Inhibition of miR-21-5p suppresses high glucose-induced proliferation and angiogenesis of human retinal microvascular endothelial cells by the regulation of AKT and ERK pathways via maspin

The aim of the present study is to investigate the role of miR-21-5p in angiogenesis of human retinal microvascular endothelial cells (HRMECs). HRMECs were incubated with 5 mM glucose, 30 mM glucose or 30 mM mannitol for 24 h, 48 h or 72 h. Then, HRMECs exposed to 30 mM glucose were transfected with miR-21-5p inhibitor. We found that high glucose increased the expression of miR-21-5p, VEGF, VEGFR2 and cell proliferation activity. Inhibition of miR-21-5p reduced high glucose-induced proliferation, migration, tube formation of HRMECs, and reversed the decreased expression of maspin as well as the abnormal activation of PI3K/AKT and ERK pathways. Down-regulation of maspin by siRNA significantly increased the activities of PI3K/AKT and ERK pathways. In conclusion, inhibition of miR-21-5p could suppress high glucose-induced proliferation and angiogenesis of HRMECs, and these effects may partly dependent on the regulation of PI3K/AKT and ERK pathways via its target protein maspin.     

Advanced glycation alters expression of the 67kDa laminin receptor in retinal microvascular endothelial cells

The 67kDa laminin receptor (67LR) plays an important role in vascular cell function and dysfunction. The present study has examined 67LR expression in retinal microvascular endothelial cells after exposure to AGEs. Retinal microvascular endothelial cells were exposed to either AGE-BSA, or were grown on methylglyoxal-modified laminin or Matrigel and expression of 67LR analysed by Western Blotting and RT-PCR/Southern blotting. Western blotting of plasma membrane and RT-PCR/Southern blotting revealed a significant upregulation of 67LR protein/mRNA expression after exposure to AGEs (p<0.05-0.01). The results show that 67LR is upregulated in cells exposed to AGEs and suggests a previously unrecognised role for this receptor in retinal microvascular endothelial cell interaction with glycated substrates.     

Hypoxia enhances CXCR4 expression in human microvascular endothelial cells and human melanoma cells

The influence of environmental factors (cytokines, matrix components, serum factors and O(2) level) on expression of receptors for angiogenic versus angiostatic CXC chemokines in human microvascular endothelial cells has not been extensively investigated. Our semi-quantitative RT-PCR analysis demonstrated that TNF-alpha and IFN-gamma repressed CXCR4 mRNA levels in immortalized human microvascular endothelial HMEC-1 cells after 4 h, whereas only TNF-alpha displayed inhibitory activity in primary human microvascular endothelial cells (HMVEC). CXCR4 mRNA expression was not affected by VEGF, GM-CSF, IL-1beta or various basal membrane matrix components, but was significantly up-regulated after serum starvation and/or hypoxic treatment of the microvascular endothelial cells. The alternative CXCL12 receptor, CXCR7/RDC1, was also up-regulated by hypoxia in HMEC-1 cells, although less consistently than CXCR4. Furthermore, hypoxia and serum starvation were required for cell surface display of CXCR4 and CXCL12 induction of ERK activation in HMEC-1 cells. In contrast, CXCR2 and CXCR3 mRNA levels remained, respectively, low and undetectable under all the conditions tested, and surface expression of CXCR2, CXCR3 and CXCR7 on the HMEC- 1 cells could not be demonstrated by FACS. In the human SK-MEL-5 melanoma cell line, CXCR4 mRNA expression was also increased under hypoxic conditions, whereas CXCR2 mRNA levels remained low and levels of CXCR3 and CXCR7 were undetectable. However, immunohistochemical staining of human metastatic melanoma sections demonstrated that CXCR2, CXCR3, CXCR4 and CXCR7 are expressed on tumor cells and, to a lesser extent, on endothelial cells. These results demonstrate that the tumor microenvironment regulates chemokine receptor expression through both cytokine and oxygen levels.     

