Circulating microparticles as biomarkers of stroke: A focus on the value of endothelial- and platelet-derived microparticles

Stroke is one of the leading causes of mortality and disability worldwide. Numerous pathophysiological mechanisms involving blood vessels, coagulation and inflammation contribute to the vascular occlusion. Perturbations in these pathways can be detected by numerous methods including changes in endoplasmic membrane remodeling and rearrangement leading to the shedding of microparticles (MPs) from various cellular origins in the blood. MPs are small membrane-derived vesicles that are shed from nearly all cells in the body in resting state or upon stimulation. MPs act as biological messengers to transfer information to adjacent and distant cells thus regulating various biological processes. MPs may be important biomarkers and tools for the identification of the risk and diagnosis of cerebrovascular diseases. Endothelial activation and dysfunction and altered thrombotic responses are two of the main features predisposing to stroke. Endothelial MPs (EMPs) have been recognized as both biomarkers and effectors of endothelial cell activation and injury while platelet-derived MPs (PMPs) carry a strong procoagulant potential and are activated in thrombotic states. Therefore, we reviewed here the role of EMPs and PMPs as biomarkers of stroke. Most studies reported high circulating levels of EMPs and PMPs in addition to other cell origins in stroke patients and have been linked to stroke severity, the size of infarction, and prognosis. The identification and quantification of EMPs and PMPs may thus be useful for the diagnosis and management of stroke.     

Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells

Platelet-derived microparticles (PMP) bind and modify the phenotype of many cell types including endothelial cells. Recently, we showed that PMP were internalized by human brain endothelial cells (HBEC). Here we intend to better characterize the internalization mechanisms of PMP and their intracellular fate. Confocal microscopy analysis of PKH67-labelled PMP distribution in HBEC showed PMP in early endosome antigen 1 positive endosomes and in LysoTracker-labelled lysosomes, confirming a role for endocytosis in PMP internalization. No fusion of calcein-loaded PMP with HBEC membranes was observed. Quantification of PMP endocytosis using flow cytometry revealed that it was partially inhibited by trypsin digestion of PMP surface proteins and by extracellular Ca(2+) chelation by EDTA, suggesting a partial role for receptor-mediated endocytosis in PMP uptake. This endocytosis was independent of endothelial receptors such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and was not increased by tumour necrosis factor stimulation of HBEC. Platelet-derived microparticle internalization was dramatically increased in the presence of decomplemented serum, suggesting a role for PMP opsonin-dependent phagocytosis. Platelet-derived microparticle uptake was greatly diminished by treatment of HBEC with cytochalasin D, an inhibitor of microfilament formation required for both phagocytosis and macropinocytosis, with methyl-β-cyclodextrin that depletes membrane cholesterol needed for macropinocytosis and with amiloride that inhibits the Na(+)/H(+) exchanger involved in macropinocytosis. In conclusion, PMP are taken up by active endocytosis in HBEC, involving mechanisms consistent with both phagocytosis and macropinocytosis. These findings identify new processes by which PMP could modify endothelial cell phenotype and functions.     

肌肽对低氧所致大鼠血管内皮细胞损伤的保护作用

目的：研究肌肽对低氧所致大鼠血管内皮细胞损伤的影响。方法：建立低氧条件下大鼠血管内皮细胞损伤模型,用MTT法观察肌肽对低氧损伤的血管内皮细胞活性的影响,测定细胞培养基中LDH活力,并对细胞骨架进行考马斯亮蓝R-250染色观测其细胞结构。结果：浓度为10mmol/L～20mmol/L肌肽孵育血管内皮细胞6h后,可以抑制缺氧12h和24h引起的血管内皮细胞活性下降,同时减少LDH的释放,保持细胞骨架完整。结论：肌肽对低氧所致的血管内皮细胞损伤具有保护作用。     

Significance of neutrophil microparticles in ischaemia‐reperfusion: Pro‐inflammatory effectors of endothelial senescence and vascular dysfunction

