肝细胞生长因子对骨髓内皮祖细胞的动员作用

目的: 分析肝细胞生长因子(HGF)能否动员骨髓内皮祖细胞,以及动员的内皮祖细胞能否参与创伤修复时的血管新生和内皮修复.方法: 将腺病毒HGF载体(adenovirus vector encoding HGF gene, Ad-HGF)经尾静脉注射到Balb/c小鼠体内,用ELISA方法检测血浆HGF水平的变化;用流式细胞术检测外周血CD34 细胞含量变化;对外周血单个核细胞进行分离、培养,并对生长的细胞克隆进行内皮细胞表面标志Tie-2、vW因子的免疫组化检测.建立雌性小鼠CCl4肝损伤模型,静脉移植HGF处理后雄性小鼠外周血单个核细胞到其体内,4 W后利用原位杂交技术检测新生肝组织中是否存在雄性细胞.结果: 注射Ad-HGF能明显提高小鼠血浆的HGF水平,并使外周血中以CD34、Tie-2和vW因子等为标志的内皮祖细胞的数量显著增多.这些细胞参与肝损伤修复时的血管新生.结论: HGF对骨髓内皮祖细胞具有明显的动员作用.     

Control of vascular endothelial growth factor angiogenic activity by the extracellular matrix

In adult vessels the proliferation rate of differentiated endothelial cells is very low. In response to several environmental stimuli the expression of so-called ‘angiogenic factors’ is upregulated and the messenger RNAs are actively translated in secreted factors which induce the proliferation of endothelial cells; the digestion of their basement membrane then allows their migration and differentiation. Considerable progress has been made during the past years in elucidating the molecular actors of angiogenesis. Vascular endothelial growth factor turned out to represent the major inducer of angiogenesis. Optional splicing of its pre-messenger RNA generates various isoforms which differ not only by their storage in the extracellular matrix but also by their signaling pathways.     

Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells

Growth factor-induced signaling by receptor tyrosine kinases (RTKs) plays a central role in embryonic development and in pathogenesis and, hence, is tightly controlled by several regulatory proteins. Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered. Subsequently, four mammalian Sprouty homologues (Spry-1-4) have been identified. Here, we report the functional characterization of two of them, Spry-1 and -2, in endothelial cells. Overexpressed Spry-1 and -2 inhibit fibroblast growth factor- and vascular endothelial growth factor-induced proliferation and differentiation by repressing pathways leading to p42/44 mitogen-activating protein (MAP) kinase activation. In contrast, although epidermal growth factor-induced proliferation of endothelial cells was also inhibited by Spry-1 and -2, activation of p42/44 MAP kinase was not affected. Biochemical and immunofluorescence analysis of endogenous and overexpressed Spry-1 and -2 reveal that both Spry-1 and -2 are anchored to membranes by palmitoylation and associate with caveolin-1 in perinuclear and vesicular structures. They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane. The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.     

The involvement of endothelial progenitor cells in tumor angiogenesis

Endothelial progenitor cells (EPCs) have been isolated from peripheral blood CD34, VEGFR-2, or AC 133 (CD133) antigen-positive cells, which may home to site of neovascularization and differentiate into endothelial cells in situ. Endothelial cells contribute to tumor angiogenesis, and can originate from sprouting or co-option of neighbouring pre-existing vessels. Emerging evidence indicate that bone marrow-derived circulating EPCs can contribute to tumor angiogenesis and growth of certain tumors. This review article will summarize the literature data concerning this new role played by EPCs in tumor angiogenesis.     

Platelet-derived endothelial cell growth factor.

Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45 kDa single chain polypeptide which stimulates endothelial cell growth and chemotaxis in vitro and angiogenesis in vivo. Analysis of a full length PD-ECGF cDNA revealed an open reading frame coding for 482 amino acids without homology to other known proteins. No signal sequence was observed, and analysis of the biosynthesis and processing of PD-ECGF in a thyroid carcinoma cell line revealed that PD-ECGF is released only very slowly. PD-ECGF becomes covalently associated with nucleotide triphosphates (e.g., ATP) in vivo, as well as in vitro. The physiological significance of this posttranslational modification remains to be elucidated. The tissue distribution and target cell specificity of PD-ECGF suggest roles in angiogenesis (e.g., during wound healing and in the developing placenta), as well as in the maintenance of the integrity of the endothelial cell lining of large vessels.     

