衰逝波探测川芎嗪与剪应力对内皮细胞膜PS异位的影响

为探讨川芎嗪和剪应力对血管内皮细胞凋亡的影响,以锥-板旋转式剪切装置提供剪应力,与川芎嗪共同作用于大鼠微血管内皮细胞(rCMEC),采用Annexin V-FITC染色标记,利用全内反射衰逝波激发ICCD成像技术,探测川芎嗪和剪应力对rCMEC磷脂酰丝氨酸(PS)异位的影响。结果表明,培养的内皮细胞膜表面PS异位具有普遍性,静态培养内皮细胞的PS异位率(异位程度)为9.97％。作为单因素,剪应力(6×10~(-5)、12×10~(-5)、24×10~(-5)N/cm~2)或川芎嗪(31.5、63.0、126.0μg/ml)均可使PS异位率明显下降(P<0.001)。在同一剪应力水平条件下,川芎嗪的给予可使内皮细胞的PS异位率进一步降低。中等水平的两因素组合降低异位率的效果较突出。结果提示,适当水平的剪应力与川芎嗪联合作用有可能明显抑制rCMEC凋亡。     

脉冲电场对粒细胞内Ca2+浓度影响的动态变化

利用激光共聚焦扫描显微镜和装有CCD系统的荧光显微镜,研究在单脉冲电场作用下经fluo-3/AM标记的鸡胚小脑粒细胞内自由Ca2+浓度(i)的动态变化过程.结果表明:在单个电脉冲作用下,细胞内Ca2+浓度立刻升高并达到其最大值.Ca2+浓度升高的幅度以及升高的速率具有电场强度的依赖性.当细胞外Ca2+被过量的EGTA络合或细胞膜上的Ca2+通道被La3+堵塞后,细胞内的Ca2+浓度仍然升高.细胞内不同区域的Ca2+浓度同时升高;两极内的Ca2+浓度早于胞体的Ca2+浓度达到最大值并迅速恢复.     

炎性刺激下中性粒细胞血管内皮细胞粘附的单细胞定量研究

炎性刺激下中性粒细胞血管内皮细胞粘附的单细胞定量研究周向东龙勉１（第三军医大学大坪医院,重庆６３００３８;重庆大学生物工程研究院１）中性粒细胞（ＰＭＮ）粘附于血管内皮细胞（ＶＥＣ）是炎性过程的第一步,故此两种细胞在致炎因素作用下其粘附特性的改变情况...     

水分胁迫及复水过程中小麦幼苗叶片内Ca2+的定位

展现了冬小麦幼苗在干旱胁迫及干旱后复水过程中叶肉细胞内Ca2 的动态分布 :在正常水分条件下生长的小麦幼苗 ,其细胞中的Ca2 主要位于液泡内 ,同时 ,细胞间隙中有大量的Ca2 分布。在水分胁迫下 ,随着胁迫时间的加长 ,液泡和细胞间隙的Ca2 逐渐进入细胞质 ,导致细胞质中自由Ca2 浓度过高 ,并对细胞造成伤害。复水后 ,细胞质中高浓度Ca2 迅速排入液泡和细胞间隙 ,细胞质中Ca2 浓度又基本恢复正常水平 ,形象地展示了细胞内Ca2 的稳态调控机制     

内皮细胞代谢与剪切作用时间的相关性

应用平行平板流动腔装置研究毛细血管内皮细胞内皮素（ＥＴ）代谢与剪切作用时间的相关关系。将培养的人胚肾小球血管单层内皮细胞置于剪应力分别为０,５×１０＾－５,１×１０＾－４和１．５×１０＾－４Ｎ／ｃｍ＾２的定常层流中切作用２５小时,样品的ＥＴ分泌量用放射免疫法测定,结果表明,剪切作用时间与内皮细胞ＥＴ的代谢活动有密切相关,ＥＴ分泌量高低不仅限决于剪应力大小,而且还取决于剪切作用时间长短,ＥＴ分泌量随     