Endothelial senescence is an emerging cause of vascular dysfunction. Because microparticles are effectors of endothelial inflammation and vascular injury after ischaemia‐reperfusion, we examined leucocyte‐derived microparticles of spleen origin as possible contributors. Microparticles were generated from primary rat splenocytes by either lipopolysaccharide or phorbol‐myristate‐acetate/calcium ionophore, under conditions mimicking innate and adaptive immune responses. Incubation of primary porcine coronary endothelial cells with either type of microparticles, but not with those from unstimulated splenocytes, leads to a similar threefold raise in senescence‐associated β‐galactosidase activity within 48 hours, indicating accelerated senescence, to endothelial oxidative stress, and a fivefold and threefold increase in p21 and p16 senescence markers after 24 hours. After 12‐hour incubation, the endothelial‐dependent relaxation of coronary artery rings was reduced by 50%, at distinct optimal microparticle concentration. In vitro, microparticles were pro‐thrombotic by up‐regulating the local angiotensin system, by prompting tissue factor activity and a secondary generation of pro‐coagulant endothelial microparticles. They initiated an early pro‐inflammatory response by inducing phosphorylation of NF‐κB, MAP kinases and Akt after 1 hour, and up‐regulated VCAM‐1 and ICAM‐1 at 24 hours. Accordingly, VCAM‐1 and COX‐2 were also up‐regulated in the coronary artery endothelium and eNOS down‐regulated. Lipopolysaccharide specifically favoured the shedding of neutrophil‐ and monocyte‐derived microparticles. A 80% immuno‐depletion of neutrophil microparticles reduced endothelial senescence by 55%, indicating a key role. Altogether, data suggest that microparticles from activated splenocytes prompt early pro‐inflammatory, pro‐coagulant and pro‐senescent responses in endothelial cells through redox‐sensitive pathways. The control of neutrophil shedding could preserve the endothelium at site of ischaemia‐reperfusion–driven inflammation and delay its dysfunction.     

Vascular endothelial growth factor and substrate mechanics regulate in vitro tubulogenesis of endothelial progenitor cells

Endothelial progenitor cells (EPCs) in the circulatory system have been suggested to maintain vascular homeostasis and contribute to adult vascular regeneration and repair. These processes require that EPCs break down the extracellular matrix (ECM), migrate, differentiate and undergo tube morphogenesis. Evidently, the ECM plays a critical role by providing biochemical and biophysical cues that regulate cellular behaviour. Using a chemically and mechanically tunable hydrogel to study tube morphogenesis in vitro, we show that vascular endothelial growth factor (VEGF) and substrate mechanics co‐regulate tubulogenesis of EPCs. High levels of VEGF are required to initiate tube morphogenesis and activate matrix metalloproteinases (MMPs), which enable EPC migration. Under these conditions, the elasticity of the substrate affects the progression of tube morphogenesis. With decreases in substrate stiffness, we observe decreased MMP expression while increased cellular elongation, with intracellular vacuole extension and coalescence to open lumen compartments. RNAi studies demonstrate that membrane type 1‐MMP (MT1‐MMP) is required to enable the movement of EPCs on the matrix and that EPCs sense matrix stiffness through signalling cascades leading to the activation of the RhoGTPase Cdc42. Collectively, these results suggest that coupled responses for VEGF stimulation and modulation of substrate stiffness are required to regulate tube morphogenesis of EPCs.     

带鱼下脚料水解物对大鼠血管内皮损伤的影响

通过检测带鱼下脚料蛋白酶水解物的药理作用,为带鱼下脚料的开发利用提供理论依据。用带鱼下脚料三酶混合水解物喂养盐酸肾上腺素造成血管内皮损伤模型的大鼠,检测大鼠血清中血管内皮生长因子(VEGF)和血管性血友病因子(vWF)的含量变化。结果表明:实验不给药组与空白对照组及实验给药组相比,VEGF和vWF的含量存在显著差异。注射盐酸肾上腺素成功制造了血管内皮损伤模型,带鱼下脚料蛋白酶水解物对内皮损伤没有显著影响,但对损伤后的内皮修复有明显的促进作用。     

The extracellular matrix and the control of proliferation of vascular endothelial and vascular smooth muscle cells

In this short review we describe the observations which have led us to conclude that one of the most important components involved in modulating cell proliferation in vitro, and probably in vivo as well, may be the extrac-cellular matrix upon which cells rest.     

Evaluation of bone-derived and marrow-derived vascular endothelial cells by microarray analysis

This study focused on the differential expression levels of proteins that may exist between bone-derived and marrow-derived vascular endothelial cells (BVEC and MVEC). The vascular cells were isolated from trabecular bone regions and central marrow cavity regions of mouse long bones. Cells were cultured for 1 week to expand the population then separated from non-vascular cells using biotinylated isolectin B4, streptavidin-coated metallic microbeads, and a magnetic column. After an additional week of culture time, RNA was isolated from both cell types and compared using microarray analysis. RT-PCR was used to confirm and relatively quantitate the RNA messages. The bone-derived cells expressed more aldehyde dehydrogenase 3A1 (ALDH3A1), Secreted Modular Calcium-2 (SMOC-2), CCAAT enhancer binding protein (C/EBP-beta), matrix metalloproteinase 13 (MMP-13), and annexin 8 (ANX8) than the marrow-derived cells. Spalpha and matrix GLA-protein (MGP) were produced in greater abundance by the marrow-derived cells. This study reveals that there are profound and unique differences between the vasculature of the metaphysis as compared to that of the central marrow cavity. The unique array of proteins expressed by the bone-derived endothelial cells may support growth of tumors from cancer cells that frequently metastasize and lodge in the trabecular bone regions.     