Directing migration of endothelial progenitor cells with applied DC electric fields

Naturally-occurring, endogenous electric fields (EFs) have been detected at skin wounds, damaged tissue sites and vasculature. Applied EFs guide migration of many types of cells, including endothelial cells to migrate directionally. Homing of endothelial progenitor cells (EPCs) to an injury site is important for repair of vasculature and also for angiogenesis. However, it has not been reported whether EPCs respond to applied EFs. Aiming to explore the possibility to use electric stimulation to regulate the progenitor cells and angiogenesis, we tested the effects of direct-current (DC) EFs on EPCs. We first used immunofluorescence to confirm the expression of endothelial progenitor markers in three lines of EPCs. We then cultured the progenitor cells in EFs. Using time-lapse video microscopy, we demonstrated that an applied DC EF directs migration of the EPCs toward the cathode. The progenitor cells also align and elongate in an EF. Inhibition of vascular endothelial growth factor (VEGF) receptor signaling completely abolished the EF-induced directional migration of the progenitor cells. We conclude that EFs are an effective signal that guides EPC migration through VEGF receptor signaling in vitro. Applied EFs may be used to control behaviors of EPCs in tissue engineering, in homing of EPCs to wounds and to an injury site in the vasculature.     

Vascular endothelial growth factor‐D modulates oxidant–antioxidant balance of human vascular endothelial cells

Vascular endothelial growth factor‐D (VEGF‐D) is an angiogenic and lymphangiogenic glycoprotein that facilitates tumour growth and distant organ metastasis. Our previous studies showed that VEGF‐D stimulates the expression of proteins involved in cell–matrix interactions and promoting the migration of endothelial cells. In this study, we focused on the redox homoeostasis of endothelial cells, which is significantly altered in the process of tumour angiogenesis. Our analysis revealed up‐regulated expression of proteins that form the antioxidant barrier of the cell in VEGF‐D‐treated human umbilical endothelial cells and increased production of reactive oxygen and nitrogen species in addition to a transient elevation in the total thiol group content. Despite a lack of changes in the total antioxidant capacity, modification of the antioxidant barrier induced by VEGF‐D was sufficient to protect cells against the oxidative stress caused by hypochlorite and paraquat. These results suggest that exogenous stimulation of endothelial cells with VEGF‐D induces an antioxidant response of cells that maintains the redox balance. Additionally, VEGF‐D‐induced changes in serine/threonine kinase mTOR shuttling between the cytosol and nucleus and its increased phosphorylation at Ser‐2448, lead us to the conclusion that the observed shift in redox balance is regulated via mTOR kinase signalling.     

血小板活化因子对大鼠黄体细胞孕酮分泌及血管内皮生长因子表达的作用

本文旨在研究血小板活化因子(platelet-activating factor,PAF)对大鼠黄体细胞孕酮分泌及血管内皮生长因子(vascularendothelial growth factor,VEGF)mRNA表达的作用.将未成年(25～28 d)Sprague-Dawley雌性大鼠颈部皮下注射50 IU孕马血清促性腺激素(pregnant mare serum gonadotrophin,PMSG),48 h后注射25 IU人绒毛膜促性腺激素(human chorionicgonadotrophin.hCG)诱导卵泡发育和黄体生成,第6天(hCG注射日为第1天)收集卵巢黄体细胞,体外培养24 h后,不加或加入不同剂量(0.1 μg/mL、1 μg/mL、10 μg/mL)PAF,37℃、5%CO2培养箱内培养24 h.用放射免疫方法测定培养液中孕酮的含量,流式细胞仪和RT-PCR方法检测黄体细胞凋亡以及VEGF mRNA的表达.结果显示,PAF促进黄体细胞孕酮分泌,1 μg/mL PAF作用最强(P<0.05);PAF促进黄体细胞凋亡无明显剂量依赖性,但10 μg/mL PAF显著促进大鼠黄体细胞凋亡(P<0.05):PAF刺激黄体细胞VEGF mRNA表达,1 μg/mL PAF效果最显著(P<0.01).结果提示,PAF可通过调节黄体细胞孕酮的分泌和VEGF mRNA的表达来促进黄体形成.     

