While the classical view is that the autonomic nervous system consists largely of antagonistic cholinergic and adrenergic nerves, about sixteen putative neurotransmitters in the autonomic nervous system have been proposed in the past few years, including various monoamines, polypeptides, purines and amino acids. Modulatory transmitter mechanisms have also been recognised, including prejunctional inhibition or enhancement of transmitter release, postjunctional modulation of transmitter action, and the secondary involvement of locally synthesised hormones and prostaglandins. The existence of more than one transmitter substance in some nerves is now widely recognised, and suggestions are made about the ways that this can lead to further peripheral control mechanisms at nerve terminals themselves. The cotransmitters always have synergistic actions on postjunctional effector cells, but two different mechanisms are postulated. (1) If both substances are stored in the same vesicles (for example, ACh or NA with ATP), release is closely parallel at all impulse frequencies. Upon release, the cotransmitter, in addition to having a direct action on postjunctional cells, may facilitate the action of the other transmitter and/or act as an inhibitor of its release. (2) If the two substances are stored in separate and different vesicle types (for example ACh or NA with some peptides), then differential release is possible at different impulse frequencies; the peptides released at higher frequencies modulate the role of the classical transmitters, by both prejunctional enhancement of its release and postjunctional facilitation of its action. The long-term effects by 'trophic' factors released by excitable cells including NGF, purine nucleosides and polypeptides, on development and regeneration were considered. The part played by peripheral neuroeffector control mechanisms has been underestimated. These are additional to central and ganglionic control mechanisms and are much more elaborate than originally thought.     

Increased ischemia-reperfusion blood flow impairs the skeletal muscle contractile function.

BACKGROUND: The ultimate aim of replantations and transplantations of skeletal muscle is to improve impaired function. The purpose of this study was to examine the contribution of varying durations of ischemia to postischemic blood flow in the skeletal muscle and the contribution of modulation of postischemic blood flow to skeletal muscle function and viability, using an ischemic revascularized hind limb model in rats. MATERIALS AND METHODS: Warm ischemia produced by vascular pedicle clamping was sustained for 90 min, 3 h, or 6 h. Postischemic blood flow was measured by a Doppler flowmeter or microsphere technique. In another series of experiments of 3-h ischemia, either saline or N(G)-methyl-l-arginine acetate (l-NMMA) was infused for the first 2 h of reperfusion. Postischemic blood flow was also measured. Muscle contractile function and viability were determined after 24 h of reperfusion. RESULTS: Postischemic blood flow was significantly increased during the first 10 min of reperfusion in the 90-minute ischemic group and during the first 2 h in the 3-h ischemic group compared with contralateral control blood flow. No significant increase in postischemic blood flow was noted in the 6-h ischemic group. Postischemic blood flow was significantly decreased by the l-NMMA infusion. Contractile function and viability of the tibialis anterior muscle and contractile function of the gastrocnemius muscle in the l-NMMA group were significantly increased. CONCLUSION: Reperfusion blood flow increased time dependently until 3 h of warm ischemia. Hyperemia deteriorated skeletal muscle contractile function, although it was well preserved by l-NMMA infusion to restrict the postischemic hyperemia.     

Antagonist drugs and bone vascular smooth muscle.

An ex vivo canine tibia model was used to quantitate the specific adrenergic subtype contribution in bone vasculature. Tibiae were obtained from mongrel dogs, the nutrient artery was catheterized, and the bone was placed in an ex vivo perfusion apparatus at constant flow. Perfusion was accomplished using oxygenated Krebs-Ringer solution. A norepinephrine dose-response curve was obtained by using incremental single bolus doses. Each bone was perfused with a vasoactive drug at a standard physiologic dosage. After 30 min of perfusion, a second norepinephrine dose-response curve was generated. The degree of attenuation of the norepinephrine dose-response curve, as determined by the total area under the curve, was interpreted as the relaxation effect of the drug on the smooth muscle of the vascular bed. Prazosin (alpha 1-receptor antagonist), rauwolszin (alpha 2-receptor antagonist), propranolol (beta-receptor antagonist), and diltiazem (calcium-entry inhibitor) were evaluated. Our data suggest that alpha 1 and alpha 2 adrenergic receptor antagonism results in a quantitatively similar attenuation of norepinephrine-induced vascular smooth muscle contraction. Calcium-entry antagonism produced less, but significant, attenuation of smooth muscle contractility. Beta-adrenergic receptor blockade yielded only a slight, although consistent, reduction in reactivity. Simple perfusion with Krebs-Ringer solution had no effect.     