Suppression of protein kinase C-ζ attenuates vascular leakage via prevention of tight junction protein decrease in diabetic retinopathy

To investigate the effect of protein kinase C (PKC)-ζ inhibition on vascular leakage in diabetic retinopathy, streptozotocin-induced diabetic mice were intravitreously injected with siPKC-ζ. According to the fluorescein angiography of the retinal vessels, suppression of PKC-ζ effectively attenuated vascular leakage in diabetic retina. Further evaluation on the retina with western blot analysis and immunohistochemistry revealed accompanying restoration of tight junction proteins on retinal vessels. As two major contributors to vascular leakage in diabetic retinopathy, vascular endothelial growth factor (VEGF) and advanced glycation end products (AGEs) were investigated on the tight junction protein expression in endothelial cells. Inhibition of PKC-ζ attenuated VEGF-induced decrease of tight junction proteins and accompanying hyperpermeability in human retinal microvascular endothelial cells (HRMECs). PKC-ζ inhibition also attenuated AGE-induced decrease of tight junction proteins in HRMECs. Our findings suggest that inhibition of PKC-ζ could be an alternative treatment option for compromised blood-retinal barrier in diabetic retinopathy.     

Inhibitory role of miR-203 in the angiogenesis of mice with pathological retinal neovascularization disease through downregulation of SNAI2

BackgroundPathological retinal neovascularization is a disease characterized by abnormal angiogenesis in retina that is a major cause of blindness in humans. Previous reports have highlighted the involvement of microRNAs (miRNAs) in retinal angiogenesis. Therefore, we aimed at exploring the mechanism underlying miR-203 regulating the progression of pathological retinal neovascularization.MethodsInitially, the mouse model of pathological retinal neovascularization disease was established and the hypoxia-induced human retinal microvascular endothelial cells (HRMECs) were generated. Then, miR-203 and SNAI2 expression in HRMECs and retinal tissues was examined. Subsequently, the effects of miR-203 and SNAI2 on viability, migration, apoptosis and angiogenesis of HRMECs were investigated, with the expression of Bax, Ki-67, MMP-2, MMP-9, VEGF and CD34 measured. Finally, the regulation of miR-203 or SNAI2 on GSK-3β/β-catenin pathway was determined through examining the levels of phosphorylated p-GSK-3β and β-catenin.ResultsPoorly expressed miR-203 and highly expressed SNAI2 were found in HRMECs and retinal tissues of pathological retinal neovascularization. Importantly, overexpressed miR-203 or silencing SNAI2 inhibited viability, migration and angiogenesis but promoted apoptosis of HRMECs, evidenced by elevated Bax expression but reduced expression of Ki-67, MMP-2, MMP-9, VEGF and CD34. Moreover, overexpression of miR-203 was found to repress the GSK-3β/β-catenin pathway by downregulating SNAI2.ConclusionCollectively, this study demonstrated that overexpression of miR-203 suppressed the angiogenesis in mice with pathological retinal neovascularization disease via the inactivation of GSK-3β/β-catenin pathway by inhibiting SNAI2, which provided a novel therapeutic insight for pathological retinal neovascularization disease.     

Oxidative stress upregulates Wnt signaling in human retinal microvascular endothelial cells through activation of disheveled

Abnormal retinal neovascularization associated with various retinopathies can result in irreversible vision loss. Although the mechanisms involved in this occurrence is unclear, increasing evidence suggests that aberrant Wnt signaling participates in the pathogenesis of abnormal neovascularization. Because Wnt signaling upregulation can be induced by oxidative stress through the activation of disheveled (DVL), a key molecule in the Wnt signaling pathway, we investigated whether oxidative stress can activate Wnt signaling and induce angiogenic phenotypes in human retinal microvascular endothelial cells (HRMECs). We found that increased Wnt signaling activity, as well as enhanced angiogenic phenotypes, such as tube formation and cell migration, were detected in the hydrogen peroxide-treated HRMECs. Moreover, these effects were effectively suppressed by a small-molecule Wnt inhibitor targeting the PDZ domain of DVL. Therefore, we propose that targeting abnormal Wnt signaling at the DVL level with a small-molecule inhibitor may represent a novel approach in retinal neovascularization treatment and prevention.     