p38丝裂原素激活的蛋白激酶在调节低氧诱导人内皮细胞分泌血管内皮生长因子过程中的作用

血管内皮细胞中血管内皮生长因子(vascular endothelial growthfactor,VEGF)的合成增加在促进血管新生的过程中起着非常重要的作用.然而低氧诱导VEGF分泌的细胞内信号转导机制还不是很清楚.人脐静脉内皮细胞系(ECV304)在低氧或常氧的状态下培养12～24 h后分别用实时定量PCR和Western blot的方法来检测VEGF mRNA的表达及ERK1/2和p38激酶的磷酸化水平.分泌到培养液中的VEGF蛋白用酶联免疫吸附(ELISA)的方法来检测.业已报道,ERK的抑制剂PD98059能够抑制低氧诱导的VEGF基因的表达,根据这个报道,我们发现在低氧情况下,ECV304细胞的ERK1/2磷酸化水平增高以及VEGF的合成增加等这些变化也能被PD98059所抑制.本次实验的新发现是p38激酶的激活在低氧诱导VEGF合成增加中的作用.p38激酶的抑制剂SB202190能抑制低氧诱导的VEGF合成增加.这些数据首次直接证实了p38激酶在低氧诱导人内皮细胞分泌VEGF增加过程中的作用.     

SPARC antagonizes the effect of basic fibroblast growth factor on the migration of bovine aortic endothelial cells.

Migration of endothelial cells is requisite to wound repair and angiogenesis. Since the glycoprotein SPARC (secreted protein, acidic and rich in cysteine) is associated with remodeling, cellular migration, and angiogenesis in vitro, we questioned whether SPARC might influence the motility of endothelial cells. In this study we show that, in the absence of serum, exogenous SPARC inhibits the migration of bovine aortic endothelial cells induced by bFGF. Similar results were obtained from two different assays, in which cell migration was measured in a Boyden chamber and in monolayer culture after an experimental wound. Without bFGF, the migration of endothelial cells was unaffected by SPARC. The inhibitory effect of SPARC on cell motility was dose-dependent, required the presence of Ca2+, was mimicked by synthetic peptides from the N- and C-terminal Ca(2+)-binding domains of the protein, and was not seen in the presence of serum. Modulation of the activities of secreted and cell-associated proteases, including plasminogen activators and metalloproteinases, appeared not to be responsible for the effects that we observed on the motility of endothelial cells. Moreover, a molecular interaction between SPARC and bFGF was not detected, and SPARC did not interfere with the binding of bFGF to high-affinity receptors on endothelial cells. Finally, in culture medium that contained serum, SPARC inhibited the incorporation of [3H]-thymidine into newly synthesized DNA, both in the absence and presence of bFGF. However, DNA synthesis was not affected by SPARC when the cells were plated on gelatin or fibronectin in serum-free medium. We propose that the combined action of a serum factor and SPARC regulates both endothelial cell proliferation and migration and coordinates these events during morphogenetic processes such as wound repair and angiogenesis.     

Culture of endothelial cells by transfection with plasmid harboring vascular endothelial growth factor

Vascular endothelial cells (ECs) are usually difficult to culture in a large scale because of their complicated requirements for cell growth. As the vascular endothelial growth factor (VEGF) is a key growth factor in the EC culture, we transfected human umbilical vein endothelial cells (HUVEC) using a plasmid containing VEGF gene and let them grow in a culture medium eliminated an important supplement, endothelail cell growth supplement (ECGS). The expression of VEGF by HUVEC tansfected with VEGF gene was not enough to stimulate the growth of HUVEC, only 40% of maximum cell density obtainable in the presence of ECGS., However, when the culture medium was supplied with 2.5 ng/mL of basic fibroblast growth factor (bFGF), a synergistic effect of VEGF and bFGF was observed. In this case, the final cell density was recovered up to about 78% of maxium value.     