内皮祖细胞在炎症损伤修复中的作用和机制

内皮祖细胞（endothelial progenitor cells,EPCs）是出生后,可以在机体内分化为成熟内皮细胞的一种前体细胞,主要来源于骨髓。多种伴有血管内皮细胞损伤的疾病都可引起外周血EPCs数量变化。有研究显示EPCs参与炎性损伤修复,并且外周血EPCs数量与血管内皮损伤程度和疾病预后存在一定的相关关系。EPCs。通过动员、迁移、归巢和分化等步骤修复内皮。炎症反应中受损组织释放的基质细胞衍生因子、血管内皮生长因子可与EPCs相应的受体结合,通过内皮型一氧化氮合酶、基质金属蛋白酶9等途径调节内皮修复过程,这是EPCs分化为内皮细胞过程的主要调控机制。此外,EPCs还可通过旁分泌机制促进相邻的内皮细胞增殖分化。目前,EPCs在炎症领域仅用于内皮炎性损伤和疾病预后评估,但是EPCs在心血管疾病和组织工程领域应用研究的成功,为EPCs在炎症反应的诊断和治疗提供了新的思路。     

A comparison of the tube forming potentials of early and late endothelial progenitor cells

The identification of circulating endothelial progenitor cells (EPCs) has revolutionized approaches to cell-based therapy for injured and ischemic tissues. However, the mechanisms by which EPCs promote the formation of new vessels remain unclear. In this study, we obtained early EPCs from human peripheral blood and late EPCs from umbilical cord blood. Human umbilical vascular endothelial cells (HUVECs) were also used. Cells were evaluated for their tube-forming potential using our novel in vitro assay system. Cells were seeded linearly along a 60 μm wide path generated by photolithographic methods. After cells had established a linear pattern on the substrate, they were transferred onto Matrigel. Late EPCs formed tubular structures similar to those of HUVECs, whereas early EPCs randomly migrated and failed to form tubular structures. Moreover, late EPCs participate in tubule formation with HUVECs. Interestingly, late EPCs in Matrigel migrated toward pre-existing tubular structures constructed by HUVECs, after which they were incorporated into the tubules. In contrast, early EPCs promote sprouting of HUVECs from tubular structures. The phenomena were also observed in the in vivo model. These observations suggest that early EPCs cause the disorganization of pre-existing vessels, whereas late EPCs constitute and orchestrate vascular tube formation.     

Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells

Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization, but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line, where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter, we screened for > 60 bioactive small molecules that would promote endothelial differentiation, and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated > 50% conversion of hPSCs to endothelial cells (ECs), specifically VEC⁺CD31⁺CD34⁺CD14⁻KDR^high endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling, in combination with VEGF-A treatment, resulted in efficient formation of EPs via KDR⁺ mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods, which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs.     

CRBP-III:lacZ expression pattern reveals a novel heterogeneity of vascular endothelial cells

Vascular endothelial cells are structurally and functionally heterogeneous. However, the molecular basis of this heterogeneity remains poorly defined. We used subtractive and differential screening to identify genes that exhibit heterogeneous expression patterns among vascular endothelial cells. One such gene is cellular retinol binding protein III (CRBP-III/Rbp7). Analysis of the lacZ knockin line for this gene (CRBP-III:lacZ) revealed a novel organ-specific vascular endothelial expression pattern. LacZ was expressed in vascular endothelial cells in heart, skeletal muscle, adipose tissues, thymus, and salivary gland. However, it was not detected in other tissues such as brain, liver, and lung. Furthermore, the expression within each organ was primarily restricted to small capillary endothelial cells, but could not be detected in larger vessels. This organ-specific vascular endothelial expression of CRPB:lacZ is relatively resistant to the changes of organ microenvironment. However, the level of expression can be modified by vitamin A deficiency. Therefore, our results provide novel molecular evidence for the heterogeneity of vascular endothelial cells.     