Hypercholesterolemia impairs endothelium-dependent relaxation of rabbit corpus cavernosum smooth muscle

The majority of cases of impotence are associated with vascular risk factors such as diabetes, hypercholesterolemia, hypertension and smoking. These factors induce impairment of endothelium-dependent relaxation of blood vessels in man and in experimental animals. In this study the effects of hypercholesterolemia on the reactivity of rabbit corpus cavernosum smooth muscle strips to endothelium-dependent and endothelium-independent agents were investigated. New Zealand White rabbits (n = 14) were randomly divided into control and treatment groups. The control group (n = 7) received a regular diet while the treatment group (n = 7) was fed a diet of 0.5% cholesterol and 4% peanut oil for 10 weeks. Animals were then sacrificed and the corporal tissue studied in organ chambers for isometric tension measurement. Tissue was contracted with phenylephrine and concentration-dependent relaxation to acetylcholine, in the presence and absence of indomethacin, and to nitroprusside were examined. Blood level of cholesterol in the cholesterol-fed group was significantly higher compared to the control group. Contractions to phenylephrine were similar in both groups. Hypercholesterolemia, however, inhibited relaxation to acetylcholine but did not alter relaxation to nitroprusside, a cyclic guanosine monophosphate (cGMP)-dependent, direct smooth muscle dilator. Indomethacin enhanced the relaxations to acetylcholine in both control and cholesterol-fed groups but did not correct the difference in the relaxation to acetylcholine between both groups. It is concluded that hypercholesterolemia impairs endothelium-mediated relaxation of rabbit corpus cavernosum smooth muscle. The mechanism for the endothelial dysfunction does not appear to involve alteration in cyclooxygenase products of arachidonate or the cGMP-dependent relaxation of corporal smooth muscle. Impairment of endothelium-dependent relaxation of corporal smooth muscle may contribute to the pathophysiology of impotence associated with hypercholesterolemia in man.     

血管平滑肌细胞凋亡与尿毒症血管钙化

目的 探讨尿毒症患者血管平滑肌细胞（VSMC）凋亡与血管钙化之间的关系及其与临床指标的相关性。 方法 在43例尿毒症患者行动-静脉内瘘手术时留取桡动脉0.5~1 cm。用茜素红染色判断血管中膜钙化，并根据茜素红染色结果将患者分为钙化组和无钙化组。以肾功能正常、年龄相匹配的因外周血管疾病行血管手术的4例患者的动脉为对照组。采用DNA 片段原位缺口末端标记技术(TUNEL 法)检测上述3组血管平滑肌细胞凋亡情况，结果用凋亡指数表示。用免疫组化的方法检测凋亡相关基因p53、Bcl-2的表达情况。临床指标包括血钙、血磷、钙磷乘积、全段甲状旁腺素（iPTH）、血浆白蛋白（Alb）、血红蛋白（Hb）、总胆固醇（TC）、三酰甘油（TG）、低密度脂蛋白（LDL）、C反应蛋白（CRP）、丙二醛（MDA）、超氧化物歧化酶（SOD）。用Pearson相关分析计算凋亡指数与上述临床指标的相关性。 结果 尿毒症患者的血管中膜均可见到明显的VSMC凋亡，钙化组VSMC凋亡指数显著高于无钙化组（48.13±17.81比15.90±8.25，P ＜ 0.01）。而在对照组血管中膜只有极少量凋亡的VSMC。钙化组p53表达比无钙化组和对照组明显升高（31.22±14.25比20.67±7.35、4.17±2.61，P ＜ 0.01），Bcl-2表达比对照组明显降低（4.29±2.16比14.87±5.62，P ＜ 0.01)。VSMC凋亡指数与临床指标中的钙磷乘积、血磷和LDL水平呈正相关（P ＜ 0.05）。 结论 尿毒症患者桡动脉中膜存在VSMC凋亡，且VSMC凋亡发生在血管钙化之前。VSMC凋亡可能参与了尿毒症患者血管钙化的发生和发展。     