Steroid Inhibition of Neural Micro vessel Morphogenesis In Vitro: Receptor Mediation and Astroglial Dependence

Steroid hormones alter several aspects of microvascular function within the CNS. Both microvessel formation and blood-brain barrier expression appear to be influenced by interactions between astrocytes and endothelial cells. To determine if steroids alter astrocyte-endothelial interactions, we studied their effects on astroglial-induced microvessel morphogenesis in vitro. C6 astroglial cells induce bovine retinal microvascular endothelial cells to differentiate into capillary-like structures. Dexamethasone, hydrocortisone, and progesterone at 10 nM inhibited C6-induced microvessel morphogenesis by 75, 35, and 30%, respectively. Inhibition by dexamethasone was both time and concentration dependent, reaching 80-100% at 1 microM. Tetrahydrocortisone and 17 alpha-hydroxyprogesterone had only marginal inhibitory effects. Cortexolone, a glucocorticoid receptor antagonist, blocked inhibition by dexamethasone. Progesterone receptors were expressed in C6 but not bovine retinal microvascular endothelial cells, identifying the astroglial cell as the likely effector of progesterone-mediated inhibition. Astroglial cells were further implicated as the effectors of steroid-mediated inhibition because none of the steroids inhibited astroglial-independent capillary-like structure formation in response to a reconstituted extracellular matrix, Matrigel. These findings are evidence that steroids modulate neural microvascular endothelial cell functions indirectly through perivascular astrocytes via a receptor-mediated mechanism.     

HIV-1 Tat-Mediated Apoptosis in Human Blood-Retinal Barrier-Associated Cells

HIV-1-associated ocular complications, such as microvasculopathies, can lead to the loss of vision in HIV-1-infected patients. Even in patients under highly active antiretroviral therapy, ocular lesions are unavoidable. Ocular complications have been demonstrated to be closely related to the breakdown of the blood-retinal-barrier (BRB); however, the underlying mechanism is not clear. The data from this study indicated that the HIV-1 Tat protein induced the apoptosis of human retinal microvascular endothelial cells (HRMECs) and retinal pigmen epithelium (RPE) cells, which compose the inner BRB and the outer BRB, respectively. In addition, this study found that the activation of N-methyl-D-aspartate receptors (NMDARs) was involved in the apoptosis of RPE cells, but it caused no changes in HRMECs. Furthermore, both cell types exhibited enhanced expression of Bak, Bax and Cytochrome c. The inhibition of Tat activity protected against the apoptosis induced by NMDAR activation and prevented the dysregulation of Bak, Bax and Cytochrome c, revealing an important role for the mitochondrial pathway in HIV-1 Tat-induced apoptosis. Together, these findings suggest a possible mechanism and may identify a potential therapeutic strategy for HIV-1-associated ocular complications.     

m6A transferase METTL3 regulates endothelial-mesenchymal transition in diabetic retinopathy via lncRNA SNHG7/KHSRP/MKL1 axis

Diabetic retinopathy is one of the microvascular complications in diabetic patients and the leading cause of blindness worldwide. The levels of METTL3, lncRNA SNHG7, KHSRP, MKL1, endothelial and mesenchymal markers were determined by RT-qPCR or western blot assays in vitro and in vivo. H&E staining was used to observe the retinal structure in a mouse model of DR. The expression levels of METTL3 and SNHG7 were significantly downregulated in DR patients, DR mice and high glucose-induced HRMECs cells. Notably, METTL3 installed the m6A modification and enhanced the stability of SNHG7. Besides, METTL3 inhibited HRMECs EndoMT by promoting the expression of SNHG7. Additionally, SNHG7 was found to weaken MKL1 mRNA stability by binding to the RNA-binding protein KHSRP. Furthermore, we verified that METTL3 regulated EndoMT in DR through the SNHG7/MKL1 axis. We conclude that METTL3 regulates endothelial-mesenchymal transition in DR via the SNHG7/KHSRP/MKL1 axis, providing a new target for DR treatment.     

Calpain 1 and -2 play opposite roles in cord formation of lymphatic endothelial cells via eNOS regulation

Calpains are a family of calcium-dependent proteases. Two isoforms, calpain 1 and 2, have been implicated in angiogenesis and endothelial cell adhesion and migration. Calpains regulate the function of eNOS;however, the relation of calpains and eNOS to lymphangiogenesisis still unclear. In the present study, we evaluated the role of calpain and eNOS in the formation of cords by lymphatic endothelial cells on Matrigel. Human lymphatic microvascular dermal-derived endothelial cells were transfected with siRNA against calpain 1 or 2. Calpain 2 knockdown, but not calpain 1 knockdown, significantly reduced cord formation, adhesion, and migration on Matrigel. These decreases correlated with a reduction in eNOS, and phosphorylated eNOS and Hsp90 levels, as assayed by immunoprecipitation and western blotting. In contrast, the knockdown of calpain 1, but not calpain 2,increased cell adhesion, enhanced migration, and stabilized late-stage cord formation by increasing cord length compared to the control. These differences correlated with an increase in the level of phosphorylated eNOS. The results indicated that the functions of calpains and eNOS are important for cord formation by lymphatic endothelial cells. For the first time, we have found different functions of calpain 1 and 2. Calpain 1 is involved in the degradation of eNOS and Hsp90 and the phosphorylation of eNOS,while calpain 2 regulates eNOS phosphorylation during cord formation by lymphatic endothelial cells on Matrigel.     

Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium

Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca²⁺]_i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca²⁺-signals were completely inhibited by removal of extracellular Ca²⁺, confirming their dependence on influx of extracellular Ca²⁺. The 4-αPDD Ca²⁺-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca²⁺-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca²⁺-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months’ streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the molecular and functional expression of TRPV4 channels in retinal microvascular endothelial cells. These changes may contribute to diabetes induced endothelial dysfunction and retinopathy.     

CXCL12 mediates trophic interactions between endothelial and tumor cells in glioblastoma

Emerging evidence suggests endothelial cells (EC) play a critical role in promoting Glioblastoma multiforme (GBM) cell proliferation and resistance to therapy. The molecular basis for GBM-EC interactions is incompletely understood. We hypothesized that the chemokine CXCL12 and its receptor CXCR4 could mediate direct interactions between GBM cells and tumor-associated endothelial cells and that disruption of this interaction might be the molecular basis for the anti-tumor effects of CXCR4 antagonists. We investigated this possibility in vivo and in an in vitro co-culture model that incorporated extracellular matrix, primary human brain microvascular ECs (HBMECs) and either an established GBM cell line or primary GBM specimens. Depletion of CXCR4 in U87 GBM cells blocked their growth as intracranial xenografts indicating that tumor cell CXCR4 is required for tumor growth in vivo. In vitro, co-culture of either U87 cells or primary GBM cells with HBMECs resulted in their co-localization and enhanced GBM cell growth. Genetic manipulation of CXCL12 expression and pharmacological inhibition of its receptors CXCR4 and CXCR7 revealed that the localizing and trophic effects of endothelial cells on GBM cells were dependent upon CXCL12 and CXCR4. These findings indicate that the CXCL12/CXCR4 pathway directly mediates endothelial cell trophic function in GBMs and that inhibition of CXCL12-CXCR4 signaling may uniquely target this activity. Therapeutic disruption of endothelial cell trophic functions could complement the structural disruption of anti-angiogenic regimens and, in combination, might also improve the efficacy of radiation and chemotherapy in treating GBMs.     

The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR+ CXC chemokine-induced angiogenic activity

We have previously shown that members of the ELR(+) CXC chemokine family, including IL-8; growth-related oncogenes alpha, beta, and gamma; granulocyte chemotactic protein 2; and epithelial neutrophil-activating protein-78, can mediate angiogenesis in the absence of preceding inflammation. To date, the receptor on endothelial cells responsible for chemotaxis and neovascularization mediated by these ELR(+) CXC chemokines has not been determined. Because all ELR(+) CXC chemokines bind to CXC chemokine receptor 2 (CXCR2), we hypothesized that CXCR2 is the putative receptor for ELR(+) CXC chemokine-mediated angiogenesis. To test this postulate, we first determined whether cultured human microvascular endothelial cells expressed CXCR2. CXCR2 was detected in human microvascular endothelial cells at the protein level by both Western blot analysis and immunohistochemistry using polyclonal Abs specific for human CXCR2. To determine whether CXCR2 played a functional role in angiogenesis, we determined whether this receptor was involved in endothelial cell chemotaxis. We found that microvascular endothelial cell chemotaxis in response to ELR(+) CXC chemokines was inhibited by anti-CXCR2 Abs. In addition, endothelial cell chemotaxis in response to ELR(+) CXC chemokines was sensitive to pertussis toxin, suggesting a role for G protein-linked receptor mechanisms in this biological response. The importance of CXCR2 in mediating ELR(+) CXC chemokine-induced angiogenesis in vivo was also demonstrated by the lack of angiogenic activity induced by ELR(+) CXC chemokines in the presence of neutralizing Abs to CXCR2 in the rat corneal micropocket assay, or in the corneas of CXCR2(-/-) mice. We thus conclude that CXCR2 is the receptor responsible for ELR(+) CXC chemokine-mediated angiogenesis.     

Difference in proangiogenic potential of systemic and pulmonary endothelium: role of CXCR2

The systemic vasculature in and surrounding the lung is proangiogenic, whereas the pulmonary vasculature rarely participates in neovascularization. We studied the effects of the proangiogenic ELR+ CXC chemokine MIP-2 (macrophage inflammatory protein-2) on endothelial cell proliferation and chemotaxis. Mouse aortic, pulmonary arterial, and lung microvascular endothelial cells were isolated and subcultured. Proliferation ([3H]thymidine uptake) and migration (Transwell chemotaxis) were evaluated in each cell type at baseline and upon exposure to MIP-2 (1-100 ng/ml) without and with exposure to hypoxia (24 h)-reoxygenation. Baseline proliferation did not vary among cell types, and all cells showed increased proliferation after MIP-2. Aortic cell chemotaxis increased markedly upon exposure to MIP-2; however, neither pulmonary artery nor lung microvascular endothelial cells responded to this chemokine. Assessment of CXCR2, the G protein-coupled receptor through which MIP-2 signals, displayed no baseline difference in mRNA, protein, or cell surface expression among cell types. Exposure to hypoxia increased expression of CXCR2 of aortic endothelial cells only. Additionally, aortic cells, compared with pulmonary cells, showed significantly greater protein and activity of cathepsin S, a proteolytic enzyme important for cell motility. Thus the combined effects of increased cathepsin S activity, providing increased motility and enhanced CXCR2 expression after hypoxia, both contribute to the proangiogenic phenotype of systemic arterial endothelial cells.     

CXC and CC chemokine receptors on coronary and brain endothelia

BACKGROUND: Chemokine receptors on leukocytes play a key role in inflammation and HIV-1 infection. Chemokine receptors on endothelia may serve an important role in HIV-1 tissue invasion and angiogenesis. MATERIALS AND METHODS: The expression of chemokine receptors in human brain microvascular endothelial cells (BMVEC) and coronary artery endothelial cells (CAEC) in vitro and cryostat sections of the heart tissue was determined by light and confocal microscopy and flow cytometry with monoclonal antibodies. Chemotaxis of endothelia by CC chemokines was evaluated in a transmigration assay. RESULTS: In BMVEC, the chemokine receptors CCR3 and CXCR4 showed the strongest expression. CXCR4 was localized by confocal microscopy to both the cytoplasm and the plasma membrane of BMVEC. In CAEC, CXCR4 demonstrated a strong expression with predominantly periplasmic localization. CCR5 expression was detected both in BMVEC and CAEC but at a lower level. Human umbilical cord endothelial cells (HUVEC) expressed strongly CXCR4 but only weakly CCR3 and CCR5. Two additional CC chemokines, CCR2A and CCR4, were detected in BMVEC and CAEC by immunostaining. Immunocytochemistry of the heart tissues with monoclonal antibodies revealed a high expression of CXCR4 and CCR2A and a low expression of CCR3 and CCR5 on coronary vessel endothelia. Coronary endothelia showed in vitro a strong chemotactic response to the CC chemokines RANTES, MIP-1alpha, and MIP-1beta. CONCLUSIONS: The endothelia isolated from the brain display strongly both the CCR3 and CXCR4 HIV-1 coreceptors, whereas the coronary endothelia express strongly only the CXCR4 coreceptor. CCR5 is expressed at a lower level in both endothelia. The differential display of CCR3 on the brain and coronary endothelia could be significant with respect to the differential susceptibility of the heart and the brain to HIV-1 invasion. In addition, CCR2A is strongly expressed in the heart endothelium. All of the above chemokine receptors could play a role in endothelial migration and repair.     

