Irisin Induces Angiogenesis in Human Umbilical Vein Endothelial Cells In Vitro and in Zebrafish Embryos In Vivo via Activation of the ERK Signaling Pathway

As a link between exercise and metabolism, irisin is assumed to be involved in increased total body energy expenditure, reduced body weight, and increased insulin sensitivity. Although our recent evidence supported the contribution of irisin to vascular endothelial cell (ECs) proliferation and apoptosis, further research of irisin involvement in the angiogenesis of ECs was not conclusive. In the current study, it was found that irisin promoted Human Umbilical Vein Endothelial Cell (HUVEC) angiogenesis via increasing migration and tube formation, and attenuated chemically-induced intersegmental vessel (ISV) angiogenic impairment in transgenic TG (fli1: GFP) zebrafish. It was further demonstrated that expression of matrix metalloproteinase (MMP) 2 and 9 were also up-regulated in endothelial cells. We also found that irisin activated extracellular signal–related kinase (ERK) signaling pathways. Inhibition of ERK signaling by using U0126 decreased the pro-migration and pro-angiogenic effect of irisin on HUVEC. Also, U0126 inhibited the elevated expression of MMP-2 and MMP-9 when they were treated with irisin. In summary, these findings provided direct evidence that irisin may play a pivotal role in maintaining endothelium homeostasis by promoting endothelial cell angiogenesis via the ERK signaling pathway.     

Irisin Promotes Human Umbilical Vein Endothelial Cell Proliferation through the ERK Signaling Pathway and Partly Suppresses High Glucose-Induced Apoptosis

Irisin is a newly discovered myokine that links exercise with metabolic homeostasis. It is involved in modest weight loss and improves glucose intolerance. However, the direct effects and mechanisms of irisin on vascular endothelial cells (ECs) are not fully understood. In the current study, we demonstrated that irisin promoted Human Umbilical Vein Endothelial Cell (HUVEC) proliferation. It was further demonstrated that this pro-proliferation effect was mediated by irisin-induced activation of extracellular signal–related kinase (ERK) signaling pathways. Inhibition of ERK signaling with U0126 decreased the pro-proliferation effect of irisin on HUVECs. It was also demonstrated that irisin reduced high glucose-induced apoptosis by up-regulating Bcl-2 expression and down-regulating Bax, Caspase-9 and Caspase-3 expression. In summary, these results suggested that irisin plays a novel role in sustaining endothelial homeostasis by promoting HUVEC proliferation via the ERK signaling pathway and protects the cell from high glucose-induced apoptosis by regulating Bcl-2,Bax and Caspase expression.     

Effect of human endothelial cells on human bone marrow stromal cell phenotype: role of VEGF?

Angiogenesis is a tightly regulated process involved in growth, repair, and bone remodeling. Several studies have shown that there is a reciprocal regulation and functional relationship between endothelial cells and osteoblast-like cells during osteogenesis, where systemic hormones and paracrine growth factors play an active role. Angiogenesis is induced by a variety of growth factors; among them vascular endothelial growth factor (VEGF) may be an important mediator for the angiogenic process involved in bone physiology. We studied the VEGF effect on osteoblast progenitor cells (Human Bone Marrow Stromal Cells: HBMSE) cultured alone or associated with endothelial cells (Human Umbilical Vein Endothelial Cells: HUVEC) in different co-culture models (co-culture with or without direct contact, conditioned medium), to determine the influence of VEGF on these cells and on their relationship. In agreement with other studies, we show that HBMSC express and synthesize VEGF, HUVEC conditioned medium has a proliferative effect on them, and early osteoblastic marker (Alkaline phosphatase activity) levels increase when these cells are co-cultured with HUVEC only in direct contact. However, unlike previous studies, we did not find that VEGF increased these processes. These results suggest that the intercommunication between endothelial cells and osteoblastic-like cells requires not only diffusible factors, but also involving cell membrane proteins.     

Differential effects of Bartonella henselae on human and feline macro- and micro-vascular endothelial cells

Bartonella henselae, a zoonotic agent, induces tumors of endothelial cells (ECs), namely bacillary angiomatosis and peliosis in immunosuppressed humans but not in cats. In vitro studies on ECs represent to date the only way to explore the interactions between Bartonella henselae and vascular endothelium. However, no comparative study of the interactions between Bartonella henselae and human (incidental host) ECs vs feline (reservoir host) ECs has been carried out because of the absence of any available feline endothelial cell lines.To this purpose, we have developed nine feline EC lines which allowed comparing the effects of Bartonella strains on human and feline micro-vascular ECs representative of the infection development sites such as skin, versus macro-vascular ECs, such as umbilical vein.Our model revealed intrinsic differences between human (Human Skin Microvascular ECs -HSkMEC and Human Umbilical Vein ECs - iHUVEC) and feline ECs susceptibility to Bartonella henselae infection.While no effect was observed on the feline ECs upon Bartonella henselae infection, the human ones displayed accelerated angiogenesis and wound healing.Noticeable differences were demonstrated between human micro- and macro-vasculature derived ECs both in terms of pseudo-tube formation and healing. Interestingly, Bartonella henselae effects on human ECs were also elicited by soluble factors.Neither Bartonella henselae-infected Human Skin Microvascular ECs clinically involved in bacillary angiomatosis, nor feline ECs increased cAMP production, as opposed to HUVEC.Bartonella henselae could stimulate the activation of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) in homologous cellular systems and trigger VEGF production by HSkMECs only, but not iHUVEC or any feline ECs tested.These results may explain the decreased pathogenic potential of Bartonella henselae infection for cats as compared to humans and strongly suggest that an autocrine secretion of VEGF by human skin endothelial cells might induce their growth and ultimately lead to bacillary angiomatosis formation.     

Effect of HUVEC on human osteoprogenitor cell differentiation needs heterotypic gap junction communication

Bone development and remodeling depend oncomplex interactions between bone-forming osteoblasts and other cellspresent within the bone microenvironment, particularly vascularendothelial cells that may be pivotal members of a complex interactivecommunication network in bone. Our aim was to investigate theinteraction between human umbilical vein endothelial cells (HUVEC) andhuman bone marrow stromal cells (HBMSC). Cell differentiation analysisperformed with different cell culture models revealed that alkalinephosphatase activity and type I collagen synthesis were increased onlyby the direct contact of HUVEC with HBMSC. This "juxtacrinesignaling" could involve a number of different heterotypic connexionsthat require adhesion molecules or gap junctions. A dyecoupling assay with Lucifer yellow demonstrated a functional couplingbetween HUVEC and HBMSC. Immunocytochemistry revealed that connexin43 (Cx43), a specific gap junction protein, is expressed not only in HBMSCbut also in the endothelial cell network and that these two cell typescan communicate via a gap junctional channel constituted at least byCx43. Moreover, functional inhibition of the gap junction by18-glycyrrhetinic acid treatment or inhibition of Cx43 synthesis with oligodeoxyribonucleotide antisense decreased the effect of HUVECcocultures on HBMSC differentiation. This stimulation could be mediatedby the intercellular diffusion of signaling molecules that permeate thejunctional channel.

     

Endothelial progenitor cells for regeneration

Endothelial progenitor cells (EPCs) have been recently isolated from peripheral blood and bone marrow (BM), and shown to be incorporated into sites of physiological and pathological neovascularization in vivo. In contrast to differentiated endothelial cells (ECs), transplantation of EPCs successfully enhanced vascular development by in situ differentiation and proliferation within ischemic organs. Based on such a novel concept of closed up function on EPCs in postnatal neovascularization, the beneficial property of EPC is attractive for cell therapy as well as cell-mediated gene therapy applications targeting regeneration of ischemic tissue.     

Pleiotrophin induces angiogenesis: involvement of the phosphoinositide-3 kinase but not the nitric oxide synthase pathways

Pleiotrophin (PTN) is a developmentally regulated protein that has been shown to be involved in tumor growth and metastasis presumably by activating tumor angiogenesis. To clarify the potential angiogenic activity of PTN and to analyze the signaling pathways involved in this process, we used an in vitro model of Human Umbilical Vein Endothelial Cells (HUVEC). We show that PTN was mitogenic toward a variety of endothelial cells including HUVEC, stimulated HUVEC migration across a reconstituted basement membrane and induced the formation of capillary-like structures by HUVEC grown as 3D-cultures in Matrigel or collagen. The signaling pathways triggered following endothelial cell stimulation by PTN were studied by using pharmacological inhibitors of the Phosphoinositide-3 kinase (PI3K) and endothelial Nitric Oxide Synthase (eNOS), two enzymes that have been shown to be crucial in the angiogenic response to Vascular Endothelial Growth Factor (VEGF). Whereas wortmannin (a PI3K inhibitor) and L-NAME (an eNOS inhibitor) dramatically reduced HUVEC growth induced by VEGF, only the former inhibitor reduced the growth induced by PTN and to a lesser extent that stimulated by basic Fibroblast Growth Factor. Thus, our results indicate that PTN induces angiogenesis and utilizes PI3K- but not eNOS-dependent pathways for its angiogenic activity.     

Effects of Ca2+-activated potassium and inward rectifier potassium channel on the differentiation of endothelial progenitor cells from human peripheral blood

Endothelial progenitor cells (EPCs) are bone marrow-derived cells that have the propensity to differentiate into mature endothelial cells (ECs). The transplantation of EPCs has been shown to enhance in vivo postnatal neo-vasculogenesis, as well as repair infarcted myocardium. Via the whole-cell patch clamp technique, numerous types of ion channels have been detected in EPCs, including the inward rectifier potassium channel (IKir), Ca²⁺-activated potassium channel (IKCa), and volume-sensitive chloride channel, but their influence on the differentiation of EPCs has yet to be characterized. The present study was designed to investigate: (1) which ion channels have the most significant impact on the differentiation of EPCs; (2) what role ion channels play in the functional development of EPCs; (3) the mRNA and protein expression levels of related ion channel subunits in EPCs. In our study, EPCs were obtained from the peripheral blood of healthy adults and cultured with endothelial growth factors. When EPCs differentiate into mature ECs, they lose expression of the stem cell/progenitor marker CD133, as analyzed by flow cytometry (0.44 ± 0.20 %). However, treatment with the potassium channel inhibitor, tetraethylammonium (TEA) results in an increase in CD133⁺ cells (25.50 ± 7.55 %). In a functional experiment, we observed a reduction in the capacity of TEA treated ECs (differentiated from EPCs) to form capillary tubes when seeded in Matrigel. At the mRNA and protein levels, we revealed several K⁺ subtypes, including KCNN4 for IKCa, KCNNMA1 for BKCa and Kir3.4 for IKir. These results demonstrate for the first time that potassium channels play a significant role in the differentiation of EPCs. Moreover, inhibition of potassium channels may depress the differentiation of EPCs and the significant potassium channel subunits in EPCs appear to be IKCa, BKCa and Kir3.4.     

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。     

Successful in vitro expansion and differentiation of cord blood derived CD34+ cells into early endothelial progenitor cells reveals highly differential gene expression

Endothelial progenitor cells (EPCs) can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU) assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP), PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15) or pro-angiogenic (galectin-3) properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP) was the most up-regulated gene.     

Alloimmunity to human endothelial cells derived from cord blood progenitors

There is considerable interest in exploiting circulating endothelial progenitor cells (EPCs) for therapeutic organ repair. Such cells may be differentiated into endothelial cells (ECs) in vitro and then expanded for use in tissue engineering. Vessel-derived ECs are variably immunogenic, depending upon tissue source, and it is unknown whether ECs derived from cord blood EPCs are able to initiate an allogeneic response. In this study, we compare the phenotype and alloantigenicity of human cord blood progenitor cell-derived ECs with HUVECs isolated from the same donors. Human cord blood progenitor cell-derived ECs are very similar to HUVECs in the expression of proteins relevant for alloimmunity, including MHC molecules, costimulators, adhesion molecules, cytokines, chemokines, and IDO, and in their ability to initiate allogeneic CD4(+) and CD8(+) memory T cell responses in vitro and in vivo. These findings have significant implications for the use of cord blood EPCs in regenerative medicine or tissue engineering.     

Decursin inhibits vasculogenesis in early tumor progression by suppression of endothelial progenitor cell differentiation and function

Endothelial progenitor cells (EPCs) contribute to the tumor vasculature during tumor progression. Decursin isolated from the herb Angelica gigas is known to possess potent anti‐inflammatory activities. Recently, we reported that decursin is a novel candidate for an angiogenesis inhibitor [Jung et al., 2009 ]. In this study, we investigated whether decursin regulates EPC differentiation and function to inhibit tumor vasculogenesis. We isolated AC133+ cells from human cord blood and decursin significantly decreased the number of EPC colony forming units of human cord blood‐derived AC133+ cells that produce functional EPC progenies. Decursin dose‐dependently decreased the cell number of EPC committing cells as demonstrated by EPC expansion studies. Decursin inhibited EPC differentiation from progenitor cells into spindle‐shaped EPC colonies. Additionally, decursin inhibited proliferation and migration of early EPCs isolated from mouse bone marrow. Furthermore, decursin suppressed expression of angiopoietin‐2, angiopoietin receptor Tie‐2, Flk‐1 (vascular endothelial growth factor receptor‐2), and endothelial nitric oxide synthase in mouse BM derived EPCs in a dose‐dependent manner. Decursin suppressed tube formation ability of EPCs in collaboration with HUVEC. Decursin (4 mg/kg) inhibited tumor‐induced mobilization of circulating EPCs (CD34 + /VEGFR‐2+ cells) from bone marrow and early incorporation of Dil‐Ac‐LDL‐labeled or green fluorescent protein (GFP)+ EPCs into neovessels of xenograft Lewis lung carcinoma tumors in wild‐type‐ or bone‐marrow‐transplanted mice. Accordingly, decursin attenuated EPC‐derived endothelial cells in neovessels of Lewis lung carcinoma tumor masses grown in mice. Together, decursin likely affects EPC differentiation and function, thereby inhibiting tumor vasculogenesis in early tumorigenesis. J. Cell. Biochem. 113: 1478–1487, 2012. © 2012 Wiley Periodicals, Inc.     

Collagen type I together with fibronectin provide a better support for endothelialization

Endothelialization of vascular implants is limited by the inability of cells to retain adhesion when exposed to flow. Extracellular matrix proteins, including fibronectin and collagen, enhance cell adherence on materials. This study investigated the behaviour of Human Umbilical Vein Endothelial Cells (HUVEC) on extracellular matrix coated polystyrene. Collagen and fibronectin were coated as single and double layers to analyse differences in cell proliferation, morphology, and cell-protein interactions. Significantly higher endothelial cell proliferation and migration rates were observed on the collagen and collagen+fibronectin coating compared to the uncoated or fibronectin-coated sample. Immmunofluorescent microscopy showed evidence of extracellular matrix remodelling in the double, collagen+fibronectin coating. These results strongly suggest that a double coating of collagen+fibronectin provides a better support structure for endothelial cell growth and contributes to improve the ability of vascular implants to become and remain endothelialized.     

PLGA支架降解对血管化功能影响的体外研究

目的:研究聚乳酸-羟基乙酸(PLGA)支架材料降解产物对血管内皮细胞增殖、迁移和小管样结构形成的影响。方法:将PLGA支架材料放入磷酸盐缓冲液(PBS)中体外无菌降解1、2、4周。用降解液处理人脐静脉内皮细胞株HUVEC,采用Brdu ELISA法、Transwell小室法和小管形成实验检测PLGA支架材料降解液对血管内皮细胞增殖、迁移和小管样结构形成的影响。结果:PLGA支架材料1周降解液对内皮细胞的迁移和小管形成无明显影响,对内皮细胞增殖有一定的促进作用。随着降解时间的延长,2周降解液抑制内皮细胞的迁移和小管形成,4周的降解液对内皮细胞增殖、迁移和小管形成均有抑制作用。结论:PLGA支架材料降解初期有对血管内皮细胞的增殖有促进作用,降解后期可能由于降解过程中产生的酸性物质累积增多,影响了血管内皮细胞的生长和功能,从而抑制新生血管的形成。     

Hela l-CaD Is Implicated in the Migration of Endothelial Cells/Endothelial Progenitor Cells in Human Neoplasms

Caldesmon (CaD) is a major actin-binding protein distributed in a variety of cell types. No functional differences among the isoforms in in vitro studies were found so far. In a previous study we found that the low molecular caldesmon isoform (Hela l-CaD) is expressed in endothelial cells (ECs)/endothelial progenitor cells (EPCs) in tumor vasculature of various human tumors. Activation of cell motility is necessary for the navigation of the tip ECs during angiogenesis, and migration of EPCs from the bone marrow during vasculogenesis. In the present study we searched for features of motility and the intracellular expression sites of Hela l-CaD in ECs/EPCs of various human tumors under histologically preserved microenviroment. We discovered a variety of motility-related cell protrusions like filopodia, microspikes, lamellipodia, podosomes, membrane blebs and membrane ruffles in the activated ECs/EPCs. Hela l-CaD appeared to be invariably expressed in the subregions of these cell protrusions. The findings suggest that Hela l-CaD is implicated in the migration of ECs/EPC in human neoplasms where they contribute to tumor vasculogenesis and angiogenesis.     

