The Two Endocytic Pathways Mediated by the Carbohydrate Recognition Domain and Regulated by the Collagen-like Domain of Galectin-3 in Vascular Endothelial Cells

Galectin-3 plays an important role in endothelial morphogenesis and angiogenesis. We investigated the endocytosis of galectin-3 in human vascular endothelial cells and showed that galectin-3 could associate with and internalized into the cells in a carbohydrate-dependent manner. Our work also revealed that galectin-3 was transported to the early/recycling endosomes and then partitioned into two routes – recycling back to the plasma membrane or targeting to the late endosomes/lysosomes. Various N- and C-terminal truncated forms of galectin-3 were constructed and compared with the full-length protein. These comparisons showed that the carbohydrate-recognition domain of galectin-3 was required for galectin-3 binding and endocytosis. The N-terminal half of the protein, which comprises the N-terminal leader domain and the collagen-like internal repeating domain, could not mediate binding and endocytosis alone. The collagen-like domain, although it was largely irrelevant to galectin-3 trafficking to the early/recycling endosomes, was required for targeting galectin-3 to the late endosomes/lysosomes. In contrast, the leader domain was irrelevant to both binding and intracellular trafficking. The data presented in this study correlate well with different cellular behaviors induced by the full-length and the truncated galectin-3 and provide an alternative way of understanding its angiogenic mechanisms.     

Double-stranded RNA induces galectin-9 in vascular endothelial cells: involvement of TLR3, PI3K, and IRF3 pathway

Galectin-9 is a member of the galectin family, which induces various biological reactions such as chemotaxis of eosinophils and apoptosis of T cells. We previously reported that polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA (dsRNA), induces the expression of galectin-9 in human umbilical vein endothelial cells (HUVECs). In the present study, we addressed the possible involvement of two potential receptors for dsRNA, Toll-like receptor (TLR) 3 and retinoic acid-inducible gene-I (RIG-I), in the expression of galectin-9 in HUVECs. Poly IC-induced galectin-9 expression was almost completely suppressed by RNA interference (RNAi) against TLR3, but not against RIG-I. LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited the induction of galectin-9 by poly IC. RNAi against interferon regulatory factor 3 (IRF3) also inhibited poly IC-induced galectin-9 expression. We conclude that TLR3, PI3K, and IRF3 are involved in the poly IC-induced galectin-9 expression in HUVECs.     

Synthesis of an endogeneous lectin, galectin-1, by human endothelial cells is up-regulated by endothelial cell activation

The pattern of expression of an endogenous lectin, galectin-1, was examined in human lymphoid tissue. Galectin-1 was detected in the endothelial cells lining specialized vessels, termed high endothelial venules, in activated lymphoid tissue, but not in a resting lymph node. Cultured endothelial cells (human aortic and umbilical vein endothelial cells (HAECs and HUVECs)) expressed galectin-1. Activation of the cultured endothelial cells increased the level of galectin-1 expression, as determined by ELISA, Northern blot analysis and high throughput cDNA sequencing. These results suggest that galectin-1 expressed by endothelial cells may bind to and affect the trafficking of cells emigrating from blood into tissues.     

Agkistin, a snake venom-derived glycoprotein Ib antagonist, disrupts von Willebrand factor-endothelial cell interaction and inhibits angiogenesis

Glycoprotein (GP) Ib, an adhesion receptor expressed on both platelets and endothelial cells, mediates the binding of von Willebrand factor (vWF). Platelet GPIb plays an important role in platelet adhesion and activation, whereas the interaction of vWF and endothelial GPIb is not fully understood. We report here that agkistin, a snake venom protein, selectively blocks the interaction of vWF with human endothelial GPIb and inhibits angiogenesis in vivo. Agkistin specifically blocked human umbilical vein endothelial cell (HUVEC) adhesion to immobilized vWF in a concentration-dependent manner. Fluorescein isothiocyanate (FITC)-conjugated agkistin bound to HUVECs in a saturable manner. AP1, a monoclonal antibody (mAb) raised against GPIb, specifically inhibited the binding of FITC-conjugated agkistin to HUVECs in a dose-dependent manner, but other anti-integrin mAbs raised against alpha(v)beta(3), alpha(2)beta(1), and alpha(5)beta(1) did not affect this binding reaction. However, neither agkistin (2 microgram/ml) nor AP1 (40 microgram/ml) apparently reduced HUVEC viability. Both agkistin and AP1 exhibited a profound anti-angiogenic effect in vivo when assayed by using the 10-day-old embryo chick chorioallantoic membrane model. These results suggest endothelial GPIb plays a role in spontaneous angiogenesis in vivo, and the anti-angiogenic effect of agkistin may be because of disruption of the interaction of endogenous vWF with endothelial GPIb.     

