Homocysteine inhibits angiogenesis in vitro and in vivo

Homocysteine has been reported to inhibit endothelial cell proliferation, which is closely related to angiogenesis. However, the relationship between homocysteine and angiogenesis is unknown. To clarify whether homocysteine would inhibit angiogenesis in vitro and in vivo, we examined the effect of homocysteine on tube formation by bovine aortic endothelial cells (BAECs) and by human microvessel endothelial cell-1 (HMEC-1) in vitro, and on angiogenesis in vivo using the chorioallantoic membrane (CAM) assay, as well as on BAEC proliferation and migration. Homocysteine, but not cysteine, inhibited BAEC proliferation, migration, and tube formation in a dose-dependent manner at concentrations from 0 to 10 mM. Homocysteine also inhibited tube formation by HMEC-1s. In these assay, 50% inhibition was induced by about 1 mM homocysteine. In the in vivo CAM assay, 0, 10, 100, 500, and 1000 microgram homocysteine induced an avascular zone by 0, 0, 16.7, 53.3 and 76.5%, respectively, also showing a dose-dependent effect. It was suggested that homocysteine inhibited angiogenesis by preventing proliferation and migration of endothelial cells.     

原核表达Arresten蛋白抑制血管生成作用

目的:研究原核表达的Arresten蛋白纯化品对血管内皮细胞及血管生成的抑制作用。方法:MTT法检测Arresten蛋白对人脐静脉内皮细胞(HUVEC)增殖的影响;流式细胞仪分析Arresten蛋白作用下HUVEC凋亡的情况;细胞迁移实验观察Arresten蛋白对HUVEC迁移能力的影响;鸡胚绒毛尿囊膜(CAM)实验观察Arresten蛋白对新生血管的抑制情况。结果:原核表达的Arresten蛋白纯化品能特异性地抑制 HUVEC的增殖、迁移,诱导HUVEC的凋亡,并在一定范围内呈现出剂量—效应关系。Arresten蛋白能有效抑制鸡胚尿囊膜血管的生长(P<0.01)。结论:原核表达的Arresten蛋白纯化品对内皮细胞有特异的抑制作用,能有效抑制血管生成。     

Anti-angiogenic effects of conjugated docosahexaenoic acid in vitro and in vivo

The anti-angiogenic effects of conjugated docosahexaenoic acid (CDHA), which was prepared by an alkaline treatment of docosahexaenoic acid and contained conjugated double bonds, were investigated in vitro and in vivo. CDHA inhibited tube formation by the bovine aortic endothelial cell (BAEC), and also inhibited the proliferation of BAEC at a concentration of CDHA that suppressed tube formation, but did not influence cell migration. The inhibition of BAEC growth caused by CDHA was accompanied by a marked change in cellular morphology. Nuclear condensation and brightness were observed in Hoechst 33342-stained cells treated with CDHA, indicating that CDHA induced apoptosis in BAEC. We also evaluated the angiogenesis inhibition of CDHA in vivo. The vessel formation which was triggered by tumor cells was clearly suppressed in mice orally given CDHA. Our findings suggest that CDHA has potential use as a therapeutic dietary supplement for minimizing tumor angiogenesis.     

合欢皮提取物抑制血管生成的体内药效学研究

为了探讨合欢皮提取物体内对血管生成的抑制作用。本文采用MTT法和Matrigel plug方法考察合欢皮提取物对人血管内皮细胞(HMEC)增殖和体内Matrigel胶小管形成的影响,运用Lewis肺癌转移小鼠模型,观察合欢皮提取物对小鼠Lewis肺癌的疗效。结果表明合欢皮提取物可明显抑制HMEC细胞的体外增殖(IC50为30μg/mL),并且呈明显的剂量依赖性,同时具有抑制Matrigel plug中的血管新生的作用。合欢皮提取物还能够明显抑制Lewis肺癌肿瘤细胞生长,减少转移病灶。提示合欢皮提取物具有明显抑制血管生成的作用,有可能成为有效的血管生成抑制剂。     

Discovery of a benzoxazine derivative promoting angiogenesis in vitro and in vivo