T17b murine embryonal endothelial progenitor cells can be induced towards both proliferation and differentiation in a fibrin matrix

Endothelial progenitor cells (EPC) may enhance blood vessel formation in a variety of clinical settings such as ischaemia and tumour angiogenesis as well as in tissue-engineered matrices. In the present study, we cultured a murine endothelial progenitor cell line, T17b, in vitro in cell culture as well as in an FDA-approved fibrin matrix and investigated cell proliferation, differentiation and secretion patterns of the angiogenic growth factor VEGF under hypoxia and differentiation. We show that T17b EPC remain viable for at least 8 days in the fibrin matrix where they proliferate and form clusters including lumen-like structures. Proliferation in fibrin clots overlayed with basal medium (BM) was confirmed morphologically and immunohistochemically by positive Ki67 staining, indicating mitotic activity. Significant cell proliferation and Ki-67 expression were absent when cells were incubated with dibutyryl-cAMP and retinoic acid (RA). Incubation with dibutyryl-cAMP and RA stimulated the expression of the EPC differentiation markers von Willebrand Factor (vWF) and VEGF receptor 2 (VEGFR-2), indicating successful differentiation in the fibrin clot. EPC differentiation induced by dibutyryl-cAMP and RA was confirmed in 2-D chamber slide cultures by positive vWF immunostaining, which was absent in BM controls. EPC chamber slides also displayed positive vWF staining when exposed to hypoxia under BM conditions, indicating EPC activation and differentiation could also be induced by hypoxia. Taken together, T17b EPC secrete increased levels of VEGF when submitted to either hypoxia or differentiation and can be differentiated into mature endothelial cells not only in cell and matrigel cultures but also in a fibrin matrix that is FDA approved for clinical application.     

Loss of chromosome 13 in cultured human vascular endothelial cells

In the vascular endothelium of human beings, telomere length is negatively related while the frequency of aneuploidy is positively related to donor age. Both in culture and in vivo the frequency of aneuploidy increases as telomere length is shortened. In this study we explored the relation between telomere length and aneuploidy in cultured human umbilical vein endothelial cells (HUVEC) by: (a) karyotype analysis and fluorescent in situ hybridization (FISH), (b) measurement of the terminal restriction fragments (TRF), and (c) assessment of replicative senescence by the expression of beta-galactosidase. Of 8 HUVEC strains, 7 cell strains lost chromosome 13, as shown by metaphase analysis and FISH of interphase cells. Five strains gained chromosome 11. In addition, five HUVEC strains became hypotetraploid shortly after the loss of chromosome 13. The loss of chromosome 13 was observed as early as PD 20, when mean TRF length was greater than 9 kb and the percentage of cells positive for beta-galactosidase was relatively low. The almost uniform loss of chromosome 13 suggests that this unique type of aneuploidy of HUVEC is the result of a progressive expression of clones with survival advantage.     

急性低氧条件下大鼠循环血中内皮细胞及肺小动脉内皮细胞的改变

目的:探讨急性低氧对血管内皮细胞(VEC)和循环血液中内皮细胞(CEC)的影响.方法:用光镜观察急性低氧情况下血中CEC的形态并记数,用组织化学染色和显微分光光度计对血中年轻型CEC及肺小动脉VEC的乳酸脱氢酶(LDH)活性进行定量分析.结果:与低氧前相比,低氧后各组VEC的LDH活性均下降,且随低氧时间延长,LDH活性下降越明显.而低氧前后各组年轻型CEC的LDH活性是一致的,且明显低于各组VEC的LDH活性.低氧前CEC多为衰老型,低氧后CEC数量增多且多为年轻型,随低氧时间延长年轻型CEC增多越明显.结论:CEC的形态和数量的变化可以反映血管的损伤情况;VEC的LDH活性可以反映VEC向CEC转化的进程;年轻型CEC的LDH活性可以反映VEC脱落时的损伤程度.     

淋巴细胞功能相关抗原-1在冻融PMN介导的VEC损伤中的作用

目的：探讨组织冻结融化造成血管内皮细胞（VEC）损伤的机理。方法：采用密度梯度离心法分离大鼠外周血嗜中性粒细胞（PMN）,速率冷冻仪冷冻PMN后水浴复温建立冻融PMN模型。冻融后4、12和24h,测定PMN表面淋巴细胞功能相关抗原－1（LFA－1）的表达;将冻融PMN与正常VEC共同孵育后测定培养液中乳酸脱氢酶活性及冻融PMN与VEC的粘附。结果：冻融后24h内,冻融PMN表面LFA－1的表面呈时间依赖性增强。（2）冻融PMN与VEC相互作用后,PMN－VEC粘附明显增强、VEC受到明显损伤。（3）抗LFA－1单克隆抗体明显抑制冻融PMN与VEC粘附的增强、明显减轻VEC损伤。结论：冻融可诱发PMN表面LFA－1的表达,进而增强PMN－VEC粘附而导致VEC损伤。