Acetate relaxation of isolated vascular smooth muscle

The vasorelaxant effects of acetate in arginine vasopressin (AVP)-contracted rat tail artery strips were examined in order to study mechanism of action. Dose-dependent relaxation by acetate was found in the clinically important range of 4 to 16 mM. Relaxation was not due to complexing of ionized calcium, persisted after mechanical removal of the endothelium, and was not altered by pretreatment with indomethacin. Although acetate also inhibited contraction by alpha-1 and alpha-2 agonists, the relaxant effect was not altered by destruction of sympathetic nerve terminals using 6-hydroxydopamine. The degree of relaxation in this model by various anions correlated with their lyotropic properties; however, the vasorelaxant effect of acetate exceeded that which would be expected on the basis of its position in the lyotropic series. The vasorelaxant effect of acetate was shared by other short-chain fatty acids that can be conjugated with coenzyme A (CoA), such as propionate and malonate. In contrast, a much lesser or absent relaxant effect was found with nonfatty-acid precursors of acetyl CoA, such as pyruvate, lactate, and alanine. The vasorelaxant effect of acetate was abolished by pretreatment with DIDS, an inhibitor of organic anion uptake, suggesting that cellular uptake of acetate is essential to its vasorelaxant action. The results suggest that the relaxant effect of acetate in vascular smooth muscle is non-specific, is not mediated by prostaglandins, does not depend upon the presence of either endothelium or the sympathetic nervous system, and may be due to metabolism of acetate to acetyl CoA with attendant conversion of ATP to AMP.     

Cellular stress inhibits vascular smooth muscle relaxation

PURPOSE: Cellular stress has been shown to induce a group of proteins called heat shock proteins (HSPs). Recent evidence suggests that a group of small HSPs may modulate vascular smooth muscle contraction (HSP27) and/or relaxation (HSP20). In this investigation, we hypothesized that cellular stress would alter contraction and/or relaxation of intact vascular smooth muscles and would lead to changes in the induction and/or phosphorylation of the small HSPs. METHODS: Bovine carotid arteries were obtained from an abattoir, and physiologic contractile responses were determined in a muscle bath. Phosphorylation state-specific antibodies were produced and characterized against HSP27. Phosphorylation events were determined with phosphorylation state-specific antibodies or whole-cell phosphorylation and two-dimensional gel electrophoresis. RESULTS: Cellular stress induced by arsenite or heat shock did not alter basal tone or the magnitude of contractions induced by serotonin or high extracellular potassium chloride. However, cellular stress led to inhibition of forskolin and sodium nitroprusside-induced vasorelaxation. This impaired vasorelaxation was associated with increases in the phosphorylation of HSP27 and decreases in forskolin-induced phosphorylation of HSP20. CONCLUSION: Cellular stress, which leads to increases in the phosphorylation of HSP27, inhibits cyclic nucleotide-dependent vascular relaxation and cyclic nucleotide-dependent increases in the phosphorylation of HSP20.     

血管平滑肌细胞在糖尿病血管病变中的作用

糖尿病血管病变是常见的糖尿病并发症之一,可危及全身各个器官,是糖尿病患者死亡的重要原因。目前常规治疗手段对严重的糖尿病血管病变的效果有限。血管平滑肌细胞(VSMC)在糖尿病血管病变的进程中发挥重要作用,因此,通过调控VSMC来逆转糖尿病血管病变有广泛的应用前景。笔者就VSMC的生物学特征及其在糖尿病中的表型转换特点与VSMC调控应用于糖尿病的治疗策略进行综述。     

Modulation of lymphatic smooth muscle contractile responses by the endothelium.

The endothelium regulates smooth muscle tone in blood vessels through the release of endothelium-deprived relaxing factor (EDRF). We hypothesized that the lymphatics possess endothelium-dependent relaxant properties analogous to those in blood vessels. Fresh porcine tracheobronchial lymphatic vessel rings were mounted in organ baths and connected to force-displacement transducers. Rings were submaximally precontracted with 10(-5) M histamine and exposed to cumulative addition of acetylcholine (ACH; 10(-7)-3 x 10(-4) M) or bradykinin (BRD; 10(-8)-3 x 10(-6) M), both of which are known to promote release of EDRF. ACH caused concentration-dependent relaxation (maximum effect = -2.3 +/- 2.6% of initial histamine-induced active tension), while a similar but less pronounced effect was seen with BRD (39.6 +/- 5.4%). The relaxant effects of ACH and BRD were inhibited by collagenase pretreatment and mechanical endothelial denudation. The results confirm our hypothesis that lymphatic vessels possess endothelium-dependent relaxant properties and suggest that lymph vessels can regulate lymph flow through processes similar to those found in blood vessels.