Netrin-4 promotes mural cell adhesion and recruitment to endothelial cells.

Netrins are secreted molecules involved in axon guidance and angiogenesis. We previously showed that Netrin-4 acts as an anti-angiogenic factor by inhibiting endothelial cell (EC) functions. In this study, we investigated the effects of Netrin-4 on vascular smooth muscle cell (VSMC) activity in vitro and in vivo. We show that exogenous Netrin-4 stimulated VSMC adhesion and migration, and increased their coverage on EC tubes (grown on a Matrigel substrate). siRNA knock-down of endogenous Netrin-4 expression in VSMC decreased their recruitment to EC tubes. VSMC expressed Netrin-4 and three of the six Netrin-1 cognate receptors: DCC, Neogenin, and Unc5B. Silencing of these receptors reduced Netrin-4 adhesion to VSMC, strongly suggesting that these receptors were involved in the recruitment process. We previously showed that Netrin-4 overexpression in PC3 cancer cells delayed tumor growth in a model of subcutaneous xenograft by reducing tumor vessel density. Here, we show that Netrin-4 overexpression improved tumor blood vessel structure and increased VSMC coverage. Thus, Netrin-4 induced mural cell recruitment may play a role in the inhibition of tumor growth. Our data suggest that Netrin-4 is important for blood vessel normalization through the regulation of both endothelial and perivascular cells.

Brain pericytes from stress-susceptible pigs increase blood-brain barrier permeability in vitro.

BACKGROUND: The function of pericytes remains questionable but with improved cultured technique and the use of genetically modified animals, it has become increasingly clear that pericytes are an integral part of blood-brain barrier (BBB) function, and the involvement of pericyte dysfunction in certain cerebrovascular diseases is now emerging. The porcine stress syndrome (PSS) is the only confirmed, homologous model of malignant hyperthermia (MH) in veterinary medicine. Affected animals can experience upon slaughter a range of symptoms, including skeletal muscle rigidity, metabolic acidosis, tachycardia and fever, similar to the human syndrome. Symptoms are due to an enhanced calcium release from intracellular stores. These conditions are associated with a point mutation in ryr1/hal gene, encoding the ryanodine receptor, a calcium channel. Important blood vessel wall muscle modifications have been described in PSS, but potential brain vessel changes have never been documented in this syndrome. METHODS: In the present work, histological and ultrastructural analyses of brain capillaries from wild type and ryr1 mutated pigs were conducted to investigate the potential impairment of pericytes, in this pathology. In addition, brain pericytes were isolated from the three porcine genotypes (wild-type NN pigs; Nn and nn pigs, bearing one or two (n) mutant ryr1/hal alleles, respectively), and tested in vitro for their influence on the permeability of BBB endothelial monolayers. RESULTS: Enlarged perivascular spaces were observed in ryr1-mutant samples, corresponding to a partial or total detachment of the astrocytic endfeet. These spaces were electron lucent and sometimes filled with lipid deposits and swollen astrocytic feet. At the ultrastructural level, brain pericytes did not seem to be affected because they showed regular morphology and characteristics, so we aimed to check their ability to maintain BBB properties in vitro. Our results indicated that pericytes from the three genotypes of pigs had differing influences on the BBB. Unlike pericytes from NN pigs, pericytes from Nn and nn pigs were not able to maintain low BBB permeability. CONCLUSIONS: Electron microscopy observations demonstrated brain capillary modifications in PSS condition, but no change in pericyte morphology. Results from in vitro experiments suggest that brain pericytes from ryr1 mutated pigs, even if they are not affected by this condition at the ultrastructural level, are not able to maintain BBB integrity in comparison with pericytes from wild-type animals.

Cross-talk between the VEGF-A and HGF signalling pathways in endothelial cells.