Nobiletin, a polymethoxylated flavonoid from citrus, shows anti-angiogenic activity in a zebrafish in vivo model and HUVEC in vitro model

Traditional Chinese medicinal herbs are a rich source of compounds with reported anti-inflammatory and anti-carcinogenic effects. Growing evidence shows the codependence of chronic inflammation and angiogenesis, and the potential benefits of targeting angiogenesis in the treatment of chronic inflammation and targeting inflammation in the treatment of diseases with impaired angiogenesis. We hypothesized that the anti-inflammatory activity of the natural compounds may owe at least some of its efficacy to their anti-angiogenic activity and hence we investigated the anti-angiogenic activity of these compounds in vivo in zebrafish embryos and in vitro in human umbilical vein endothelial cells (HUVECs). Nobiletin, a polymethoxylated flavonoid from citrus fruits, showed anti-angiogenic activity in both assays. Nobiletin inhibited the formation of intersegmental vessels (ISVs) in live transgenic zebrafish embryos expressing green fluorescent protein (GFP) in the vasculature. Cell cycle analysis of dissociated zebrafish embryo cells showed that nobiletin induced G0/G1 phase accumulation in a dose-dependent manner in GFP-positive endothelial cells. Nobiletin also dose-dependently induced VEGF-A mRNA expression. In HUVECs, nobiletin inhibited endothelial cell proliferation and, to a greater extent, tube formation in a dose-dependent manner. As in the in vivo study, nobiletin induced G0/G1 cell cycle arrest in HUVECs. However, this arrest was not accompanied by an increase in apoptosis, indicating a cytostatic effect of nobiletin. This study, for the first time, identifies nobiletin as having potent anti-angiogenic activity and suggests that nobiletin has a great potential for future research and development as a cytostatic anti-proliferative agent.     

VEGF-A promotes angiogenesis after acute myocardial infarction through increasing ROS production and enhancing ER stress-mediated autophagy

Proangiogenesis is generally regarded as an effective approach for treating ischemic heart disease. Vascular endothelial growth factor (VEGF)-A is a strong and essential proangiogenic factor. Reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, and autophagy are implicated in the process of angiogenesis. This study is designed to clarify the regulatory mechanisms underlying VEGF-A, ROS, ER stress, autophagy, and angiogenesis in acute myocardial infarction (AMI). A mouse model of AMI was successfully established by occluding the left anterior descending coronary artery. Compared with the sham-operated mice, the microvessel density, VEGF-A content, ROS production, expression of vascular endothelial cadherin, positive expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/Bip), and LC3 puncta in CD31-positive endothelial cells of the ischemic myocardium were overtly elevated. Moreover, VEGF-A exposure predominantly increased the expression of beclin-1, autophagy-related gene (ATG) 4, ATG5, inositol-requiring enzyme-1 (IRE-1), GRP78/Bip, and LC3-II/LC3-I as well as ROS production in the human umbilical vein endothelial cells (HUVECs) in a dose and time-dependent manner. Both beclin-1 small interfering RNA and 3-methyladenine treatment predominantly mitigated VEGF-A-induced tube formation and migration of HUVECs, but they failed to elicit any notable effect on VEGF-A-increased expression of GRP78/Bip. Tauroursodeoxycholic acid not only obviously abolished VEGF-A-induced increase of IRE-1, GRP78/Bip, beclin-1 expression, and LC3-II/LC3-I, but also negated VEGF-A-induced tube formation and migration of HUVECs. Furthermore, N-acetyl- l -cysteine markedly abrogated VEGF-A-increased ROS production, IRE-1, GRP78/Bip, beclin-1 expression, and LC3-II/LC3-I in the HUVECs. Taken together, our data demonstrated that increased spontaneous production of VEGF-A may induce angiogenesis after AMI through initiating ROS–ER stress-autophagy axis in the vascular endothelial cells.     

