内皮依赖性超极化因子的研究进展

内皮依赖性超极化因子是由内皮细胞释放并作用于平滑肌细胞的非一氧化氮、非前列环素途径的血管舒张因子,其作用机制复杂,可通过钙激活钾通道开放、缝隙连接传递电化学信号以及内皮释放等方式作用于平滑肌细胞,最终导致血管舒张。高血压、糖尿病等血管性疾病的发生发展可能与内皮依赖性超极化因子相关,其有望成为心血管疾病治疗的新靶点。     

冠状动脉内皮功能的研究进展

血管内皮产生各种血管活性物质 ,可以调节平滑肌细胞舒缩活动 ,具有维持血管紧张 ,抑制血小板积聚和平滑肌细胞增生的功能[1] 。如果内皮功能受损 ,将会促使动脉粥样硬化的发生发展[2 ] 。研究发现 ,动脉粥样硬化的各种危险因素都可引起不同程度的内皮功能减低。而冠状动脉是发生动脉粥样硬化最常见和最重要的部位 ,因此检测冠状动脉内皮功能对于早期评价冠状动脉粥样硬化性心脏病具有重要临床价值。1　血管内皮依赖性舒张功能的发生机理1.1　一氧化氮 (ＮＯ) :血管内皮细胞分泌多种物质 ,参与血管紧张性的调节。其中发现ＮＯ是最重要的内皮…     

n-3多不饱和脂肪酸改善高胆固醇血症内皮依赖性舒张

为研究ｎ-３多不饱和脂肪酸对内皮的保护功能，本文在血管环内皮依赖性舒张、内皮细胞一氧化氮合成酶和平滑肌细胞功能等三个方面进行了研究。结果表明高胆固醇血症下的内皮依赖性舒张明显减弱是由于一氧化氨合成酶受损，ｎ-３多不饱和脂肪酸通过恢复一氧化氮合成酶功能，大幅度改善内皮依赖性舒张。说明ｎ-３多不饱和脂肪酸对动脉粥样硬化的有益作用机制之一是保护内皮功能。     

活血化瘀药对血管内皮功能的影响

目前人们已经开始认识到血管内皮细胞（VECs）不仅是血液和血管平滑肌的屏障，而且是高度活跃的代谢库，参与凝血及抗血栓形成，调节血管张力，影响血管通透性。目前许多学者发现血瘀证与血管内皮损伤有着密切联系，并开始利用活血化瘀药调节血管内皮细胞的内环境及其分泌功能，从而逆转内皮功能，已成为一个新的研究方向。现对近年来活血化瘀药对血管内皮功能的影响综述如下。     

衰老与内皮依赖血流介导的血管扩张功能异常

内皮是血流与血管平滑肌之间的生理屏障。由于其解剖部位 ,内皮是心血管危险因子作用的最先目标。正常内皮细胞可释放血管活性物质调控血管张力。内皮功能异常可导致血管矛盾运动。内皮细胞可产生许多缩血管舒血管物质 ,如 :一氧化氮 ,Ｐ物质 ,内皮素 ,血管紧张素 ,前列腺素 ,血栓素Ａ2及自由基 ,参与心血管病理生理改变。由于一氧化氮扩张血管及抑制血小板作用 ,其对血管功能具有重要的保护作用。内皮细胞可通过其自分泌 ,旁分泌甚至内分泌途径而发挥其作用。完整的内皮细胞通过其分泌的活性物质可影响血管细胞的生长增殖 ,血小板粘附聚集…     

L-精氨酸能改善血管内皮依赖性舒张和抗动脉粥样硬化的损伤

内皮细胞功能不良、血小板和粒细胞的粘附和聚集、机械损伤的炎症反应、中膜平滑肌细胞向内腊下迁移增殖等在动脉粥样硬化发生中起重要作用，而内皮细胞持续产生释放的一氧化氮除介导血管内皮依赖性舒张作用外，补充Ｌ－精氨酸（一氧化氮的前体），将增加一氧化氮的产生和改进内皮依赖性舒张功能。本实验将２４只新西兰兔分成４组：①未损伤组；②右侧髂动脉损伤＋２％胆固醇组；③２％精氨酸组；④右侧髂动脉损伤＋２％胆固醇＋２％     