BACKGROUND INFORMATION: Endothelial cells play a major role in angiogenesis, the process by which new blood vessels arise from a pre-existing vascular bed. VEGF-A (vascular endothelial growth factor-A) is a key regulator of angiogenesis during both development and in adults. HGF (hepatocyte growth factor) is a pleiotropic cytokine that may promote VEGF-A-driven angiogenesis, although the signalling mechanisms underlying this co-operation are not completely understood. RESULTS: We analysed the effects of the combination of VEGF-A and HGF on the activation of VEGFR-2 (VEGF receptor-2) and c-met receptors, and on the stimulation of downstream signalling pathways in endothelial cells. We found that VEGFR-2 and c-met do not physically associate and do not transphosphorylate each other, suggesting that co-operation involves signalling events more distal from receptor activation. We demonstrate that the VEGF isoform VEGF-A(165) and HGF stimulate a similar set of MAPKs (mitogen-activated protein kinases), although the kinetics and strengths of the activation differ depending on the growth factor and pathway. An enhanced activation of the signalling was observed when endothelial cells were stimulated by the combination of VEGF-A(165) and HGF. Moreover, the combination of VEGF-A and HGF results in a statistically significant synergistic activation of ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 kinases. We demonstrated that VEGF-A(165) and HGF activate FAK (focal adhesion kinase) with different kinetics and stimulate the recruitment of phosphorylated FAK to different subsets of focal adhesions. VEGF-A(165) and HGF regulate distinct morphogenic aspects of the cytoskeletal remodelling that are associated with the preferential activation of Rho or Rac respectively, and induce structurally distinct vascular-like patterns in vitro in a Rho- or Rac-dependent manner. CONCLUSIONS: Under angiogenic conditions, combining VEGF-A with HGF can promote neovascularization by enhancing intracellular signalling and allowing more finely regulated control of the signalling molecules involved in the regulation of the cytoskeleton and cellular migration and morphogenesis.

[Effects of dihydroartiminisin on the adhesion, migration, and invasion of epithelial ovarian cancer cells].

OBJECTIVE: To investigate the effects of dihydroartiminisin (DHA) on the adhesion, migration, and invasion ovarian cancer cells. METHODS: Human ovarian cancer cells of the lines SKOV3 and OVCAR3 were cultured. Suspensions of SKOV3 and OVCAR3 cells were treated with DHA of the concentrations of 0.5, 2.5, 12.5, and 62.5 micromol/L respectively, and then inoculated on the plate coated with Matrigel. MTT method was used to -determine the adhesion rate. Transwell membrane chamber model was used to evaluate the effect of DHA on the migration and invasion of the SKOV3 and OVCAR3 cells. Western blotting and reverse transcriptase polymerase chain reaction were used to detect the effect of DHA on the phosphorylation of focal adhesion kinase (FAK) and on the effect of expression of metal matrix proteinases (MMPs) and their tissue inhibitors (TIMPs) respectively. RESULTS: (1) Compared to the cells without DHA treatment, the cell adhesion ability levels of the SKOV3 and OVCAR3 cells treated with 12.5 micromol/L DHA decreased by 76.1% and 57.9% respectively (P < 0.05), while their migration ability levels decreased by 59.3% and 69.7% respectively (P < 0.05). (2) Both SKOV3 and OVCAR3 showed weak invasion ability, and DHA only showed a slight inhibitory effect on the cell invasion of these 2 lines (both P > 0.05). (3) Compared to the cells without DHA treatment, the phosphorylation level of FAK of the SKOV3 and OVCAR3 cells treated with 12.5 micromol/L DHA decreased by 42.9% and 44.8% respectively (both P < 0.05). (4) RT-PCR showed mRNA expression of MMP2, TIMP1, and TIMP2, but not mRNA expression of MMP9 in both SKOV3 and OVCAR3 cells. The mRNA expression levels of the SKOV3 and OVCAR3 cells treated with 12.5 micromol/L DHA increased by 1.5 and 2.6 times respectively (both P < 0.05). CONCLUSION: DHA has inhibitory effects on the adhesion and migration of epithelial ovarian cancer cells, which may be related to its down-regulation of the phosphorylation of FAK in these cells.