CXCL8和CXCR2在血管内皮细胞趋化外周血循环纤维细胞中的作用

目的:周皮细胞的分化在血管新生过程中具有重要作用,没有周皮细胞及其分泌组建的基底膜的支撑,毛细血管就没有正常的功能.作者以前的工作证明周皮细胞可能来源于外周血循环纤维细胞(PBFC),但血管内皮细胞如何趋化PBFC还不清楚.本实验重点观察CXCL8及其受体CXCR2在血管内皮细胞趋化PBFC中的作用.方法:分离纯化人PBFC后与人微血管内皮细胞(HDMEC)共培养,观察共培养条件下PBFC的形态学改变,并检测PBFC细胞内CXCR2 mRNA表达和HDMEC内CXCL8mRNA的表达.结果:与HDMEC共培养后,PBFC由梭形向菱形改变;HDMEC内的CXCL8 mRNA水平与PBFC共培养24小时后增高约10倍,培养后48小时仍维持在高水平;PBFC内的CXCR2 mRNA水平在共培养后24小时增高约3倍,且在培养后24小时仍维持在较高水平.结论:CXCL8/CXCR2可能参与了血管内皮细胞趋化PBFC的过程.     

剪切应力对毛细血管内皮细胞代谢的影响

建立的平行平板流动腔装置适用于研究血管内皮细胞代谢对剪切流场的响应。将培养的人胚肾小球血管单层内皮细胞置于剪应力分别为５×１０－５Ｎ／ｃｍ２,１×１０－４Ｎ／ｃｍ２和１．５×１０－４Ｎ／ｃｍ２的定常层流中剪切２５小时,样品中的内皮素分泌量用放射免疫法测定。结果表明,剪应力水平对内皮细胞内皮素的代谢活动有显著影响。与静态培养对照,低水平的剪应力（５×１０－５Ｎ／ｃｍ２、１×１０－４Ｎ／ｃｍ２）促进内皮素的分泌,而较高水平的剪应力（１．５×１０－４Ｎ／ｃｍ２）抑制内皮素的分泌;剪应力对内皮素累积含量的影响比之分泌速率更大     

剪切力诱导微血管内皮细胞K~ 通道的开放

利用膜片钳及内皮细胞流动小室方法对大鼠脑微血管内皮细胞在剪切力作用下内皮细胞膜K 通道的开放进行了初步研究 ,结果提示脑微血管内皮细胞膜上存在剪切力敏感的K 通道 ,剪切力作用后 ,内皮细胞膜上K 电流明显增大 ,此电流有明显的短暂延迟现象 ,也可以被胞外施加的TEA抑制 ,符合IKv特征。流动剪切力可以通过影响内皮细胞膜上的K 通道的开放引起穿细胞的离子通透性的增加 ,进而引起细胞内Ca2 的变化。在K 、Ca2 等离子浓度改变的诱导下可以促使G -Actin装配为F -Actin。同时诱导内皮细胞内钙库调节机制的激活 ,这些变化都可以进一步引起细胞信号转导机制的激活。该工作为进一步开展剪切力对微血管内皮细胞信号转导机制的影响提供了实验数据。     

流体剪应力作用对内皮细胞变形的影响

血液流动和内皮的耦合是重要的生物医学问题,引起了学者们的广泛兴趣。目前已知流场剪应力对内皮细胞的形态和功能有重要影响,流体剪应力被认为是引起内皮细胞重建的始发信号。所以,了解流体剪应力与内皮细胞之间的相互作用机制是十分重要的。建立了一个理论模型来模拟流场剪应力与内皮细胞之间的相互作用。根据二维计算流体动力学方法研究了流体剪应力作用下内皮细胞表面的应力、压力分布。模拟结果表明:(1) 内皮细胞的变形随琢(对应于流体作用于细胞表面的剪应力)的变化而变化。当琢很小时(<0.02),流场剪应力对细胞变形的影响很小;随着琢的增大,细胞的变形也相应增大;当琢达到0.20以上时,细胞的变形变化很小,即细胞的形态保持相对稳定。(2) 流动引起了细胞表面应力和压力分布的不均匀,从而导致了细胞的变形,但内皮细胞的最大应力总是位于细胞的顶点。同时,用流室系统提供剪切流动,测量了不同剪应力作用下培养的人主动脉内皮细胞的变形,所得到的实验结果与数值模拟结果吻合。结果提示,由于剪切流动引起细胞表面应力分布的不均一,可能在细胞激活和细胞功能的调节(如细胞骨架的调节、粘附分子的表达与分布等)机制上具有特殊的作用。为应用流体动力学理论研究细胞(内皮细胞、白细胞等)变形以及细胞-基质粘     

内质网应激条件下血管内皮细胞生长因子在人脑微血管内皮细胞中的表达

为观察内质网应激条件下血管内皮细胞生长因子的表达情况,用不同浓度的衣霉素处理体外培养的人脑微血管内皮细胞,建立内质网应激模型,采用RT—PCR、蛋白质免疫印迹以及免疫细胞化学的方法检测了细胞内血管内皮细胞生长因子的表达。结果发现血管内皮细胞生长因子在人脑微血管内皮细胞中存在一定的表达;内质网应激可诱导血管内皮细胞生长因子表达升高,随着衣霉素浓度的增高,血管内皮细胞生长因子的表达逐渐增加,与mRNA水平相比,血管内皮细胞生长因子蛋白量的增加更明显。实验结果提示人脑微血管内皮细胞中存在血管内皮细胞生长因子自分泌,血管内皮细胞生长因子可能是内质网应激的靶基因。     

Effect of fluid force on vascular cell function

Endothelial cells (ECs) that line the inner surface of blood vessels are continuously exposed to fluid frictional force (shear stress) induced by blood flow, and shear stress affects the intracellular calcium ([Ca2+]i), which initiates cellular responses. Here, we studied the effect of long-term exposure of shear stress on [Ca2+]i responses in cultured ECs by using a confocal laser microscope and calcium indicator. At the initiation of shear stress of 20 dyn/cm2 (0 hr), 27% of the cells exhibited [Ca2+]i responses. This percentage gradually decreased with increasing exposure time, reaching about 4% after 24 hr of exposure. These data indicate that long-term shear-stress exposure affects [Ca2+]i responses in cultured ECs. Furthermore, we studied the effect of magnitude of shear stress on macromolecule uptake. For the low shear-stress, the uptake was enhanced, whereas the uptake was inhibited for higher shear-stress.     

Selective modulation of endothelial cell [Ca2+]i response to flow by the onset rate of shear stress

The response of endothelial cells (ECs) to their hemodynamic environment strongly influences normal vascular physiology and the pathogenesis of atherosclerosis. Unique responses to the complex flow patterns in lesion-prone regions imply that the temporal and spatial features of the mechanical stimuli modulate the cellular response to flow. We report the first systematic study of the effects of temporal gradients of shear stress on ECs. Flow was applied to cultured ECs using a novel cone-and-plate device allowing precise and independent control of the shear stress magnitude and the onset rate. Intracellular free calcium concentration ([Ca2+]i) increased rapidly following the onset of flow, and the characteristics of the transient were modulated by both the shear stress magnitude and onset rate. ECs were most sensitive to shear stress applied at physiological onset rates. Furthermore, the relative contribution of extracellular calcium and IP3-mediated release were dependent upon the specific flow regime.     

Interleukin-6-induced JAK2/STAT3 signaling pathway in endothelial cells is suppressed by hemodynamic flow

Endothelial cells (ECs) are constantly exposed to shear stress, the action of which triggers signaling pathways and cellular responses. During inflammation, cytokines such as IL-6 increase in plasma. In this study, we examined the effects of steady flow on IL-6-induced endothelial responses. ECs exposed to IL-6 exhibited STAT3 activation via phosphorylation of Tyr705. However, when ECs were subjected to shear stress, shear force-dependent suppression of IL-6-induced STAT3 phosphorylation was observed. IL-6 treatment increased the phosphorylation of JAK2, an upstream activator of STAT3. Consistently, shear stress significantly reduced IL-6-induced JAK2 activation. Pretreatment of ECs with an inhibitor of MEK1 did not alter this suppression by shear stress, indicating that extracellular signal-regulated kinase (ERK1/2) was not involved. However, pretreatment of ECs with an endothelial nitric oxide synthase inhibitor (nitro-L-arginine methyl ester) attenuated this inhibitory effect of shear stress on STAT3 phosphorylation. Shear stress-treated ECs displayed decreased nuclear transmigration of STAT3 and reduced STAT3 binding to DNA. Intriguingly, ECs exposed to IL-6 entered the cell cycle, as evidenced by increasing G₂/M phase, and shear stress to these ECs significantly reduced IL-6-induced cell cycle progression. STAT3-mediated IL-6-induced cell cycle was confirmed by the inhibition of the cell cycle in ECs infected with adenovirus carrying the inactive mutant of STAT3. Our study clearly shows that shear stress exerts its inhibitory regulation by suppressing the IL-6-induced JAK2/STAT3 signaling pathway and thus inhibits IL-6-induced EC proliferation. This shear force-dependent inhibition of IL-6-induced JAK2/STAT3 activation provides new insights into the vasoprotective effects of steady flow on ECs against cytokine-induced responses. shear stress; nitric oxide; cell cycle     

Laminar high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in endothelium. A quantitative analysis

Remodeling of endothelial basement membrane is important in atherogenesis. Since little is known about the actual relationship between type IV collagen and matrix metalloprotease−2 (MMP-2) in endothelial cells (ECs) under shear stress by blood flow, we performed quantitative analysis for type IV collagen and MMP-2 in ECs under high shear stress. The mRNA of type IV collagen from ECs exposed to high shear stress (10 and 30 dyn/cm²) had a higher expression compared to ECs exposed to a static condition or low shear stress (3 dyn/cm²) (P < 0.01). ³H-proline uptake analysis and fluorography revealed a remarkable increase of type IV collagen under high shear stress (P < 0.01). In contrast, zymography revealed that exposing to high shear stress, however similar positivity was leveled in the intracellular MMP-2 in the control and high shear stress-exposed ECs, reduced the secretion of MMP-2 in ECs. The results of Northern blotting, gelatin zymography and monitoring the intracellular trafficking of GFP-labeled MMP-2 revealed that MMP-2 secretion by ECs was completely suppressed by high shear stress, but the intracellular mRNA expression, protein synthesis, and transport of MMP-2 were not affected. In conclusion, we suggest that high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in ECs, which plays an important role in remodeling of the endothelial basement membrane and may suppress atherogenesis.     

Morphological differences between guinea pig aortic and venous endothelial cells in situ

Endothelial cells (ECs) respond to fluid shear stress. They reveal shear stress related morphological changes in both their cell shape and cytoskeletal organization. Little is known about the cytoskeletal organization of ECs in situ. We studied, together with the living ultrasound high resolution imaging system, the distribution of stress fibers (SFs), certain focal adhesion (FA) and signal transduction associated proteins in guinea pig aortic and venous ECs. Although SFs present in the basal portion of venous ECs ran along the direction of the blood flow, their size was smaller and their number was fewer than those of aortic ECs. Venous ECs were elongated to the direction of flow than in aortic ECs exposed over normal shear stress (SS). Since fluid SS in the vein is low, a sustained and uni-directional low SS over a long period might thus cause these structural features observed in venous ECs.     

Endogenously released ATP mediates shear stress-induced Ca2+ influx into pulmonary artery endothelial cells

The mechanisms by which flow-imposed shear stress elevates intracellular Ca2+ in cultured endothelial cells (ECs) are not fully understood. Here we report finding that endogenously released ATP contributes to shear stress-induced Ca2+ responses. Application of flow of Hanks' balanced solution to human pulmonary artery ECs (HPAECs) elicited shear stress-dependent increases in Ca2+ concentrations. Chelation of extracellular Ca2+ with EGTA completely abolished the Ca2+ responses, whereas the phospholipase C inhibitor U-73122 or the Ca2+-ATPase inhibitor thapsigargin had no effect, which thereby indicates that the response was due to the influx of extracellular Ca2+. The Ca2+ influx was significantly suppressed by apyrase, which degrades ATP, or antisense oligonucleotide targeted to P2X4 purinoceptors. A luciferase luminometric assay showed that shear stress induced dose-dependent release of ATP. When the ATP release was inhibited by the ATP synthase inhibitors angiostatin or oligomycin, the Ca2+ influx was markedly suppressed but was restored by removal of these inhibitors or addition of extracellular ATP. These results suggest that shear stress stimulates HPAECs to release ATP, which activates Ca2+ influx via P2X4 receptors.     

A model for studying the effect of shear stress on interactions between vascular endothelial cells and smooth muscle cells

Vascular endothelial cells (ECs) are constantly subjected to blood flow-induced shear stress and the influences of neighboring smooth muscle cells (SMCs). In the present study, a coculture flow system was developed to study the effect of shear stress on EC-SMC interactions. ECs and SMCs were separated by a porous membrane with only the EC side subjected to the flow condition. When ECs were exposed to a shear stress of 12 dynes/cm2 for 24 h, the cocultured SMCs tended to orient perpendicularly to the flow direction. This perpendicular orientation of the cocultured SMCs to flow direction was not observed when ECs were exposed to a shear stress of 2 dynes/cm2. Under the static condition, long and parallel actin bundles were observed in the central regions of the cocultured SMCs, whereas the actin filaments localized mainly at the periphery of the cocultured ECs. After 24 h of flow application, the cocultured ECs displayed very long, well-organized, parallel actin stress fibers aligned with the flow direction in the central regions of the cells. Immunostaining of platelet endothelial cell adhesion molecule-1 confirmed the elongation and alignment of the cocultured ECs with the flow direction. Coculture with SMCs under static condition induced EC gene expressions of growth-related oncogene-alpha and monocyte chemotactic protein-1, and shear stress was found to abolish these SMC-induced gene expressions. Our results suggest that shear stress may serve as a down-regulator for the pathophysiologically relevant gene expression in ECs cocultured with SMCs.     

IQ Domain GTPase-Activating Protein 1 is Involved in Shear Stress-Induced Progenitor-Derived Endothelial Cell Alignment

Shear stress is one of mechanical constraints which are exerted by blood flow on endothelial cells (ECs). To adapt to shear stress, ECs align in the direction of flow through adherens junction (AJ) remodeling. However, mechanisms regulating ECs alignment under shear stress are poorly understood. The scaffold protein IQ domain GTPase activating protein 1 (IQGAP1) is a scaffold protein which couples cell signaling to the actin and microtubule cytoskeletons and is involved in cell migration and adhesion. IQGAP1 also plays a role in AJ organization in epithelial cells. In this study, we investigated the potential IQGAP1 involvement in the endothelial cells alignment under shear stress. Progenitor-derived endothelial cells (PDECs), transfected (or not) with IQGAP1 small interfering RNA, were exposed to a laminar shear stress (1.2 N/m²) and AJ proteins (VE-cadherin and β-catenin) and IQGAP1 were labeled by immunofluorescence. We show that IQGAP1 is essential for ECs alignment under shear stress. We studied the role of IQGAP1 in AJs remodeling of PDECs exposed to shear stress by studying cell localization and IQGAP1 interactions with VE-cadherin and β-catenin by immunofluorescence and Proximity Ligation Assays. In static conditions, IQGAP1 interacts with VE-cadherin but not with β-catenin at the cell membrane. Under shear stress, IQGAP1 lost its interaction from VE-cadherin to β-catenin. This “switch” was concomitant with the loss of β-catenin/VE-cadherin interaction at the cell membrane. This work shows that IQGAP1 is essential to ECs alignment under shear stress and that AJ remodeling represents one of the mechanisms involved. These results provide a new approach to understand ECs alignment under to shear stress.     

DNA microarray study on gene expression profiles in co-cultured endothelial and smooth muscle cells in response to 4- and 24-h shear stress

Shear stress, a major hemodynamic force acting on the vessel wall, plays an important role in physiological processes such as cell growth, differentiation, remodelling, metabolism, morphology, and gene expression. We investigated the effect of shear stress on gene expression profiles in co-cultured vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Human aortic ECs were cultured as a confluent monolayer on top of confluent human aortic SMCs, and the EC side of the co-culture was exposed to a laminar shear stress of 12 dyn/cm² for 4 or 24 h. After shearing, the ECs and SMCs were separated and RNA was extracted from the cells. The RNA samples were labelled and hybridized with cDNA array slides that contained 8694 genes. Statistical analysis showed that shear stress caused the differential expression (p ≤ 0.05) of a total of 1151 genes in ECs and SMCs. In the co-cultured ECs, shear stress caused the up-regulation of 403 genes and down-regulation of 470. In the co-cultured SMCs, shear stress caused the up-regulation of 152 genes and down-regulation of 126 genes. These results provide new information on the gene expression profile and its potential functional consequences in co-cultured ECs and SMCs exposed to a physiological level of laminar shear stress. Although the effects of shear stress on gene expression in monocultured and co-cultured EC are generally similar, the response of some genes to shear stress is opposite between these two types of culture (e.g., ICAM-1 is up-regulated in monoculture and down-regulated in co-culture), which strongly indicates that EC–SMC interactions affect EC responses to shear stress.     

Gαq/11-mediated intracellular calcium responses to retrograde flow in endothelial cells

Disturbed flow patterns, including reversal in flow direction, are key factors in the development of dysfunctional endothelial cells (ECs) and atherosclerotic lesions. An almost immediate response of ECs to fluid shear stress is the increase in cytosolic calcium concentration ([Ca(2+)](i)). Whether the source of [Ca(2+)](i) is extracellular, released from Ca(2+) intracellular stores, or both is still undefined, though it is likely dependent on the nature of forces involved. We have previously shown that a change in flow direction (retrograde flow) on a flow-adapted endothelial monolayer induces the remodeling of the cell-cell junction along with a dramatic [Ca(2+)](i) burst compared with cells exposed to unidirectional or orthograde flow. The heterotrimeric G protein-α q and 11 subunit (Gα(q/11)) is a likely candidate in effecting shear-induced increases in [Ca(2+)](i) since its expression is enriched at the junction and has been previously shown to be activated within seconds after onset of flow. In flow-adapted human ECs, we have investigated to what extent the Gα(q/11) pathway mediates calcium dynamics after reversal in flow direction. We observed that the elapsed time to peak [Ca(2+)](i) response to a 10 dyn/cm(2) retrograde shear stress was increased by 11 s in cells silenced with small interfering RNA directed against Gα(q/11). A similar lag in [Ca(2+)](i) transient was observed after cells were treated with the phospholipase C (PLC)-βγ inhibitor, U-73122, or the phosphatidylinositol-specific PLC inhibitor, edelfosine, compared with controls. Lower levels of inositol 1,4,5-trisphosphate accumulation seconds after the onset of flow correlated with the increased lag in [Ca(2+)](i) responses observed with the different treatments. In addition, inhibition of the inositol 1,4,5-trisphosphate receptor entirely abrogated flow-induced [Ca(2+)](i). Taken together, our results identify the Gα(q/11)-PLC pathway as the initial trigger for retrograde flow-induced endoplasmic reticulum calcium store release, thereby offering a novel approach to regulating EC dysfunctions in regions subjected to the reversal of blood flow.