Angiogenesis is a multi‐step process that refers to the growth of new vessels from pre‐existing ones. Endothelial proliferation, migration, and tube formation constitute a critical step in angiogenesis. Recently, we demonstrated that a novel benzoxazine derivative, 6‐amino‐2,3‐dihydro‐3‐hydroxymethyl‐1,4‐benzoxazine (ABO) could improve the proliferation of human umbilical vein endothelial cells (HUVECs) without basic fibroblast growth factor (bFGF) and serum. In this study, we further tested its effect on endothelial angiogenesis with Matrigel assay, migration assay, and in vivo chick chorioallantoic membrane (CAM) assay. Our results showed that ABO effectively facilitated cell migration and promoted capillary‐like tube formation in vitro and in vivo. To elucidate the underlying mechanisms, we examined intracellular reactive oxygen species (ROS) level/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and superoxide dismutase (SOD) activities, nitric oxide (NO) level/endothelial nitric oxide synthase (eNOS) activity, and mitochondrial membrane potential (MMP). Our data indicated that ABO depressed ROS with inhibition of NADPH oxidase instead of SOD activity, stimulated NO production and eNOS activation, and restored MMP in HUVECs. Our findings suggest that ABO is a promising tool for exploring the mechanisms of angiogenesis and may have a therapeutic potential in ischemic pathologies. J. Cell. Physiol. 223: 202–208, 2010. © 2009 Wiley‐Liss, Inc.     

Angiomotin: an angiostatin binding protein that regulates endothelial cell migration and tube formation

Angiostatin, a circulating inhibitor of angiogenesis, was identified by its ability to maintain dormancy of established metastases in vivo. In vitro, angiostatin inhibits endothelial cell migration, proliferation, and tube formation, and induces apoptosis in a cell type-specific manner. We have used a construct encoding the kringle domains 1--4 of angiostatin to screen a placenta yeast two-hybrid cDNA library for angiostatin-binding peptides. Here we report the identification of angiomotin, a novel protein that mediates angiostatin inhibition of migration and tube formation of endothelial cells. In vivo, angiomotin is expressed in the endothelial cells of capillaries as well as larger vessels of the human placenta. Upon expression of angiomotin in HeLa cells, angiomotin bound and internalized fluorescein-labeled angiostatin. Transfected angiomotin as well as endogenous angiomotin protein were localized to the leading edge of migrating endothelial cells. Expression of angiomotin in endothelial cells resulted in increased cell migration, suggesting a stimulatory role of angiomotin in cell motility. However, treatment with angiostatin inhibited migration and tube formation in angiomotin-expressing cells but not in control cells. These findings indicate that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.     

Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth

We isolated and identified an endogenous 24-kDa human basement membrane-derived inhibitor of angiogenesis and tumor growth, termed canstatin. Canstatin, a fragment of the alpha2 chain of type IV collagen, was produced as a recombinant molecule in Escherichia coli and 293 embryonic kidneys cells. Canstatin significantly inhibited human endothelial cell migration and murine endothelial cell tube formation. Additionally, canstatin potently inhibited 10% fetal bovine serum-stimulated endothelial cell proliferation and induced apoptosis, with no inhibition of proliferation or apoptosis observed on non-endothelial cells. Inhibition of endothelial proliferation was not concomitant with a change in extracellular signal-regulated kinase activation. We demonstrate that apoptosis induced by canstatin was associated with a down-regulation of the anti-apoptotic protein, FLIP. Canstatin also suppressed in vivo growth of large and small size tumors in two human xenograft mouse models with histology revealing decreased CD31-positive vasculature. Collectively, these results suggest that canstatin is a powerful therapeutic molecule for suppressing angiogenesis.     

Irisin attenuates oxidized low-density lipoprotein impaired angiogenesis through AKT/mTOR/S6K1/Nrf2 pathway

It is known that irisin increases total body energy expenditure, decreases body weight, and enhances insulin sensitivity. Although previous studies have demonstrated that irisin induces vascular endothelial cell (EC) angiogenesis, the molecular mechanisms underlying irisin-induced angiogenesis under conditions reflecting atherosclerosis are not known. The aim of the present study is to investigate whether irisin could inhibit oxidized low-density lipoprotein (oxLDL) impaired angiogenesis. We investigated the effect of irisin on angiogenesis in vitro by evaluating cell viability, cell migration, and the capacity to form capillary-like tubes using human umbilical vein endothelial cells and human microvascular endothelial cells (HUVECs and HMEC-1) that were treated with oxLDL. We also evaluated the effects of irisin on angiogenesis in vivo by Matrigel plug angiogenesis assay and in a chicken embryo membrane (CAM) model. Our results demonstrated that irisin increased oxLDL-treated EC viability as well as migration and tube formation. Moreover, oxLDL inhibited angiogenic response in vivo, both in the Matrigel plug angiogenesis assay and in the CAM model, and was attenuated by irisin. Furthermore, irisin decreased apoptosis, inflammatory cytokines, and intracellular reactive oxygen species (ROS) levels in oxLDL-treated EC. In addition, we found that irisin upregulated pAkt/mTOR/Nrf2 in oxLDL-treated EC. Both mTOR/Nrf2 shRNA and LY294002 could inhibit the protective effect of irisin. Taken together these results, they suggested that irisin attenuates oxLDL-induced vascular injury by activating the Akt/mTOR/Nrf2 pathway. Our findings suggest that irisin attenuates oxLDL-induced blood vessel injury.     

Coupling in vitro and in vivo paradigm reveals a dose dependent inhibition of angiogenesis followed by initiation of autophagy by C6-ceramide

The activity of N-hexanoyl-D-erythro-sphingosine, a C6-ceramide against angiogenesis was tested in vitro and in vivo. The effect of ceramide in inhibiting MCF-7 cancer cells was also determined. The aim of this study was to potentiate the effect of ceramide as anti-angiogenic compound that can regulate tumor induced angiogenesis.C6-ceramide inhibited vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVEC) tube formation in a dose-dependent manner within 24 hours. Ceramide at concentrations between 12.5 and 25 μM inhibited the viability of MCF-7 cells and reduced VEGF-induced cell migration in 24 hours. At 50 μM, ceramide induced MCF-7 cell death via autophagy as demonstrated by accumulation of MDC in ceramide-treated MCF-7 vacuoles. The expression of VEGF was reduced and the levels of cathepsin D in MCF-7 increased. In vivo, 50 μM ceramide caused a 40% reduction of new vessel formation in the CAM assay within 24 hours. Zebrafish exposed to 100 - 400 μM ceramide had a distinct disruption of blood vessel development at 48 hours post-fertilization. Ceramide-exposed embryos also had primary motoneurons exhibiting abnormal axonal trajectories and ectopic branching. Ceramide induced cell-death was not detected in the zebrafish assay. Collectively, these data indicate that ceramide is a potent anti-angiogenic compound and that the mechanism underlying its anti-angiogenic capabilities does not rely upon the induction of apoptosis.     

新型埃博霉素衍生物对血管新生抑制作用的研究

研究新合成的新型埃博霉素单甲基衍生物对血管新生的影响,以期阐明埃博霉素在肿瘤细胞和血管新生中的效应及其所作用的重要途径,为药物新靶点的开发提供理论依据.首先,以人脐静脉内皮细胞为研究对象,利用MTT法检测细胞增殖,Transwell法检测细胞迁移和侵润,明胶酶谱分析金属基质蛋白酶MMP2的活性,RT PCR检测MMP2 RNA表达水平|然后以鸡胚绒毛尿囊膜为模型,研究该衍生物对血管新生的影响.结果表明:1）新型埃博霉素单甲基衍生物可以显著抑制人脐静脉内皮细胞HUVEC的增殖、迁移和侵润以及Ⅳ型胶原酶分泌; 2）新型埃博霉素单甲基衍生物可以使鸡胚尿囊膜产生明显的无血管区,对血管新生产生明显的抑制作用. 结果说明,新型埃博霉素单甲基衍生物可以通过抑制血管内皮细胞的增殖、迁移、侵润以及Ⅳ型胶原酶分泌等显著抑制诱导血管新生过程的步骤,最终抑制血管新生.     