[Effects of dihydroartiminisin on proliferation and phosphorylation of mitogen-activated protein kinase in epithelial ovarian cancer cell lines].

OBJECTIVE: To determine the effect of dihydroartiminisin on the proliferation and phosphorylation of mitogen-activated protein kinase (MAPK) in SKOV3 and OVCAR3 ovarian cancer cell lines. METHODS: Methyl thiazolyl tetrazolium assay was performed to evaluate the anti-proliferative effect of dihydroartiminisin in SKOV3 and OVCAR3 cells, and Western blot was used to determine its effect on phosphorylation level of MAPK, including extra-cell regulated kinase (ERK) 1/2 and p38 protein kinase, in the two cell lines. RESULTS: Dihydroartiminisin inhibited the proliferation of ovarian cancer cells in vitro, with a mean of 50% inhibition concentration (IC(50)) at 72 h of (9.0 +/- 1.4) micromol/L for SKOV3 and (5.5 +/- 1.2) micromol/L for OVCAR3 respectively. Compared to cells without dihydroartiminisin treatment, phosphorylation level of ERK 1/2 in SKOV3 and OVCAR3 cells treated with dihydroartiminisin decreased by 64.2% and 75.3% respectively (P < 0.05), while phosphorylation of p38 protein kinase in SKOV3 and OVCAR3 only decreased by 8.5%and 6.4%respectively (P > 0.05). CONCLUSION: Dihydroartiminisin can inhibit the proliferation of ovarian cancer cell in vitro, probably through down-regulation of the phosphorylation of ERK 1/2 in ovarian cancer cells.

Coadministration of endothelial and smooth muscle progenitor cells enhances the efficiency of proangiogenic cell-based therapy.

Cell-based therapy is a promising approach designed to enhance neovascularization and function of ischemic tissues. Interaction between endothelial and smooth muscle cells regulates vessels development and remodeling and is required for the formation of a mature and functional vascular network. Therefore, we assessed whether coadministration of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs) can increase the efficiency of cell therapy. Unilateral hindlimb ischemia was surgically induced in athymic nude mice treated with or without intravenous injection of EPCs (0.5 x 10(6)), SMPCs (0.5 x 10(6)) and EPCs+SMPCs (0.25 x 10(6)+0.25 x 10(6)). Vessel density and foot perfusion were increased in mice treated with EPCs+SMPCs compared to animals receiving EPCs alone or SMPCs alone (P<0.001). In addition, capillary and arteriolar densities were enhanced in EPC+SMPC-treated mice compared to SMPC and EPC groups (P<0.01). We next examined the role of Ang-1/Tie2 signaling in the beneficial effect of EPC and SMPC coadministration. Small interfering RNA directed against Ang-1-producing SMPCs or Tie2-expressing EPCs blocked vascular network formation in Matrigel coculture assays, reduced the rate of incorporated EPCs within vascular structure, and abrogated the efficiency of cell therapy. Production of Ang-1 by SMPCs activates Tie2-expressing EPCs, resulting in increase of EPC survival and formation of a stable vascular network. Subsequently, the efficiency of EPC- and SMPC-based cotherapy is markedly increased. Therefore, coadministration of different types of vascular progenitor cells may constitute a novel therapeutic strategy for improving the treatment of ischemic diseases.

Netrin-4 inhibits angiogenesis via binding to neogenin and recruitment of Unc5B.