内皮功能与血管活性物质

1976年Ross与Glomset创立了内皮细胞损伤学说,打开了血管病原学研究的新领域。Ross在最初的理论中提出长期高血压及血流冲击等切应力促使内皮损伤,引起血小板聚集及释放血管活性物质,造成血栓形成与血管收缩。1993年又阐述,内皮细胞功能障碍学说。在动脉硬化的初始病灶上,单核细胞、T细胞结合粘连分子后,游走侵入内皮,并释放活性物质,引起中膜平滑肌细胞向内膜游走和增生。同年,Rubaryi[1]提出,内皮是一种兼有感觉与效应的细胞。它作为中介器官,在感受血流压力变化、炎性信号及循环中激素水平的同时,做出调节反应;调节血管舒缩状态及细胞…     

血管内皮生长因子预防血管成形术后再狭窄的机理

为探讨血管内皮生长因子预防血管成形术后再狭窄的机理 ,构建含人血管内皮生长因子 165基因的重组腺病毒 ,感染体外培养的血管平滑肌细胞 ,将人脐静脉内皮细胞和血管平滑肌细胞分别分成对照组、H2 O2 处理组和H2 O2 +血管内皮生长因子处理组 ,采用WST 1比色法、原位末端标记法及流式细胞术检测各组细胞光密度值和凋亡发生情况。结果发现 ,人脐静脉内皮细胞中 ,与对照组和H2 O2 +血管内皮生长因子处理组比较 ,H2 O2 处理组光密度值降低 ,凋亡细胞明显增加 ;血管平滑肌细胞中上述改变相反。提示H2 O2 能促进平滑肌细胞增殖 ,诱导内皮细胞凋亡 ,抑制内皮细胞增殖及平滑肌细胞凋亡 ,促进再狭窄的发生 ,而血管内皮生长因子能拮抗H2 O2 的作用 ,有利于再狭窄的防治 ,为血管内皮生长因子预防再狭窄提供新的理论依据     

内皮祖细胞对血管紧张素Ⅱ诱导的血管平滑肌细胞表型转化的影响

目的 观察内皮祖细胞对血管紧张素Ⅱ诱导的血管平滑肌细胞表型转化的影响.方法 采用6%羟乙基淀粉沉降法和密度梯度离心法分离人脐血单个核细胞,EGM-2细胞培养基进行培养,诱导单个核细胞贴壁向内皮祖细胞分化.采用荧光显微镜双染色、流式细胞术鉴定内皮祖细胞,间接免疫荧光检测血管平滑肌细胞标志物平滑肌α-肌动蛋白、钙调节蛋白的表达,采用逆转录聚合酶链反应和免疫印迹检测早期内皮祖细胞条件培养液、晚期内皮祖细胞条件培养液以及人脐静脉内皮细胞条件培养液对血管紧张素Ⅱ诱导的血管平滑肌细胞收缩表型标志基因平滑肌α-肌动蛋白以及合成表型标志基因骨桥蛋白表达变化的影响.结果 与对照组比较,血管紧张素Ⅱ(10-6mmol/L)诱导血管平滑肌细胞增殖48 h后,平滑肌α-肌动蛋白mRNA和蛋白表达明显减少,而骨桥蛋白mRNA和蛋白表达明显增加,提示血管平滑肌细胞从收缩表型向合成表型转化;与血管紧张素Ⅱ组比较,早期内皮祖细胞条件培养液、晚期内皮祖细胞条件培养液以及人脐静脉内皮细胞条件培养液处理后均不同程度抑制血管紧张素Ⅱ诱导的平滑肌α-肌动蛋白表达减少和骨桥蛋白表达增加,其中以早期内皮祖细胞条件培养液的抑制效果最明显.结论 内皮祖细胞能够抑制血管紧张素Ⅱ诱导的血管平滑肌细胞从收缩表型向合成表型转化.     

钙通道阻滞剂与高血压血管内皮依赖性舒张功能

血管内皮细胞具有重要的生理功能,内皮功能不全与高血压病理生理过程密切相关.维护内皮功能的完整性对于预防心血管疾病事件的发生有重要的作用.由于血管内皮细胞上没有电压依赖性钙通道表达,因而钙通道阻滞剂(CCB)不是通过阻滞内皮细胞电压依赖性钙通道起作用.CCB可能通过对抗氧自由基和超氧阴离子对内皮的损伤,增加内皮细胞释放内皮源性舒张因子(EDRF),改善血管内皮依赖性舒张功能.研究表明:CCB对保护和改善内皮功能、改善心血管疾病预后方面有重要的作用.     

Incidence of myoendothelial gap junctions in the proximal and distal mesenteric arteries of the rat is suggestive of a role in endothelium-derived hyperpolarizing factor-mediated responses

Although the chemical nature of endothelium-derived hyperpolarizing factor (EDHF) remains elusive, electrophysiological evidence exists for electrical communication between smooth muscle cells and endothelial cells suggesting that electrotonic propagation of hyperpolarization may explain the failure to identify a single chemical factor as EDHF. Anatomical evidence for myoendothelial gap junctions, or the sites of electrical coupling, is, however, rare. In the present study, serial-section electron microscopy and reconstruction techniques have been used to examine the incidence of myoendothelial gap junctions in the proximal and distal mesenteric arteries of the rat where EDHF responses have been reported to vary. Myoendothelial gap junctions were found to be very small in the mesenteric arteries, the majority being <100 nm in diameter. In addition, they were significantly more common in the distal compared with the proximal regions of this arterial bed. Pentalaminar gap junctions between adjacent endothelial cells were much larger and were common in both proximal and distal mesenteric arteries. These latter junctions were frequently found near the myoendothelial gap junctions. These results provide the first evidence for the presence of sites for electrical communication between endothelial cells and smooth muscle cells in the mesenteric vascular bed. Furthermore, the relative incidence of these sites suggests that there may be a relationship between the activity of EDHF and the presence of myoendothelial gap junctions.     

Endothelial feedback and the myoendothelial projection

The endothelium plays a critical role in controlling resistance artery diameter, and thus blood flow and blood pressure. Circulating chemical mediators and physical forces act directly on the endothelium to release diffusible relaxing factors, such as NO, and elicit hyperpolarization of the endothelial cell membrane potential, which spreads to the underlying smooth muscle cells via gap junctions (EDH). It has long been known that arterial vasoconstriction in response to agonists is limited by the endothelium, but the question of how contraction of smooth muscle cells leads to activation of the endothelium (myoendothelial feedback) has, until recently, received little attention. Initial studies proposed the permissive movement of Ca(2+) ions from smooth muscle to endothelial cells to elicit release of NO. However, more recent evidence supports the notion that flux of IP(3) leading to localized Ca(2+) events within spatially restricted myoendothelial projections and activation of EDH may underlie myoendothelial feedback. In this perspective, we review recent data which supports the functional role of myoendothelial projections in smooth muscle to endothelial communication. We also discuss the functional evidence supporting the notion that EDH, as opposed to NO, is the primary mediator of myoendothelial feedback in resistance arteries.     

Heterocellular contact at the myoendothelial junction influences gap junction organization

Heterocellular communication between vascular smooth muscle cells (VSMC) and endothelial cells (EC) at the myoendothelial junction (MEJ) is a critical part of control of the arteriolar wall. We have developed an in vitro model of the MEJ composed of primary cultures of murine EC and VSMC. Immunoctytochemistry and immunoblots demonstrated Cx37 and Cx43 in both cell types, whereas Cx40 was found only in EC. Cx37 was excluded from the MEJ in both EC and VSMC. Connexin composition as well as functionality of the gap junctions at the MEJ was assessed by measuring diffusional transfer of biocytin and Cy3. Using connexin-specific blockers and manipulations of expression of individual connexin proteins, we confirmed that Cx37 is not a part of EC-VSMC coupling, and we demonstrated that heterotypic gap junctions are functional at the MEJ. We speculate that specific gap junction organization may be a vital component of EC-VSMC contact at the MEJ.     

Modulation of endothelial cell KCa3.1 channels during endothelium-derived hyperpolarizing factor signaling in mesenteric resistance arteries

Arterial hyperpolarization to acetylcholine (ACh) reflects coactivation of K(Ca)3.1 (IK(Ca)) channels and K(Ca)2.3 (SK(Ca)) channels in the endothelium that transfers through myoendothelial gap junctions and diffusible factor(s) to affect smooth muscle relaxation (endothelium-derived hyperpolarizing factor [EDHF] response). However, ACh can differentially activate K(Ca)3.1 and K(Ca)2.3 channels, and we investigated the mechanisms responsible in rat mesenteric arteries. K(Ca)3.1 channel input to EDHF hyperpolarization was enhanced by reducing external [Ca(2+)](o) but blocked either with forskolin to activate protein kinase A or by limiting smooth muscle [Ca(2+)](i) increases stimulated by phenylephrine depolarization. Imaging [Ca(2+)](i) within the endothelial cell projections forming myoendothelial gap junctions revealed increases in cytoplasmic [Ca(2+)](i) during endothelial stimulation with ACh that were unaffected by simultaneous increases in muscle [Ca(2+)](i) evoked by phenylephrine. If gap junctions were uncoupled, K(Ca)3.1 channels became the predominant input to EDHF hyperpolarization, and relaxation was inhibited with ouabain, implicating a crucial link through Na(+)/K(+)-ATPase. There was no evidence for an equivalent link through K(Ca)2.3 channels nor between these channels and the putative EDHF pathway involving natriuretic peptide receptor-C. Reconstruction of confocal z-stack images from pressurized arteries revealed K(Ca)2.3 immunostain at endothelial cell borders, including endothelial cell projections, whereas K(Ca)3.1 channels and Na(+)/K(+)-ATPase alpha(2)/alpha(3) subunits were highly concentrated in endothelial cell projections and adjacent to myoendothelial gap junctions. Thus, extracellular [Ca(2+)](o) appears to modify K(Ca)3.1 channel activity through a protein kinase A-dependent mechanism independent of changes in endothelial [Ca(2+)](i). The resulting hyperpolarization links to arterial relaxation largely through Na(+)/K(+)-ATPase, possibly reflecting K(+) acting as an EDHF. In contrast, K(Ca)2.3 hyperpolarization appears mainly to affect relaxation through myoendothelial gap junctions. Overall, these data suggest that K(+) and myoendothelial coupling evoke EDHF-mediated relaxation through distinct, definable pathways.     

Distinct endothelium-derived hyperpolarizing factors emerge in vitro and in vivo and are mediated in part via connexin 40-dependent myoendothelial coupling

The endothelium-derived hyperpolarizing factor (EDHF) contributes critically to the regulation of vascular tone. Its dependency on direct signaling through myoendothelial gap junctions composed of connexins (Cx) is controversially discussed. We studied the impact of Cx40 in EDHF-type dilations in vivo and in vitro (wire and pressure myography) in small arteries (A. gracilis) using different Cx40-deficient mouse models. Acetylcholine induced prominent EDHF-type dilations (inhibition of NO synthase and cyclooxygenase) of ≈90% (maximum effect) in wild-type and Cx40-deficient vessels (Cx40-/-) in vitro under isobaric conditions. In contrast, under isometric conditions, EDHF-type relaxations were nearly abrogated in Cx40-/- (9±3%) but only slightly reduced in wild-type vessels (45±4%; P<0.05). Vessels expressing Cx45 instead of Cx40 exhibited similarly reduced relaxations (13±1%), demonstrating that Cx45 cannot replace Cx40 functionally. The necessity of Cx40 in EDHF-type dilations under isometric conditions was verified by the attenuation in vessels being specifically deficient for Cx40 in endothelial cells (Cx40fl:TIE2-Cre: 17±3%; Cx40-floxed controls: 67±6%; P<0.05). Nevertheless, EDHF-type dilations were Cx40 independent when studied isobarically. The EDHF-type dilation in vivo resembled the isobaric situation, being virtually Cx40 independent and similar powerful. Distinct EDHF mechanisms can be distinguished by their Cx40 dependency. A powerful EDHF is present in vivo and in vitro under isobaric conditions but is lacking in wire myography (isometric conditions). Herein, a less potent EDHF depends on Cx40 and may represent signaling through myoendothelial gap junctions. We suggest that distinct EDHFs (even in the same artery) explain partially the controversy on the role of myoendothelial gap junctions in EDHF signaling.     

Expression of homocellular and heterocellular gap junctions in hamster arterioles and feed arteries

OBJECTIVES: Conduction of vasoconstrictor and vasodilator responses in the microcirculation involves electrical coupling through gap junction channels among cells of the vascular wall. The present study determined whether reported differences in the properties of conduction along the arterioles of the epithelial hamster cheek pouch (CPA) and feed arteries of its retractor skeletal muscle (RFA) result from differences in the expression profile of specific connexin (Cx) isoforms and the gap junctions they comprise. METHODS: Real-time PCR, immunohistochemistry and serial section electron microscopy were used to compare wall morphology and the distribution of gap junctions between respective vessels. RESULTS: Expression of mRNA for Cx37, 40, 43 and 45 was similar between CPA and RFA. In the endothelium, Cx37, 40 and 43 proteins were expressed abundantly between adjacent cells while Cx37 was present in the smooth muscle. In both vessels, endothelial and smooth muscle cell (SMC) layers were well connected by myoendothelial gap junctions (MEGJs), which were found near endothelial cell (EC) gap junctions. CONCLUSIONS: The absence of differential gap junctional expression between CPA and RFA, in spite of documented differences in cellular conduction pathways, supports the hypothesis that conductance of vascular gap junction channels can be differentially modulated in resistance microvessels.     

Contribution of connexins to the function of the vascular wall

Gap junction channels provide an enclosed conduit for direct exchanges of signalling molecules, including ions and small metabolites between cells. This system of communication allows cells to monitor the functional state of their neighbours, and is rapidly modulated to continuously adapt to the immediate needs of groups of coupled cells. In the major arteries, endothelial cells may express three connexins isotypes, namely Connexin 37 (Cx37), Cx40 and Cx43, whereas the underlying smooth muscle cells may express Cx37, Cx40, Cx43 and Cx45. Moreover, myoendothelial gap junctions have also been shown to be involved in the regulation of vascular tone. This review highlights the regulation of vessel connexins in response to injury, as observed during experimental hypertension or wound repair, as well as the consequences of loss of one connexin in different transgenic null mice. In view of the major endocrine role of the kidney in the control of blood pressure, we also discuss the distribution of connexins in the kidney vasculature. Cx40 is present between endothelial cells of vessels and glomeruli, as well as between renin-secreting cells, the modified smooth muscle cells which form the wall of the terminal part of afferent arterioles. Modulation of Cx40 expression in a model of renin-dependent hypertension suggests that this connexin may be implicated in the function of renin-secreting cells. Finally, to address the possible regulation of connexin expression by fluid pressure, we summarize the effects of elevated transmural urine pressure on bladder Cx43 expression.     

5-Methyltetrahydrofolate and tetrahydrobiopterin can modulate electrotonically mediated endothelium-dependent vascular relaxation