Canstatin-N fragment inhibits in vitro endothelial cell proliferation and suppresses in vivo tumor growth

Type IV collagen is one of the components of vascular basement involved in regulation of angiogenesis. Canstatin, the non-collagenous 1 (NC1) domain of alpha2 chain of type IV collagen, was identified as an inhibitor of angiogenesis and tumor growth by Kamphaus et al. Our previous studies showed that canstatin-N, the N-terminal 1-89 amino acid fragment of canstatin, inhibited the neovascularization in a dose-dependent manner as tested by CAM assay. In the present study, we demonstrate that canstatin-N produced in Escherichia coli specifically inhibited in vitro the proliferation of human umbilical vein endothelial cells (ECV304) and significantly induced apoptosis. The apoptosis-inducing activity of canstatin-N was much stronger than that of canstatin, indicating that the apoptosis-inducing activity of canstatin is likely located within its N-terminal 1-89 amino acid fragment. Canstatin-N also suppressed in vivo growth of B(16) murine melanoma in BALB/c mice at a dosage of 10mg/kg/day. These results suggest that canstatin-N is a useful candidate molecule for inhibition of tumor growth.     

Evodiamine inhibits in vitro angiogenesis: Implication for antitumorgenicity

Evodiamine, the major bioactive compound isolated from Chinese herbal drug named Wu-Chu-Yu, has been reported to exhibit anti-tumor growth and metastasis. However, the effect of evodiamine on angiogenesis remains to be investigated. We used the fresh medium containing evodiamine or human lung adenocarcinoma cell (CL1 cells) derived conditioned media free of evodiamine to test their capability to induce in vitro angiogenesis, i.e., human umbilical vein endothelial cells (HUVECs) tube formation and invasion. We demonstrated that evodiamine could directly inhibit in vitro HUVECs tube formation and invasion. Locally administered evodiamine also inhibited the in vivo angiogenesis in the chick embryo chorioallantoic membrane (CAM) assay. The gene expression of vascular endothelial growth factor (VEGF) and the p44/p42 mitogen-activated protein kinase (MAPK, ERK) that correlated with endothelial cells angiogenesis were inhibited by evodiamine. We found that the evodiamine-treated CL1 cells derived conditioned medium showed decreased VEGF release and reduced ability of inducing in vitro tube formation. After the collection of conditioned media, the VEGF expression of remaining CL1 cells were determined by Western analyses and revealed that evodiamine decreased VEGF expression. Moreover, administration of recombinant human VEGF(165) (rhVEGF(165)) induced tube formation and ERK phosphorylation by HUVECs, and partially attenuated inhibitory effect of evodiamine. From these results, we suggested that evodiamine is a potent inhibitor of angiogenesis. The mechanism might involve at least the inhibition of VEGF expression, probably through repression of ERK phosphorylation.     

DTD, an anti-inflammatory ditriazine, inhibits angiogenesis in vitro and in vivo

The ditriazine derivative DTD (4,10-dichloropyrido[5,6:4,5]thieno[3,2-d':3,2-d]-1,2,3-ditriazine) has been previously reported to reduce the degree of granulomatous inflammation and vascular density in a murine air pouch granuloma model. The aim of this study was to test whether DTD affects angiogenesis. Our results show that DTD inhibits in vivo angiogenesis in the chorioallantoic membrane (CAM) assay at doses equal or lower than 0.3 nmol/egg. Different in vitro assays were used to study the potential effects of this compound on key steps of angiogenesis, namely, a colorimetric assay of cell proliferation/viability, a morphogenesis on Matrigel assay, zymographic assays for gelatinases and nuclear morphology and cell cycle analysis for apoptosis induction. Our data indicate that DTD inhibits proliferation but does not induce apoptosis in endothelial cells in vitro. DTD suppresses the endothelial capillary-like chord formation at concentrations lower than those required to inhibit proliferation. DTD treatment inhibits the matrix metalloproteinase-2 production in endothelial and fibrosarcoma cells, but does not affect the cyclooxygenase-2 expression in endothelial cells, as assessed by western blot analysis. Taken together, results here presented indicate that DTD exhibits an anti-angiogenic activity that is independent of inflammatory processes and make it a promising drug for further evaluation in the treatment of angiogenesis-related pathologies.     

Connective tissue growth factor induces the proliferation, migration, and tube formation of vascular endothelial cells in vitro, and angiogenesis in vivo.