Netrins are secreted molecules with roles in axon guidance and angiogenesis. We identified Netrin-4 as a gene specifically overexpressed in VEGF-stimulated endothelial cells (EC) in vitro as well as in vivo. Knockdown of Netrin-4 expression in EC increased their ability to form tubular structures on Matrigel. To identify which receptor is involved, we showed by quantitative RT-PCR that EC express three of the six Netrin-1 cognate receptors: neogenin, Unc5B, and Unc5C. In contrast to Netrin-1, Netrin-4 bound only to neogenin but not to Unc5B or Unc5C receptors. Neutralization of Netrin-4 binding to neogenin by blocking antibodies abolished the chemotactic effect of Netrin-4. Furthermore, the silencing of either neogenin or Unc5B abolished Netrin-4 inhibitory effect on EC migration, suggesting that both receptors are essential for its function in vitro. Coimmunoprecipitation experiments demonstrated that Netrin-4 increased the association between Unc5B and neogenin on VEGF- or FGF-2-stimulated EC. Finally, we showed that Netrin-4 significantly reduced pathological angiogenesis in Matrigel and laser-induced choroidal neovascularization models. Interestingly, Netrin-4, neogenin, and Unc5B receptor expression was up-regulated in choroidal neovessel EC after laser injury. Moreover, Netrin-4 overexpression delayed tumor angiogenesis in a model of s.c. xenograft. We propose that Netrin-4 acts as an antiangiogenic factor through binding to neogenin and recruitment of Unc5B.

Neuropilin-1 and neuropilin-2 act as coreceptors, potentiating proangiogenic activity.

Neuropilin-1 and -2 (NRP1 and NRP2) are the transmembrane glycoproteins interacting with 2 types of ligands: class III semaphorins and several members of the VEGF family, the main regulators of blood and lymphatic vessel growth. We show here that both NRP1 and NRP2 can also bind hepatocyte growth factor (HGF). HGF is a pleiotropic cytokine and potent proangiogenic molecule that acts on its target cells by binding to the c-met receptor. We found that the N-terminal domain of HGF is involved in the interaction with neuropilins. We demonstrated that invalidation of NRP1 or NRP2 by RNA interference in human umbilical vein endothelial cells (HUVECs) decreased HGF-induced c-met phosphorylation and VEGF-A(165)- and HGF-mediated intracellular signaling. Accordingly, the disruption of NRP1 or NRP2 binding to VEGF-A(165) or HGF with a blocking antibody, decreased the proliferation and migration of endothelial cells. This effect may be further enhanced if VEGF-A(165) or HGF binding to both NRP1 and NRP2 was disrupted. Using a mouse Matrigel model, we demonstrated that NRP1 is essential for HGF-mediated angiogenesis in vivo. Our results suggest that, in endothelial cells, both NRP1 and NRP2 function as proangiogenic coreceptors, potentiating the activity of at least 2 major proangiogenic cytokines, VEGF-A(165) and HGF.

Effects of dihydroartiminisin on proliferation and phosphorylation of mitogen-activated protein kinase in epithelial ovarian cancer cell lines / 中华妇产科杂志

Objective To determine the effect of dihydroartiminisin on the proliferation and phosphorylation of mitogen-activated protein kinase (MAPK) in SKOV3 and OVCAR3 ovarian cancer cell lines.Methods Methyl thiazolyl tetrazolium assay was performed to evaluate the anti-proliferative effect of dihydroartiminisin in SKOV3 and OVCAR3 cells,and Western blot was used to determine its effect on phosphorylation level of MAPK,including extra-cell regulated kinase (ERK)1/2 and p38 protein kinase,in the two cell lines.Results Dihydroartiminisin inhibited the proliferation of ovarian cancer cells in vitro,with a mean of 50% inhibition concentration (IC50) at 72 h of (9.0 ±1.4) μmol/L for SKOV3 and (5.5 ±1.2)μmol/L for OVCAR3 respectively. Compared to cells without dihydroartiminisin treatment,phosphorylation level of ERK 1/2 in SKOV3 and OVCAR3 cells treated with dihydroartiminisin decreased by 64.2% and 75.3% respectively (P＜0.05),while phosphorylation of p38 protein kinase in SKOV3 and OVCAR3 only decreased by 8.5% and 6.4% respectively (P ＞0.05).Conclusion Dihydroartiminisin can inhibit the proliferation of ovarian cancer cell in vitro, probably through down-regulation of the phosphorylation of ERK 1/2 in ovarian cancer cells.

PSGL-1-mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells.

Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2-Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2-Fc-stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy.