Biomimetic hydrogels with VEGF induce angiogenic processes in both hUVEC and hMEC

Angiogenesis is the process by which new blood vessels arise from the pre-existing vasculature. Human endothelial cells are known to be involved in three key cellular processes during angiogenesis: increased cell proliferation, degradation of the extracellular matrix during cell migration, and the survival of apoptosis. The above processes depend upon the presence of growth factors, such as vascular endothelial growth factor isoform 165 (VEGF(165)) that is released from the extracellular matrix as it is being degraded or secreted from activated endothelial cells. Thus, the goal of the current study is to develop a system with a backbone of polyethylene glycol (PEG) and grafted angiogenic signals to compare the initial angiogenic response of human umbilical vein endothelial cells (hUVEC) or human microvascular endothelial cells (hMEC). Adhesion ligands (PEG-RGDS) for cell attachment and PEG-modified VEGF(165) (PEG-VEGF(165)) are grafted into the hydrogels to encourage the angiogenic response. Our data suggest that our biomimetic system is equally effective in stimulating proliferation, migration, and survival of apoptosis in hMEC as compared to the response to hUVEC.     

VEGF189 stimulates endothelial cells proliferation and migration in vitro and up-regulates the expression of Flk-1/KDR mRNA

The vascular endothelial growth factor (VEGF) is a critical factor for development of the vascular system in physiological and pathological angiogenesis. This growth factor exists under at least three isoforms, VEGF120/121, VEGF164/165 and VEGF188/189 which are generated by alternative splicing. VEGF isoforms have different affinities for heparan sulphate as well as for VEGF receptors, and may play distinct roles in vascular development. The role of VEGF189 as an endothelial mitogen, however, remains controversial. VEGF189 is almost entirely bound to the cell surface or extracellular matrix, and is considered active after its cleavage and release from its extracellular binding site. In the present study, we demonstrate that VEGF189 induces endothelial cell proliferation and migration in vitro. The 30-60% increase observed with VEGF189 (10 ng/ml) in HUVEC proliferation was similar to that observed with VEGF165. However, the proliferative effect observed with VEGF189 appeared dependent on the origin of the endothelial cell, since the proliferation was clearly observed with HUVEC but not with BAEC or capillary endothelial cells from dermis (HMEC). The effect of VEGF189 on endothelial cell migration was also analyzed using the wound healing and the Boyden chamber assays. The migration effect was observed with BAEC which do not proliferate with VEGF189, suggesting that different mechanisms are involved in proliferation and migration. In addition, VEGF189 as well as VEGF165 induced a 2-fold increase of Flk-1/KDR expression in HUVEC, the receptor involved in proliferation and migration of endothelial cells. In the Matrigel plug assay in vivo, both VEGF189 and 165 (100 ng/ml) increased the infiltration of endothelial cells. These data suggest that VEGF189 induced endothelial cell migration and proliferation under certain circumstances.     

The recombinant kringle domain of urokinase plasminogen activator inhibits VEGF165-induced angiogenesis of HUVECs by suppressing VEGFR2 dimerization and subsequent signal transduction

The recombinant kringle domain (UK1) of urokinase plasminogen activator was previously reported to exert antiangiogenic activity against Vascular Endothelial Growth Factor (VEGF)-induced angiogenesis in both in vitro and in vivo models. In this study, we explored the molecular signaling mechanisms involved in the antiangiogenic activity of UK1 by examining VEGF signaling proteins. VEGF165 stimulates the phosphorylation of VEGF signaling molecules, and pretreatment with UK1 blocked VEGF-induced signal transduction associated with proliferation, survival, and migration. UK1 also suppressed VEGF165-induced activation of MMP-2. Moreover, UK1 suppressed the phosphorylation and activation of VEGFR2 in VEGF-stimulated human umbilical cord vein endothelial cells (HUVECs) by blocking the dimerization of VEGFR2. Overall, our findings suggest that UK1 inhibits VEGF-induced proliferation, migration, and matrix metalloproteinase activity of HUVECs by suppressing VEGFR2 dimerization and subsequent angiogenic signals.     