Diverse signaling pathways through the SDF-1/CXCR4 chemokine axis in prostate cancer cell lines leads to altered patterns of cytokine secretion and angiogenesis

The establishment of metastatic bone lesions in prostate cancer (CaP) is a process partially dependent on angiogenesis. Previously we demonstrated that the stromal-derived factor-1 (SDF-1 or CXCL12)/CXCR4 chemokine axis is critical for CaP cell metastasis. In this investigation, cell lines were established in which CXCR4 expression was knocked down using siRNA technology. When CaP cells were co-transplanted with human vascular endothelial cells into SCID mice, significantly fewer human blood vessels were observed paralleling the reductions in CXCR4 levels. Likewise, the invasive behaviors of the CaP cells were inhibited in vitro. From these functional observations we explored angiogenic and signaling mechanisms generated following SDF-1 binding to CXCR4. Differential activation of the MEK/ERK and PI3K/AKT pathways that result in differential secretion IL-6, IL-8, TIMP-2 and VEGF were seen contingent on the cell type examined; VEGF and TIMP-2 expression in PC3 cells are dependent on AKT activation and ERK activation in LNCaP and LNCaP C4-2B cells leads to IL-6 or IL-8 secretion. At the same time, expression of angiostatin levels were inversely related to CXCR4 levels, and inhibited by SDF-1 stimulation. These data link the SDF-1/CXCR4 pathway to changes in angiogenic cytokines by different signaling mechanisms and, suggest that the delicate equilibrium between proangiogenic and antiangiogenic factors may be achieved by different signal transduction pathways to regulate the angiogenic phenotype of prostate cancers. Taken together, our results provide new information regarding expression of functional CXCR4 receptor-an essential role and potential mechanism of angiogenesis upon SDF-1 stimulation.     

AKAP12 in astrocytes induces barrier functions in human endothelial cells through protein kinase Czeta

Interactions between astrocytes and blood vessels are essential for the formation and maintenance of the blood-neural barrier (BNB). Astrocyte-derived A-kinase anchor protein 12 (AKAP12) influences BNB formation, but the mechanism of regulation of BNB functions by AKAP12 is not fully understood. We have defined a new pathway of barriergenesis in human retina microvascular endothelial cells (HRMECs) involving astrocytic AKAP12. Treatment of HRMECs with conditioned media from AKAP12-overexpressing astrocytes reduced phosphorylation of protein kinase Czeta (PKCzeta), decreased the levels of vascular endothelial growth factor (VEGF) mRNA and protein, and increased thrombospondin-1 (TSP-1) levels, which led to antiangiogenesis and barriergenesis. Transfection of a small interference RNA targeting PKCzeta decreased VEGF levels and increased TSP-1 levels in HRMECs. Rho is a putative downstream signal of PKCzeta, and inhibition of Rho kinase with a specific inhibitor, Y27632, decreased VEGF levels and increased TSP-1 levels. We therefore suggest that AKAP12 in astrocytes differentially regulates the expression of VEGF and TSP-1 via the inhibition of PKCzeta phosphorylation and Rho kinase activity in HRMECs.     

Transient receptor potential canonical channels are required for in vitro endothelial tube formation

In endothelial cells Ca(2+) entry is an essential component of the Ca(2+) signal that takes place during processes such as cell proliferation or angiogenesis. Ca(2+) influx occurs via the store-operated Ca(2+) entry pathway, involving stromal interaction molecule-1 (STIM1) and Orai1, but also through channels gated by second messengers like the transient receptor potential canonical (TRPC) channels. The human umbilical vein-derived endothelial cell line EA.hy926 expressed STIM1 and Orai1 as well as several TRPC channels. By invalidating each of these molecules, we showed that TRPC3, TRPC4, and TRPC5 are essential for the formation of tubular structures observed after EA.hy926 cells were plated on Matrigel. On the contrary, the silencing of STIM1 or Orai1 did not prevent tubulogenesis. Soon after being plated on Matrigel, the cells displayed spontaneous Ca(2+) oscillations that were strongly reduced by treatment with siRNA against TRPC3, TRPC4, or TRPC5, but not siRNA against STIM1 or Orai1. Furthermore, we showed that cell proliferation was reduced upon siRNA treatment against TRPC3, TRPC5, and Orai1 channels, whereas the knockdown of STIM1 had no effect. On primary human umbilical vein endothelial cells, TRPC1, TRPC4, and STIM1 are involved in tube formation, whereas Orai1 has no effect. These data showed that TRPC channels are essential for in vitro tubulogenesis, both on endothelial cell line and on primary endothelial cells.