Connective tissue growth factor (CTGF) is a novel cysteine-rich, secreted protein. Recently, we found that inhibition of the endogenous expression of CTGF by its antisense oligonucleotide and antisense RNA suppresses the proliferation and migration of vascular endothelial cells. In the present study, the following observations demonstrated the angiogenic function of CTGF in vitro and in vivo: (i) purified recombinant CTGF (rCTGF) promoted the adhesion, proliferation and migration of vascular endothelial cells in a dose-dependent manner under serum-free conditions, and these effects were inhibited by anti-CTGF antibodies; (ii) rCTGF markedly induced the tube formation of vascular endothelial cells, and this effect was stronger than that of basic fibroblast growth factor or vascular endothelial growth factor; (iii) application of rCTGF to the chicken chorioallantoic membrane resulted in a gross angiogenic response, and this effect was also inhibited by anti-CTGF antibodies. (iv) rCTGF injected with collagen gel into the backs of mice induced strong angiogenesis in vivo. These findings indicate that CTGF is a novel, potent angiogenesis factor which functions in multi-stages in this process.     

Synthesis and biological evaluation of scopoletin derivatives

A series of new scopoletin derivatives were designed and synthesized. Their anti-proliferative effect was initially evaluated against various human cancer cell lines. Among the tested compounds, A1, A2, and D6 showed significant anti-proliferative activities. Angiogenesis was detected by endothelial cell migration assay and tube formation study. The results showed that A1, A2, and D6 inhibited the vascular endothelial growth factor (VEGF)-stimulated proliferation, migration, and tube formation of human umbilical vein endothelial cells in vitro. Moreover, they inhibited the vessel growth in the chorioallantoic membrane in vivo. This inhibition was correlated with a significant decrease in the VEGF-triggered phosphorylated forms of ERK1/2 and Akt. In summary, these findings strongly suggested that these scopoletin derivatives might be structurally novel angiogenesis inhibitors.     

A novel non-containing-nitrogen bisphosphonate inhibits both in vitro and in vivo angiogenesis

Bisphosphonates (BP) are powerful inhibitors of bone resorption and are widely used in the treatment of patients with metastasis-induced osteolysis. In the present study, we show that a novel non-nitrogen-containing BP (BP7033) that exhibits antitumor activity is a potent inhibitor of both in vivo and in vitro angiogenesis. When administered to mice, BP7033 inhibited tumoral angiogenesis (65% at 0.06mg/injection) as well as tumor growth (65% at 0.006mg/injection) in a tumor model of A431 cells xenografted in nude mice, with no sign of toxicity. Additionally, in vivo angiogenesis induced by vascular endothelial growth factor-containing Matrigel implants was reduced by 90% in the presence of BP7033 (0.6mg/plug). In vitro, BP7033 inhibited proliferation of human umbilical vein endothelial cells (HUVEC) (IC(50) value 3x10(-4) M) and completely prevented the formation of capillary-like tubules by HUVEC in Matrigel. Moreover, treatment of A431 cells by BP7033 induced an inhibition of Ras processing and a decrease in the secretion of both vascular endothelial growth factor and matrix metalloproteinase-2, two well-known stimulators of the proliferation and migration of endothelial cells. These findings indicate that this new BP compound has marked antiangiogenic properties and thus represents a promising candidate for treatment of malignant diseases with an angiogenic component.     

Convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer

BackgroundAberrant activation of STAT3 is frequently encountered and promotes survival, cellular proliferation, migration, invasion and angiogenesis in tumor cell. Convallatoxin, triterpenoid ingredient, exhibits anticancer pharmacological properties.PurposeIn this work, we investigated the anticancer potential of convallatoxin and explored whether convallatoxin mediates its effect through interference with the STAT3 activation in colorectal cancer cells.MethodsIn vitro, the underlying mechanisms of convallatoxin at inhibiting STAT3 activation were investigated by homology modeling and molecular docking, luciferase reporter assay, MTT assay, RT-PCR, Western blotting and immunofluorescence assays. Changes in cellular proliferation, apoptosis, migration, invasion and angiogenesis were analyzed by EdU labeling assay, colony formation assay, flow cytometry assay, wound-healing assay, matrigel transwell invasion assay and tube formation assays. And in vivo, antitumor activity of convallatoxin was assessed in a murine xenograft model of HCT116 cells.ResultsConvallatoxin decreased the viability of colorectal cancer lines. Moreover, convallatoxin reduced the P-STAT3 (T705) via the JAK1, JAK2, and Src pathways and inhibited serine-727 phosphorylation of STAT3 via the PI3K-AKT-mTOR-STAT3 pathways in colorectal cancer cells. Interestingly, we discovered the crosstalk between mTOR and JAK2 in mTOR/STAT3 and JAK/STAT3 pathways, which collaboratively regulated STAT3 activation and convallatoxin play a role in it. Convallatoxin also downregulated the expression of target genes involved cell survival (e.g., Survivin, Bcl-xl, Bcl-2), proliferation (e.g., Cyclin D1), metastasis (e.g., MMP-9), and angiogenesis (e.g., VEGF). Indeed, we found that convallatoxin inhibited tube formation, migration, and invasion of endothelial cells, and inhibited the proliferation. Finally, in vivo observations were confirmed by showing antitumor activity of convallatoxin in a murine xenograft model.ConclusionThe result of the current study show that convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer cells and indicate that convallatoxin could be a valuable candidate for the development of colorectal cancer therapeutic.     