We have investigated the ability of 5-methyltetrahydrofolate (5-MTHF) and tetrahydrobiopterin (BH(4)) to modulate nitric oxide (NO)-independent vascular relaxations that are mediated by the sequential spread of endothelial hyperpolarization through the wall of the rabbit iliac artery by means of myoendothelial and homocellular smooth muscle gap junctions. Relaxations and subintimal smooth muscle hyperpolarizations evoked by cyclopiazonic acid were depressed by the gap junction inhibitor 2-aminoethoxydiphenyl borate, whose effects were prevented by 5-MTHF and BH(4), but not by their oxidized forms folic acid and 7,8-dihydrobiopterin. Analogously, 5-MTHF and BH(4), but not folic acid or 7,8-dihydrobiopterin, attenuated the depression of subintimal hyperpolarization by a connexin-mimetic peptide targeted against Cx37 and Cx40 ((37,40)Gap 26) and the depression of subadventitial hyperpolarization by a peptide targeted against Cx43 ((43)Gap 26), thus reflecting the known differential expression of Cx37 and Cx40 in the endothelium and Cx43 in the media of the rabbit iliac artery. The inhibitory effects of 2-aminoethoxydiphenyl borate and (37,40)Gap 26 against subintimal hyperpolarization were prevented by catalase, which destroys H(2)O(2). 5-MTHF and BH(4) thus appear capable of modulating electrotonic signaling by means of myoendothelial and smooth muscle gap junctions by reducing oxidant stress, potentially conferring an ability to reverse the endothelial dysfunction found in disease states through mechanisms that are independent of NO.     

Heterogeneous localization of connexin40 in the renal vasculature

The renal circulation, which treats 25% of the cardiac output, is organized and regulated in unique patterns. Gap junction channels may contribute to the control of vascular tone by transmitting intracellular signals rapidly between cells of the blood vessel. We investigated the distribution patterns of the major vascular gap junction proteins in the murine kidney by immunofluorescence staining of frozen sections, and connexin40 (Cx40) was the most prominent connexin detected. The endothelial cells of large vessels within the kidney consistently showed abundant Cx40 immunoreactivity, but small vessels showed unique distributions of Cx40 along their courses within the kidney. Cx40 immunoreactivity between endothelial cells was abundant in the interlobular arteries and the proximal portion of the afferent arterioles, but was significantly decreased when arterioles approached the glomerulus. No Cx40 immunoreactivity was detected in the region of the glomerular isthmus where the afferent and efferent arterioles join the glomeruli, although glomeruli showed very intense patchy staining for Cx40. Intense patchy staining for Cx40 was also found in the modified smooth muscle cells of the juxtaglomerular apparatus, but none was detected in smooth muscle cells elsewhere in the vasculature. Taken together, these data suggest that the abundance of Cx40-containing gap junctions may be important for coordinating function of cells within individual blood vessels, while their absence in juxtaglomerular regions of the arterioles may prevent conduction of signals between the glomerulus and afferent or efferent arterioles.     

Microscopic anatomy of the pulmonary vascular bed in the cat lung

The pulmonary microcirculation of the cat was analyzed by light and electron microscopy. In order to establish the precise structure and ultrastructure of the components of the vascular wall of each segment, special efforts were made to identify positively the pulmonary arterioles and venules by tracing their connection to small pulmonary arteries and veins, respectively. Also, the pulmonary arterioles and venules were studied with respect to their relationship to the alveolar capillary network via the precapillary sphincter areas and the postcapillary venules. It was confirmed that the small pulmonary arteries, arterioles, and precapillary sphincter areas are provided with smooth muscle cells which are present up to the point where the pulmonary capillaries branch out, although the number of smooth muscle cells decreases gradually toward the capillary bed. Cholinergic and noradrenergic nerves accompany all arterial segments. The capillary network described by many investigators in several mammalian species was studied only to the extent that a three-dimensional conceptualization could be obtained. With respect to the postcapillary venules and pulmonary venules, it was discovered that, in the cat lung, true smooth muscle cells, albeit widely scattered, are present in these segments of the pulmonary microcirculation. These smooth muscle cells display extensive areas of myoendothelial junctions. That is, the cell membranes of the endothelial cells and the smooth muscle cells make contact without an intervening basal lamina. Some myoendothelial junctions were identified also in the arterioles and precapillary sphincter areas. However, they were few in number and had points of only limited membrane contact. The functional implications of these findings are discussed in terms of possible regulatory influence on the pulmonary microcirculation and hypothetical role in the development of pulmonary hypertension.