Scutellarin promotes in vitro angiogenesis in human umbilical vein endothelial cells

Angiogenesis is critical to a wide range of physiological and pathological processes. Scutellarin, a major flavonoid of a Chinese herbal medicine Erigeron breviscapus (Vant.) Hand. Mazz. has been shown to offer beneficial effects on cardiovascular and cerebrovascular functions. However, scutellarin’s effects on angiogenesis and underlying mechanisms are not fully elucidated. Here, we studied angiogenic effects of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. Scutellarin was found by MTT assay to induce proliferation of HUVECs. In scutellarin-treated HUVECs, a dramatic increase in migration was measured by wound healing assay; Transwell chamber assay found significantly more invading cells in scutellarin-treated groups. Scutellarin also promoted capillary-like tube formation in HUVECs on Matrigel, and significantly upregulated platelet endothelial cell adhesion molecule-1 at both mRNA and protein levels. Scutellarin’s angiogenic mechanism was investigated in vitro by measuring expression of angiogenic factors associated with cell migration and invasion. Scutellarin strongly induced MMP-2 activation and mRNA expression in cultured HUVECs in a concentration-dependent manner. Taken together, these results suggest that scutellarin promotes angiogenesis and may form a basis for angiogenic therapy.     

Calcitonin gene-related peptide (CALCA) is a proangiogenic growth factor in the human placental development

Recent studies have shown that homozygous knockout of gene for calcitonin gene-related peptide (CALCA) receptor component, calcitonin receptor-like receptor (CALCRL), led to extreme hydrops fetalis and embryonic death, underlining the critical role of CALCA in embryonic development and fetal growth. The present study was designed to determine the cellular localization of CALCA and its receptor components, CALCRL and receptor activity modifying protein 1 (RAMP1), at the human implantation site during early pregnancy; to assess whether CALCA regulates in vitro angiogenesis of human endothelial cells; and to examine whether CALCA can improve angiogenic imbalance in preeclamptic placental explants. Our studies demonstrated that both protein and mRNA for CALCA were expressed by the villous and extravillous trophoblasts and decidual cells in the first-trimester villous tissues. CALCA receptor components, CALCRL and RAMP1, were expressed by both villous and extravillous trophoblast cells, as well as vascular endothelial cells. CALCA induced both endothelial proliferation and migration in a dose- and time-dependent manner, and it promoted capillarylike tube formation of human umbilical vein endothelial cells (HUVECs) on Matrigel. CALCA-induced angiogenesis of human endothelial cells was completely blocked by CALCA antagonist CALCA(8-37). Further, conditioned medium from preeclamptic placental explants significantly inhibited HUVEC capillarylike tube formation compared with gestational age-matched controls, and conditioned medium from preeclamptic placental explants incubated with CALCA significantly improved capillarylike tube formation. We conclude that CALCA induces in vitro angiogenesis by stimulating endothelial cell proliferation, migration, and capillarylike tube formation; thus, CALCA at the human implantation site may constitute a potential autocrine or paracrine mechanism that could modify placental angiogenesis and neovascularization.     