CD40-dependent activation of phosphatidylinositol 3-kinase/Akt pathway mediates endothelial cell survival and in vitro angiogenesis

CD40 has been involved in tumor and inflammatory neoangiogenesis. In this study we determined that stimulation of endothelial CD40 with sCD154 induced resistance to apoptosis and in vitro vessel-like formation by human microvascular endothelial cells (HMEC). These effects were determined to be mediated by CD40-dependent signaling because they were inhibited by a soluble CD40-muIg fusion protein. Moreover, apoptosis of HMEC was associated with an impairment of Akt phosphorylation, which was restored by stimulation with sCD154. The anti-apoptotic effect as well as in vitro vessel-like formation and Akt phosphorylation were inhibited by treatment of HMEC with two unrelated pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002. CD40 stimulation induced a rapid increase in Akt enzymatic activity that was not prevented by cycloheximide, an inhibitor of protein synthesis. The enhanced Akt activity induced by stimulation of endothelial CD40 was temporarily correlated with the association of CD40 with TRAF6, c-Cbl, and the p85 subunit of PI3K. Expression of negative-dominant Akt inhibited the activation of endogenous Akt through CD40 stimulation, despite the observation that association of CD40 with TRAF6, c-Cbl, and PI3K was intact. The defective activation of Akt abrogated not only the anti-apoptotic effect of CD40 stimulation but also the proliferative response, the enhanced motility, and the in vitro formation of vessel-like tubular structures by CD40-stimulated HMEC. In conclusion, these results suggest that endothelial CD40, through activation of the PI3K/Akt signaling pathway, regulates cell survival, proliferation, migration, and vessel-like structure formation, all steps considered critical for angiogenesis.     

Hepatoma-derived growth factor is a pulmonary endothelial cell-expressed angiogenic factor

Hepatoma-derived growth factor (HDGF) was previously identified as a developmentally regulated cardiovascular and renal gene that is mitogenic for vascular smooth muscle and aortic endothelial cells. As reciprocal interactions of smooth muscle and endothelial cells are necessary for vascular formation, we examined whether HDGF plays a role in angiogenesis. According to immunohistochemistry, HDGF was highly expressed in endothelial cells of nonmuscularized, forming blood vessels of the fetal lung. HDGF was also expressed in endothelial cells of small (20 microm) mature arteries and veins. By Western immunoblotting, HDGF was highly expressed by human pulmonary microvascular endothelial cells in vitro. Adenoviral overexpression of HDGF was mitogenic for human pulmonary microvascular endothelial cells in serum-free medium, stimulating a 1.75-fold increase in bromodeoxyuridine (BrdU) uptake and a twofold increase in cell migration. With the chick chorioallantoic membrane (CAM), a biologic assay for angiogenesis, exogenous recombinant HDGF significantly stimulated blood vessel formation and a dose-dependent reorganization of cells within the CAM into a more compact, linear alignment reminiscent of tube formation. According to double immunostaining for endothelial cells with a transforming growth factor-betaII receptor antibody and BrdU as a marker of cell proliferation, exogenous HDGF selectively stimulated endothelial cell BrdU uptake. HDGF also activated specific ERK1/2 signaling and did not overlap with VEGF SAPK/JNK, Akt-mediated pathways. We conclude that HDGF is a highly expressed vascular endothelial cell protein in vivo and is a potent endothelial mitogen and regulator of endothelial cell migration by mechanisms distinct from VEGF.