Vascular permeability factor: a unique regulator of blood vessel function.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a potent polypeptide regulator of blood vessel function. VPF promotes an array of responses in endothelium, including hyperpermeability, endothelial cell growth, angiogenesis, and enhanced glucose transport. VPF regulates the expression of tissue factor and the glucose transporter. All of the endothelial cell responses to VPF are evidently mediated by high affinity cell surface receptors. Thus, endothelial cells have a unique and specific spectrum of responses to VPF. Since each of the responses of endothelial cells to VPF are also elicited by agonists, such as bFGF, TNF, histamine and others, it remains a major challenge to determine how post-receptor signalling pathways maintain both specificity and redundancy in cellular responses to various agonists.     

Reversible acetylation regulates vascular endothelial growth factor receptor-2 activity

The tyrosine kinase receptor vascular endothelial growth factor receptor 2 （VEG FR2） is a key regulator of angiogenesis. Here we show that VEGFR2 is acetylated in endothelial cells both at four lysine residues forming a dense cluster in the kinase insert domain and at a single lysine located in the receptor activation loop. These modifications are under dynamic control of the acetyltransferase p300 and two deacetyiases HDAC5 and HDAC6. We demonstrate that VEGFR2 acetylation essentially regulates receptor phosphorylation. In par- ticular, VEGFR2 acetylation significantly alters the kinetics of receptor phosphorylation after ligand binding, allowing receptor phos- phoryiation and intraceUular signaling upon proLonged stimulation with VEGF. Molecular dynamics simulations indicate that acetylation of the lysine in the activation loop contributes to the transition to an open active state, in which tyrosine phosphorylation is favored by better exposure of the kinase target residues. These findings indicate that post-translational modification by acetyiation is a critical mechanism that directLy affects VEGFR2 function.     

Isolating and expanding endothelial progenitor cells from peripheral blood on peptide-functionalized polystyrene surfaces

The expansion of human peripheral blood endothelial progenitor cells to obtain therapeutically relevant endothelial colony-forming cells (ECFCs) has been commonly performed on xeno-derived extracellular matrix proteins. For cellular therapy applications, xeno-free culture conditions are desirable to improve product safety and reduce process variability. We have previously described a novel fluorophore-tagged RGD peptide (RGD-TAMRA) that enhanced the adhesion of mature endothelial cells in vitro. To investigate whether this peptide can replace animal-derived extracellular matrix proteins in the isolation and expansion of ECFCs, peripheral blood mononuclear cells from 22 healthy adult donors were seeded on RGD-TAMRA-modified polystyrene culture surfaces. Endothelial colony formation was significantly enhanced on RGD-TAMRA-modified surfaces compared to the unmodified control. No phenotypic differences were detected between ECFCs obtained on RGD-TAMRA compared to ECFCs obtained on rat-tail collagen-coated surfaces. Compared with collagen-coated surfaces and unmodified surfaces, RGD-TAMRA surfaces promoted ECFC adhesion, cell spreading, and clonal expansion. This study presents a platform that allows for a comprehensive in vitro evaluation of peptide-based biofunctionalization as a promising avenue for ex vivo ECFC expansion.     

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.     

Monotropein promotes angiogenesis and inhibits oxidative stress‐induced autophagy in endothelial progenitor cells to accelerate wound healing

Attenuating oxidative stress‐induced damage and promoting endothelial progenitor cell (EPC) differentiation are critical for ischaemic injuries. We suggested monotropein (Mtp), a bioactive constituent used in traditional Chinese medicine, can inhibit oxidative stress‐induced mitochondrial dysfunction and stimulate bone marrow‐derived EPC (BM‐EPC) differentiation. Results showed Mtp significantly elevated migration and tube formation of BM‐EPCs and prevented tert‐butyl hydroperoxide (TBHP)‐induced programmed cell death through apoptosis and autophagy by reducing intracellular reactive oxygen species release and restoring mitochondrial membrane potential, which may be mediated viamTOR/p70S6K/4EBP1 and AMPK phosphorylation. Moreover, Mtp accelerated wound healing in rats, as indicated by reduced healing times, decreased macrophage infiltration and increased blood vessel formation. In summary, Mtp promoted mobilization and differentiation of BM‐EPCs and protected against apoptosis and autophagy by suppressing the AMPK/mTOR pathway, improving wound healing in vivo. This study revealed that Mtp is a potential therapeutic for endothelial injury‐related wounds.