Connexin32 is expressed in vascular endothelial cells and participates in gap-junction intercellular communication

Endothelial cells (ECs) play many roles in vascular biology, including control of blood pressure, blood clotting, atherosclerosis, angiogenesis, and inflammation. Gap junctions (GJs) are channel-like assemblies of connexin (Cx) family proteins that connect neighboring cells and modulate and synchronize their intracellular environments by the transfer of intracellular mediators. It has been reported that vascular ECs express Cx37, Cx40, and Cx43, but not Cx32. Here, we showed that Cx32 mRNA and protein are expressed in various cultured human ECs. We confirmed Cx32 expression in blood vessel ECs using wild-type and Cx32 knock-out mice. We observed that dye transfer between cultured ECs through gap junctions is suppressed by an anti-Cx32 monoclonal antibody. These findings suggest that vascular ECs express Cx32, which participates in endothelial gap-junction intercellular communication.     

Proteomes of umbilical vein and microvascular endothelial cells reflect distinct biological properties and influence immune recognition

Human umbilical vein endothelial cells (HUVEC) are widely used as a source of endothelial cells (EC). However, HUVEC characteristics cannot be extrapolated to other types of EC, particularly microvascular ECs. Our objective was to compare the proteomes of microvascular ECs and HUVEC. Proteomes of HUVEC and human microvascular pulmonary EC (HMVEC-P) and dermal EC (HMVEC-D) from healthy Caucasian donors were compared by 2D DIGE and MS. Fatty acid binding proteins 4 and 5 were among the 159 and 30 proteins spots found to have at least twofold change in expression between HUVEC and HMVEC-D and between HUVEC and HMVEC-P samples, respectively. Eight protein spots showed twofold changed expression between HMVEC-D and HMVEC-P samples. Ingenuity? analysis revealed that proteins differentially expressed between HUVEC and HMVEC-D samples interact with retinoic acid. In vitro tubulogenesis assays showed a differential effect of retinoic acid between HUVEC and HMVEC. Moreover, serum IgG from patients with a rare vascular disease, systemic sclerosis, showed distinct reactivity profiles in HUVEC and HMVEC-D protein extracts. The proteome profiles of HUVEC and microvascular EC differ noticeably, which reflects distinct biological properties and influence immune recognition.     

Endothelial Progenitor Cells Promote Directional Three-Dimensional Endothelial Network Formation by Secreting Vascular Endothelial Growth Factor

Endothelial progenitor cell (EPC) transplantation induces the formation of new blood-vessel networks to supply nutrients and oxygen, and is feasible for the treatment of ischemia and cardiovascular diseases. However, the role of EPCs as a source of proangiogenic cytokines and consequent generators of an extracellular growth factor microenvironment in three-dimensional (3D) microvessel formation is not fully understood. We focused on the contribution of EPCs as a source of proangiogenic cytokines on 3D microvessel formation using an in vitro 3D network model. To create a 3D network model, EPCs isolated from rat bone marrow were sandwiched with double layers of collagen gel. Endothelial cells (ECs) were then cultured on top of the upper collagen gel layer. Quantitative analyses of EC network formation revealed that the length, number, and depth of the EC networks were significantly enhanced in a 3D model with ECs and EPCs compared to an EC monoculture. In addition, conditioned medium (CM) from the 3D model with ECs and EPCs promoted network formation compared to CM from an EC monoculture. We also confirmed that EPCs secreted vascular endothelial growth factor (VEGF). However, networks cultured with the CM were shallow and did not penetrate the collagen gel in great depth. Therefore, we conclude that EPCs contribute to 3D network formation at least through indirect incorporation by generating a local VEGF gradient. These results suggest that the location of EPCs is important for controlling directional 3D network formation in the field of tissue engineering.