Human Tumor Cells Induce Angiogenesis through Positive Feedback between CD147 and Insulin-Like Growth Factor-I

Tumor angiogenesis is a complex process based upon a sequence of interactions between tumor cells and endothelial cells. Previous studies have shown that CD147 was correlated with tumor angiogenesis through increasing tumor cell secretion of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). In this study, we made a three-dimensional (3D) tumor angiogenesis model using a co-culture system of human hepatocellular carcinoma cells SMMC-7721 and humanumbilical vein endothelial cells (HUVECs) in vitro. We found that CD147-expressing cancer cells could promote HUVECs to form net-like structures resembling the neo-vasculature, whereas the ability of proliferation, migration and tube formation of HUVECs was significantly decreased in tumor conditioned medium (TCM) of SMMC-7721 cells transfected with specific CD147-siRNA. Furthermore, by assaying the change of pro-angiogenic factors in TCM, we found that the inhibition of CD147 expression led to significant decrease of VEGF and insulin-like growth factor-I (IGF-I) secretion. Interestingly, we also found that IGF-I up-regulated the expression of CD147 in both tumor cells and HUVECs. These findings suggest that there is a positive feedback between CD147 and IGF-I at the tumor-endothelial interface and CD147 initiates the formation of an angiogenesis niche.     

Sphingosine 1-phosphate induces angiogenesis: its angiogenic action and signaling mechanism in human umbilical vein endothelial cells.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite abundantly stored in platelets and released upon platelet activation. Recently, S1P has been postulated for its potential roles in angiogenesis. In this study, we provided several lines of evidence showing that S1P has angiogenic activity. In vitro, S1P stimulated DNA synthesis and chemotactic motility of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner, reaching a near maximum at 1 microM. S1P also significantly induced tube formation of HUVECs on Matrigel. Matrigel plug assay in mice revealed that S1P promotes angiogenesis in vivo. In addition, exposure of HUVECs to S1P led to rapid activation of extracellular signal-regulated kinases (ERKs) and p38 mitogen-activated protein kinase (p38 MAPK) in a pertussis toxin (PTX)-sensitive manner. Notably, HUVEC migration and tube formation in response to S1P were completely blocked by pretreatment with PTX. Further, the MEK inhibitor U0126 markedly inhibited S1P-induced tube formation but S1P-induced migration was not affected by inhibition of ERK and p38 MAPK. Taken together, these results indicate that S1P induces angiogenesis predominantly via G(i) protein-coupled receptors in endothelial cells and suggest that S1P may act as an important modulator of platelet-induced angiogenesis.     

Critical role of c-Jun N-terminal kinase in regulating bFGF-induced angiogenesis in vitro

Angiogenesis, the process of new blood vessels formation, is a critical step for wound healing, tumour growth and metastasis, diabetic retinopathy, psoriasis, etc. The present study was designed to investigate whether c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is critical for regulating basic fibroblastic growth factor (bFGF)-induced angiogenesis in human umbilical vein endothelial cells (HUVECs). Our results showed that bFGF-induced HUVECs proliferation, migration and tube formation with a concentration-dependent manner. Further results showed that bFGF induced the phosphorylation of JNK/SAPK at 15 min. Both JNK/SAPK inhibitor SP600125 and JNK/SAPK peptide inhibitor 420116 could inhibit bFGF-induced HUVECs proliferation, migration and tube formation, so did JNK/SAPK-specific siRNA. Moreover, when HUVECs were stimulated with bFGF, upstream signals of JNK/SAPK, SEK1/MKK4 and MKK7 were both activated at 2 min. In summary, our results indicate that JNK/SAPK signal pathway plays an important role in regulating bFGF-mediated angiogenesis in HUVECs, which may therefore be a new therapeutic approach for the treatment of angiogenesis-associated diseases.     

Liraglutide restores angiogenesis in palmitate-impaired human endothelial cells through PI3K/Akt-Foxo1-GTPCH1 pathway

Glucagon-like peptide-1 (GLP-1) and its analogues have a beneficial role in cardiovascular system. Here, we aimed to investigate whether liraglutide, a GLP-1 analogue, modulated angiogenesis impaired by palmitic acid (PA) in cultured human umbilical vein endothelial cells (HUVECs). Cells were incubated with liraglutide (3–100 nmol/L) in the presence of PA (0.5 mmol/L), and endothelial tube formation was observed and quantified. The protein levels of signaling molecules were analyzed and the specific inhibitors were used to identify the signaling pathways through which liraglutide affected angiogenesis. Results showed that liraglutide ameliorated endothelial tube formation impaired by PA in HUVECs in a dose-dependent manner. Meanwhile, liraglutide increased the phosphorylation of Akt and forkhead box O1 (Foxo1), and upregulated the levels of guanosine 5′-triphosphate cyclohydrolase 1 (GTPCH1) and endothelial nitric oxide synthase (eNOS) in PA-impaired HUVECs. Notably, addition of the PI3K inhibitor LY294002, Foxo1 nuclear export inhibitor trifluoperazine dihydrochloride (TFP), GTPCH1 inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP) or NOS inhibitor N-nitro-l-arginine-methyl ester (L-NAME) eliminated the angiogenic effect of liraglutide. Moreover, either LY294002 or TFP abolished the liraglutide-induced upregulation of GTPCH1 and eNOS protein levels. In conclusion, liraglutide restores angiogenesis in PA-impaired HUVECs. The effect is mediated via upregulation of GTPCH1 and eNOS levels in a PI3K/Akt-Foxo1-dependent mechanism.     

3-O-Acetyloleanolic acid exhibits anti-angiogenic effects and induces apoptosis in human umbilical vein endothelial cells

3-O-Acetyloleanolic acid, a pentacyclic triterpenoid isolated from cowpea seeds, inhibited proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. HUVECs. The induced apoptosis was characterized by detection of cell surface annexin V and sub-G1 populations. The number of cells immunostained with annexin V-fluorescein isothiocyanate increased after treatment with 3-O-acetyloleanolic acid. The sub-G1 cell populations were also increased in treated HUVECs. 3-O-Acetyloleanolic acid induced activation of caspase 3, a critical mediator of apoptosis signaling. It also significantly inhibited angiogenesis in an in vivo Matrigel plug assay. 3-O-Acetyloleanolic acid thus exhibits anti-angiogenic effects and induces apoptosis in HUVECs and the results suggest that it has a potential use for suppression of the tumor growth stimulated by angiogenesis.     

Overexpression of hepatitis B x-interacting protein in HepG2 cells enhances tumor-induced angiogenesis

Hepatocellular carcinoma (HCC) is a common malignancy and a leading cause of cancer death worldwide. Hepatitis B x-interacting protein (HBXIP), a cofactor of survivin, was originally identified by binding with the C-terminus of the HBx and negatively regulated the activity of HBx. In this study, the effect of HBXIP on the hepatoma cells-induced angiogenesis was investigated. Proliferation and migration of human umbilical vein endothelial cells (HUVECs) were detected by MTT and transwell assay, respectively. Tube formation and chick chorioallantoic membrane model were used to observe the angiogenesis. Vascular endothelial growth factor activity was assayed using ELISA kits. Western blotting was performed to examine the protein expression. Our results indicated that overexpression of HBXIP increased HepG2 cell-induced endothelial cells migration, proliferation, and angiogenesis, which may be related to increasing phosphorylation of endothelial NO synthase in HUVECs. These results suggest that HBXIP may play an important role in tumorigenesis by enhancing angiogenesis in HCC.     

c-Jun N-Terminal Kinase Mediated VEGFR2 Sustained Phosphorylation is Critical for VEGFA-Induced Angiogenesis In Vitro and In Vivo

c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is involved in the regulation of various cellular functions including cell cycle, proliferation, apoptosis. However, whether JNK/SAPK directly regulates the angiogenesis of human umbilical vein endothelial cells (HUVECs) induced by vascular endothelial growth factor A (VEGFA) has not yet been fully elucidated. Our present study firstly demonstrated VEGFA-induced angiogenic responses including the increase of cell viability, migration, and tube formation with a concentration-dependent manner in HUVECs. Further results showed that VEGFA induced the activation of JNK/SAPK, p38 kinase and extracellular signal-regulated kinases 1 and 2 (ERK1/2), while JNK/SAPK inhibitor SP600125 and specific siRNA both blocked all those angiogenic effects induced by VEGFA. Furthermore, VEGFA induced the phosphorylation of ASK1, SEK1/MKK4, MKK7, and c-Jun, which are upstream or downstream signals of JNK/SAPK. In addition, in vivo matrigel plug assay further showed that SP600125 inhibited VEGFA-induced angiogenesis. Further results showed that SP600125 and JNK/SAPK siRNA decreased VEGFA-induced VEGFR2 (Flk-1/KDR) sustained phosphorylation in HUVECs. Taken together, all these results demonstrate that JNK/SAPK regulates VEGFA-induced VEGFR2 sustained phosphorylation, which plays important roles in VEGFA-induced angiogenesis in HUVECs.     

Endothelial LGALS9 splice variant expression in endothelial cell biology and angiogenesis

Galectins are carbohydrate binding proteins with versatile functions in tumor progression. Galectin-9, encoded by LGALS9, has been associated with metastasis and immunosuppression. We previously reported on regulation of LGALS9 expression during endothelial cell activation. Here, we show increased galectin-9 protein levels in the endothelium of different tumors, including carcinomas of the lung, liver, breast and kidney. Endothelial cells were found to express five LGALS9 splice variants, two of which have not been reported before. Splicing was found to be confined to exons 5, 6 and 10. Transfection of human microvascular endothelial cells (HMEC) with galectin-9∆5, a specific LGALS9 splice variant, induced a small but significant increase of proliferation, while migration was not affected by any LGALS9 splice variant. Application of recombinant galectin-9∆5 protein dose-dependently reduced proliferation and migration of HMEC as well as human umbilical vein endothelial cells in vitro. Enhanced sprouting and migration of human umbilical vein endothelial cell (HUVEC) towards a galectin-9∆5 gradient were observed. Interestingly, galectin-9∆5 was found to induce a small inhibitory effect on angiogenesis in vivo. Collectively, these data show that endothelial cells regulate the expression and splicing of LGALS9 during angiogenesis. The function of the dominant splice variant, i.e. galectin-9∆5, in endothelial cell biology depends on the concentration and environmental context in which it is presented to the cells.     

A fusion protein composed of receptor binding domain of vascular endothelial growth factor-A and constant region fragment of antibody: angiogenesis antagonistic activity

Vascular endothelial growth factor (VEGF) promotes the growth of solid tumor mainly via VEGF receptor-1 and receptor-2, which are expressed preferentially in proliferating endothelial cells. Therefore, a strategy for simultaneous blockage of both VEGF receptors may have a useful therapeutic effect in tumor growth. In this study, we utilized a fusion protein which is composed of receptor binding domain of VEGF-A (RBDV) and the constant region fragment (Fc) of a human immunoglobulin G1 (IgG1), to interfere with the growth of human umbilical vein endothelial cells (HUVECs) via VEGF receptors. The results showed that RBDV-IgG1 Fc was able to bind with both VEGF receptor-1 and receptor-2. In addition, RBDV-IgG1 Fc could decrease VEGF-induced proliferation and tube formation among HUVECs. Moreover, the cytotoxic test showed RBDV-IgG1 Fc could also enhance the cytotoxic activity of human natural killing cells. The data are suggesting that the fusion protein, RBDV-IgG1 Fc, may have potential as an angiogenesis antagonist for future tumor therapy.     

Surface Phosphatidylserine Is Responsible for the Internalization on Microvesicles Derived from Hypoxia-Induced Human Bone Marrow Mesenchymal Stem Cells into Human Endothelial Cells

Background

Previous data have proven that microvesicles derived from hypoxia-induced mesenchymal stem cells (MSC-MVs) can be internalized into endothelial cells, enhancing their proliferation and vessel structure formation and promoting in vivo angiogenesis. However, there is a paucity of information about how the MSC-MVs are up-taken by endothelial cells.

Methods

MVs were prepared from the supernatants of human bone marrow MSCs that had been exposed to a hypoxic and/or serum-deprivation condition. The incorporation of hypoxia-induced MSC-MVs into human umbilical cord endothelial cells (HUVECs) was observed by flow cytometry and confocal microscopy in the presence or absence of recombinant human Annexin-V (Anx-V) and antibodies against human CD29 and CD44. Further, small interfering RNA (siRNA) targeted at Anx-V and PSR was delivered into HUVECs, or HUVECs were treated with a monoclonal antibody against phosphatidylserine receptor (PSR) and the cellular internalization of MVs was re-assessed.

Results

The addition of exogenous Anx-V could inhibit the uptake of MVs isolated from hypoxia-induced stem cells by HUVECs in a dose- and time-dependent manner, while the anti-CD29 and CD44 antibodies had no effect on the internalization process. The suppression was neither observed in Anx-V siRNA-transfected HUVECs, however, addition of anti-PSR antibody and PSR siRNA-transfected HUVECs greatly blocked the incorporation of MVs isolated from hypoxia-induced stem cells into HUVECs.

Conclusion

PS on the MVs isolated from hypoxia-induced stem cells is the critical molecule in the uptake by HUVECs.     

Galectin-3 protein modulates cell surface expression and activation of vascular endothelial growth factor receptor 2 in human endothelial cells

Angiogenesis is heavily influenced by VEGF-A and its family of receptors, particularly VEGF receptor 2 (VEGF-R2). Like most cell surface proteins, VEGF-R2 is glycosylated, although the function of VEGF-R2 with respect to its glycosylation pattern is poorly characterized. Galectin-3, a glycan binding protein, interacts with the EGF and TGFβ receptors, retaining them on the plasma membrane and altering their signal transduction. Because VEGF-R2 is glycosylated and both galectin-3 and VEGF-R2 are involved with angiogenesis, we hypothesized that galectin-3 binds VEGF-R2 and modulates its signal transduction as well. Employing a Western blot analysis approach, we found that galectin-3 induces phosphorylation of VEGF-R2 in endothelial cells. Knockdown of galectin-3 and Mgat5, an enzyme that synthesizes high-affinity glycan ligands of galectin-3, reduced VEGF-A mediated angiogenesis in vitro. A direct interaction on the plasma membrane was detected between galectin-3 and VEGF-R2, and this interaction was dependent on the expression of Mgat5. Using immunofluorescence and cell surface labeling, we found an increase in the level of internalized VEGF-R2 in both Mgat5 and galectin-3 knockdown cells, suggesting that galectin-3 retains the receptor on the plasma membrane. Finally, we observed reduced suture-induced neovascularization in the corneas of Gal3(-/-) and Mgat5(-/-) mice. These findings are consistent with the hypothesis that, like its role with the EGF and TGFβ receptors, galectin-3 contributes to the plasma membrane retention and proangiogenic function of VEGF-R2.     

Piperine,a dietary phytochemical,inhibits angiogenesis

Angiogenesis plays an important role in tumor progression. Piperine, a major alkaloid constituent of black pepper, has diverse physiological actions including killing of cancer cells; however, the effect of piperine on angiogenesis is not known. Here we show that piperine inhibited the proliferation and G₁/S transition of human umbilical vein endothelial cells (HUVECs) without causing cell death. Piperine also inhibited HUVEC migration and tubule formation in vitro, as well as collagen-induced angiogenic activity by rat aorta explants and breast cancer cell-induced angiogenesis in chick embryos. Although piperine binds to and activates the cation channel transient receptor potential vanilloid 1 (TRPV1), its effects on endothelial cells did not involve TRPV1 since the antiproliferative effect of piperine was not affected by TRPV1-selective antagonists, nor did HUVECs express detectable TRPV1 mRNA. Importantly, piperine inhibited phosphorylation of Ser 473 and Thr 308 residues of Akt (protein kinase B), which is a key regulator of endothelial cell function and angiogenesis. Consistent with Akt inhibition as the basis of piperine's action on HUVECs, inhibition of the phosphoinositide-3 kinase/Akt signaling pathway with LY-294002 also inhibited HUVEC proliferation and collagen-induced angiogenesis. Taken together, these data support the further investigation of piperine as an angiogenesis inhibitor for use in cancer treatment.