The vascular endothelial growth factor VEGF165 induces perlecan synthesis via VEGF receptor-2 in cultured human brain microvascular endothelial cells

A member of the vascular endothelial growth factor (VEGF) family, VEGF165, regulates vascular endothelial cell functions in autocrine and paracrine fashions in microvessels. Proteoglycans are highly glycosylated poly-anionic macromolecules that influence cellular behaviors such as proliferation and migration by interacting with cytokines/growth factors. In the present study, we investigated the regulation of proteoglycan synthesis by VEGF165 in cultured human brain microvascular endothelial cells. The cells were exposed to recombinant human VEGF165, and the proteoglycans were then characterized using biochemical techniques. VEGF165 treatment increased the accumulation of proteoglycans 1.4- and 1.6-fold in the cell layer and conditioned medium, respectively. This effect resulted from the activation of VEGFR-2, and was mimicked by vammin, a VEGFR-2 ligand from snake venom but not placenta growth factor, which binds specifically to VEGFR-1. VEGF165 stimulated the production and secretion of perlecan, substituted with shorter heparan sulfate side chains, but with unaltered sulfated disaccharide composition. The perlecan secreted by VEGF165-stimulated endothelial cells may be involved in the regulation of cellular behavior during angiogenesis, in diseases of the brain microvessels, and in the maintenance of the endothelial cell monolayer.     

VEGF is essential for the growth and migration of human hepatocellular carcinoma cells

Vascular endothelial growth factor (VEGF) plays a crucial role in tumor angiogenesis. VEGF induces new vessel formation and tumor growth by inducing mitogenesis and chemotaxis of normal endothelial cells and increasing vascular permeability. However, little is known about VEGF function in the proliferation, survival or migration of hepatocellular carcinoma cells (HCC). In the present study, we have found that VEGF receptors are expressed in HCC line BEL7402 and human HCC specimens. Importantly, VEGF receptor expression correlates with the development of the carcinoma. By using a comprehensive approaches including TUNEL assay, transwell and wound healing assays, migration and invasion assays, adhesion assay, western blot and quantitative RT-PCR, we have shown that knockdown of VEGF165 expression by shRNA inhibits the proliferation, migration, survival and adhesion ability of BEL7402. Knockdown of VEGF165 decreased the expression of NF-κB p65 and PKCα while increased the expression of p53 signaling molecules, suggesting that VEGF functions in HCC proliferation and migration are mediated by P65, PKCα and/or p53.     

Structure and inhibitory effects on angiogenesis and tumor development of a new vascular endothelial growth inhibitor

Blocking angiogenesis is an attractive strategy to inhibit tumor growth, invasion, and metastasis. We describe here the structure and the biological action of a new cyclic peptide derived from vascular endothelial growth factor (VEGF). This 17-amino acid molecule designated cyclopeptidic vascular endothelial growth inhibitor (cyclo-VEGI, CBO-P11) encompasses residues 79-93 of VEGF which are involved in the interaction with VEGF receptor-2. In aqueous solution, cyclo-VEGI presents a propensity to adopt a helix conformation that was largely unexpected because only beta-sheet structures or random coil conformations have been observed for macrocyclic peptides. Cyclo-VEGI inhibits binding of iodinated VEGF165 to endothelial cells, endothelial cells proliferation, migration, and signaling induced by VEGF165. This peptide also exhibits anti-angiogenic activity in vivo on the differentiated chicken chorioallantoic membrane. Furthermore, cyclo-VEGI significantly blocks the growth of established intracranial glioma in nude and syngeneic mice and improves survival without side effects. Taken together, these results suggest that cyclo-VEGI is an attractive candidate for the development of novel angiogenesis inhibitor molecules useful for the treatment of cancer and other angiogenesis-related diseases.     

Marked induction of the IAP family antiapoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that has been shown to act as an endothelial cell mitogen as well as a vascular permeability factor. Several recent reports have also implicated VEGF as a major survival factor for endothelial cells during angiogenesis and vasculogenesis along with other growth factors such as bFGF and angiopoietin-1. VEGF has been shown to mediate this additional function, at least in part through the induction of bcl-2 and the activation of the PI3 kinase-Akt/PKB signaling pathway. We report here that VEGF can also mediate the induction/upregulation of members of a newly discovered family of antiapoptotic proteins, namely the Inhibitors of Apoptosis (IAP), in vascular endothelial cells. We show that VEGF(165) leads to the induction of XIAP (2.9-fold) and survivin (19.1-fold) protein in human umbilical vein endothelial cells (HUVECs). In contrast, bFGF had little effect on XIAP expression, but produced approximately a 10-fold induction on survivin. VEGF-dependent upregulation of survivin could be prevented by cell cycle arrest in the G1 and S phases. These findings implicate that the survival and mitotic functions of VEGF in an angiogenic context may be more intrinsically related than previously anticipated. Moreover, they also raise the possibility of therapeutically targeting XIAP or survivin in antiangiogenic therapy as a means of suppressing tumor growth, in addition to directly targeting tumor cells which express these survival proteins.     

Vascular endothelial growth factor upregulates follistatin in human umbilical vein endothelial cells

Vascular endothelial growth factor (VEGF), plays a key role in angiogenesis. Many endogenous factors can affect angiogenesis in endothelial cells. VEGF is known to be a strong migration, sprouting, survival, and proliferation factor for endothelial cells during angiogenesis in endothelial cells. Searching for novel genes, involved in VEGF signaling during angiogenesis, we carried out differential display polymerase chain reaction on RNA from VEGF-stimulated human umbilical vein endothelial cells (HUVECs). In this study, follistatin (FS) differentially expressed in VEGF-treated HUVECs, compared with controls. Addition of VEGF (10 ng/mL) produced an approximately 11.8-fold increase of FS mRNA. FS or VEGF produced approximately 1.8- or 2.9-fold increases, respectively, in matrix metalloproteinase-2 (MMP-2) secretion for 12 h, compared to the addition of a control buffer. We suggest that VEGF may affect the angiogenic effect of HUVECs, through a combination of the direct effects of VEGF itself, and the indirect effects mediated via induction of FSin vitro.     

Sphingosine-1-phosphate induces human endothelial VEGF and MMP-2 production via transcription factor ZNF580: Novel insights into angiogenesis

Sphingosine-1-phosphate (S1P)-induced migration and proliferation of endothelial cells are critical for angiogenesis. C2H2-zinc finger (ZNF) proteins usually play an essential role in altering gene expression and regulating the angiogenesis. The aim of this study is to investigate whether a novel human C2H2-zinc finger gene ZNF580 (Gene ID: 51157) is involved in the migration and proliferation of endothelial cells stimulated by S1P. Our study shows that EAhy926 endothelial cells express S1P1, S1P3 and S1P5 receptors. Furthermore, S1P upregulates both ZNF580 mRNA and protein levels in a concentration- and time-dependent manner. SB203580, the specific inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, blocks the S1P-induced upregulation of ZNF580. Moreover, overexpression/downexpression of ZNF580 in EAhy926 cells leads to the enhancement/decrease of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) expression as well as the migration and proliferation of EAhy926 endothelial cells. These results elucidate the important role that ZNF580 plays in the process of migration and proliferation of endothelial cells, which provides a foundation for a novel approach to regulate angiogenesis.     

CEA-related cell adhesion molecule 1: a potent angiogenic factor and a major effector of vascular endothelial growth factor

CEA-related cell adhesion molecule 1 (CEACAM1) exhibits angiogenic properties in in vitro and in vivo angiogenesis assays. CEACAM1 purified from granulocytes and endothelial cell media as well as recombinant CEACAM1 expressed in HEK293 cells stimulate proliferation, chemotaxis, and capillary-like tube formation of human microvascular endothelial cells. They increase vascularization of chick chorioallantoic membrane and potentiate the effects of vascular endothelial growth factor (VEGF)165. VEGF165 increases CEACAM1 expression both on the mRNA and the protein level. VEGF165-induced endothelial tube formation is blocked by a monoclonal CEACAM1 antibody. These data suggest that CEACAM1 is a major effector of VEGF in the early microvessel formation. Since CEACAM1 is expressed in tumor microvessels but not in large blood vessels, CEACAM1 may be a target for the inhibition of tumor angiogenesis.     

Rosiglitazone inhibits endothelial proliferation and angiogenesis

Rosiglitazone, an insulin sensitizer, is known to offer beneficial effects in retarding atherosclerotic vascular diseases. Since proliferation and angiogenesis are involved in initiation and plaque instability, two critical steps in the cardiovascular events, this study was designed to evaluate the mechanisms of rosiglitazone on endothelial proliferation and angiogenesis. Rosiglitazone-treated human umbilical vein endothelial cells were analyzed for growth rate by use of cell number counting, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay as well as 3H-thymidine incorporation. Cell cycle analysis was detected by flow cytometry and cell cycle-related proteins were measured by Western blot. Effects of rosiglitazone on angiogenesis were assessed by vascular endothelial growth factor (VEGF)-induced tube formation and wound-healing migration. Furthermore, effects of rosiglitazone on actin stress fiber were observed under confocal microscopy. Our data showed that rosiglitazone inhibits endothelial proliferation in a dose-dependent manner. Rosiglitazone caused endothelial arrest at G1 phase via affecting several cell cycle-related proteins that led to attenuate phosphorylation of retinoblastoma protein. Rosiglitazone markedly decreased VEGF-induced tube formation and endothelial cell migration, which might be explained by a disorganization of the actin cytoskeleton. Our data suggest that both anti-proliferative and anti-angiogenic activities in endothelial cells might account for the greater than expected beneficial effects of rosiglitazone for the treatment and prevention of atherosclerosis.     

Reactive oxygen and NF-kappaB in VEGF-induced migration of human vascular smooth muscle cells.

Migration and proliferation of vascular smooth muscle cells (VSMC) contribute to angiogenesis and the lesions of atherosclerosis. Since, vascular endothelial growth factor (VEGF) is overexpressed by VSMC in intima of atherosclerotic human coronary arteries, we determined if VEGF could stimulate VSMC migration and the intracellular signals involved. VEGF induced VSMC migration but had no significant activity on proliferation. VEGF increased intracellular reactive oxygen species (ROS), NF-kappaB activation and IL-6 expression. Blockade of the generation of intracellular ROS by antioxidants inhibited VEGF-induced NF-kappaB activation, IL-6 expression, and cell migration indicating that generation of ROS was required for NF-kappaB activation and the chemotactic activity of VEGF. Expression of a mutated, nondegradable form of inhibitor of NF-kappaB (IkappaB-alphaM) suppressed VEGF-triggered activation of NF-kappaB and upregulation of IL-6 as well as VSMC migration. Neutralization of IL-6 by its antibody significantly attenuated the migration stimulated by VEGF. Collectively, our data provide the first evidence that intracellular ROS and NF-kappaB are required for VEGF-mediated smooth muscle cell migration. Further, IL-6 induced by VEGF is involved in the ability of the growth factor to stimulate migration.     

Perlecan regulates developmental angiogenesis by modulating the VEGF-VEGFR2 axis

Using the zebrafish, we previously identified a central function for perlecan during angiogenic blood vessel development. Here, we explored the nature of perlecan function during developmental angiogenesis. A close examination of individual endothelial cell behavior revealed that perlecan is required for proper endothelial cell migration and proliferation. Because these events are largely mediated by VEGF-VEGFR2 signaling, we investigated the relationship between perlecan and the VEGF pathway. We discovered that perlecan knockdown caused an abnormal increase and redistribution of total VEGF-A protein suggesting that perlecan is required for the appropriate localization of VEGF-A. Importantly, we linked perlecan function to the VEGF pathway by efficiently rescuing the perlecan morphant phenotype by microinjecting VEGF-A₁₆₅ protein or mRNA. Combining the strategic localization of perlecan throughout the vascular basement membrane along with its growth factor-binding ability, we hypothesized a major role for perlecan during the establishment of the VEGF gradient which provides the instructive cues to endothelial cells during angiogenesis. In support of this hypothesis we demonstrated that human perlecan bound in a heparan sulfate-dependent fashion to VEGF-A₁₆₅. Moreover, perlecan enhanced VEGF mediated VEGFR2 activation of human endothelial cells. Collectively, our results indicate that perlecan coordinates developmental angiogenesis through modulation of VEGF-VEGFR2 signaling events. The identification of angiogenic factors, such as perlecan, and their role in vertebrate development will not only enhance overall understanding of the molecular basis of angiogenesis, but may also provide new insight into angiogenesis-based therapeutic approaches.     

Participation of PI3K and ERK1/2 pathways are required for human brain vascular smooth muscle cell migration

Human brain vascular smooth muscle cell (HBVSMC) migration contributes to angiogenesis and several pathological processes in the brain. However, the molecular mechanism of angiogenesis, in which smooth muscle cell contributes, remains unclear. Our study investigates the role of vascular endothelial growth factor (VEGF) in the HBVSMC migration and elucidates the chemotactic signaling pathway mediating this action. We used the in vitro 'scratch' wound method to detect the HBVSMC migration. VEGF(165) (1-40ng/ml) induced the HBVSMC migration in a dose-dependent manner (P<0.05). VEGF(165) does not induce HBVSMC proliferation. Wortmannin, a specific phosphatidylinositol 3-kinase (PI3K) inhibitor, significantly inhibited serine/threonine kinase Akt/protein kinase B (PKB) phosphorylation and reduced HBVSMC migration into the wound edge following VEGF(165) stimulation (P<0.05). PD98059, an extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor, also significantly inhibited ERK1/2 phosphorylation and reduced the numbers of SMC migration. Parallel distance measurement showed that VEGF(165) induced HBVSMC migration significantly reduced due to inhibition of PI3K or ERK1/2 phosphorylation (P<0.05). Our results demonstrate that VEGF(165) could induce HBVSMC migration but not proliferation in vitro. Inhibiting Akt/PKB or ERK1/2 phosphorylation could reduce VEGF(165) induced HBVSMC migration. We provide the first evidence that activation of PI3K or ERK1/2 pathways are a crucial event in VEGF(165) mediated signal transduction leading to HBVSMC migration.     

Vascular endothelial growth factor (VEGF) receptor II-derived peptides inhibit VEGF

Vascular endothelial growth factor (VEGF) directly stimulates endothelial cell proliferation and migration via tyrosine kinase receptors of the split kinase domain family. It mediates vascular growth and angiogenesis in the embryo but also in the adult in a variety of physiological and pathological conditions. The potential binding site of VEGF with its receptor was identified using cellulose-bound overlapping peptides of the extracytosolic part of the human vascular endothelial growth factor receptor II (VEGFR II). Thus, a peptide originating from the third globular domain of the VEGFR II comprising residues 247RTELNVGIDFNWEYP261 was revealed as contiguous sequence stretch, which bound 125I-VEGF165. A systematic replacement with L-amino acids within the peptide representing the putative VEGF-binding site on VEGFR II indicates Asp255 as the hydrophilic key residue for binding. The dimerized peptide (RTELNVGIDFNWEYPAS)2K inhibits VEGF165 binding with an IC50 of 0.5 microM on extracellular VEGFR II fragments and 30 microM on human umbilical vein cells. VEGF165-stimulated autophosphorylation of VEGFR II as well as proliferation and migration of microvascular endothelial cells was inhibited by the monomeric peptide RTELNVGIDFNWEYPASK at a half-maximal concentration of 3-10, 0.1, and 0.1 microM, respectively. We conclude that transduction of the VEGF165 signal can be interrupted with a peptide derived from the third Ig-like domain of VEGFR II by blockade of VEGF165 binding to its receptor.     

Oligonucleotide NX1838 inhibits VEGF165-mediated cellular responses in vitro

Summary VEGF (vascular endothelial growth factor) overproduction has been identified as a major factor underlying pathological angiogenesis in vivo, including such conditions as psoriasis, macular degeneration, and tumor proliferation. Endothelial cell tyrosine kinase receptors, KDR and Flt-1, have been implicated in VEGF responses including cellular migration, proliferation, and modulation of vascular permeability. Therefore, agents that limit VEGF-cellular interaction are likely therapeutic candidates for VEGF-mediated disease states (particularly agents blocking activity of VEGF₁₆₅, the most frequently occurring VEGF isoform). To that end, a nuclease-resistant, VEGF₁₆₅-specific aptamer NX1838 (2′-fluoropyrimidine, RNA-based oligonucleotide/40-kDa-PEG) was developed. We have assessed NX1838 inhibition of a variety of cellular events associated with VEGF, including cellular binding, signal transduction, calcium mobilization, and induction of cellular proliferation. Our data indicate that NX1838 inhibits binding of VEGF to HUVECs (human umbilical vein endothelial cells) and dose-dependently prevents VEGF-mediated phosphorylation of KDR and PLCγ, calcium flux, and ultimately VEGF-induced cell proliferation. NX1838-inhibition of VEGF-mediated cellular events was comparable to that observed with anti-VEGF monoclonal antibody, but was ineffective as an inhibitor of VEGF₁₂₁-induced HUVEC proliferation. These findings, coupled with nuclease stability of the molecule, suggest that NX1838 may provide therapeutic utility in vivo.     

NADPH oxidase 4 promotes cardiac microvascular angiogenesis after hypoxia/reoxygenation in vitro

Microvascular endothelial cell dysfunction plays a key role in myocardial ischemia/reperfusion (I/R) injury, wherein reactive oxygen species (ROS)-dependent signaling is intensively involved. However, the roles of the various ROS sources remain unclear. This study sought to investigate the role of NADPH oxidase 4 (Nox4) in the cardiac microvascular endothelium in response to I/R injury. Adult rat cardiac microvascular endothelial cells (CMECs) were isolated and subjected to hypoxia/reoxygenation (H/R). Our results showed that Nox4 was highly expressed in CMECs, was significantly increased at both mRNA and protein levels after H/R injury, and contributed to H/R-stimulated increase in Nox activity and ROS generation. Downregulation of Nox4 by small interfering RNA transfection did not affect cell viability or ROS production under normoxia, but exacerbated H/R injury as evidenced by increased apoptosis and inhibited cell survival, migration, and angiogenesis after H/R. Nox4 inhibition also increased prolyl hydroxylase 2 (PHD2) expression and blocked H/R-induced increases in HIF-1α and VEGF expression. Pretreatment with DMOG, a specific competitive PHD inhibitor, upregulated HIF-1α and VEGF expression and significantly reversed Nox4 knockdown-induced injury. However, Nox2 was scarcely expressed and played a minimal role in CMEC survival and angiogenesis after H/R, though a modest upregulation of Nox2 was observed. In conclusion, this study demonstrated a previously unrecognized protective role of Nox4, a ROS-generating enzyme and the major Nox isoform in CMECs, against H/R injury by inhibiting apoptosis and promoting migration and angiogenesis via a PHD2-dependent upregulation of HIF-1/VEGF proangiogenic signaling.     

Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin

The serine/threonine protein kinase Akt is involved in a variety of cellular processes including cell proliferation, survival, metabolism and gene expression. It is essential in vascular endothelial growth factor (VEGF)-mediated angiogenesis; however, it is not known how Akt regulates the migration of endothelial cells, a crucial process for vessel sprouting, branching and the formation of networks during angiogenesis. Here we report that Akt-mediated phosphorylation of Girdin, an actin-binding protein, promotes VEGF-dependent migration of endothelial cells and tube formation by these cells. We found that exogenously delivered adenovirus harbouring Girdin short interfering RNA in Matrigel embedded in mice, markedly inhibited VEGF-mediated angiogenesis. Targeted disruption of the Girdin gene in mice impaired vessel remodelling in the retina and angiogenesis from aortic rings, whereas Girdin was dispensable for embryonic vasculogenesis. These findings demonstrate that the Akt/Girdin signalling pathway is essential in VEGF-mediated postneonatal angiogenesis.     

Osteocyte-induced angiogenesis via VEGF–MAPK-dependent pathways in endothelial cells

Recently, it has been suggested osteocytes control the activities of bone formation (osteoblasts) and resorption (osteoclast), indicating their important regulatory role in bone remodelling. However, to date, the role of osteocytes in controlling bone vascularisation remains unknown. Our aim was to investigate the interaction between endothelial cells and osteocytes and to explore the possible molecular mechanisms during angiogenesis. To model osteocyte/endothelial cell interactions, we co-cultured osteocyte cell line (MLOY4) with endothelial cell line (HUVECs). Co-cultures were performed in 1:1 mixture of osteocytes and endothelial cells or by using the conditioned media (CM) transfer method. Real-time cell migration of HUVECs was measured with the transwell migration assay and xCELLigence system. Expression levels of angiogenesis-related genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of vascular endothelial growth factor (VEGF) and mitogen-activated phosphorylated kinase (MAPK) signaling were monitored by western blotting using relevant antibodies and inhibitors. During the bone formation, it was noted that osteocyte dendritic processes were closely connected to the blood vessels. The CM generated from MLOY4 cells-activated proliferation, migration, tube-like structure formation, and upregulation of angiogenic genes in endothelial cells suggesting that secretory factor(s) from osteocytes could be responsible for angiogenesis. Furthermore, we identified that VEGF secreted from MLOY4-activated VEGFR2–MAPK–ERK-signaling pathways in HUVECs. Inhibiting VEGF and/or MAPK–ERK pathways abrogated osteocyte-mediated angiogenesis in HUVEC cells. Our data suggest an important role of osteocytes in regulating angiogenesis.