[Epidermal growth factor-like domain 7 promotes endothelial cell migration and angiogenesis by activating ERK signaling pathway].

OBJECTIVE: To explore the effect of epidermal growth factor-like domain 7(EGFL7) on the migration and angiogenesis of endothelial cells. METHODS: EGFL7 overexpression vectors were constructed and transfected into human microvascular endothelial cells. The expression levels of EGFL7-mRNA and EGFL7 protein were examined by real-time RT-PCR and Western blot. Cell migration was analyzed by the wound healing. The capability of cell to form capillary-like tubes in vitro was evaluated on matrigel assay. Protein expression of p-AKT, AKT, p-ERK and ERK in endothelial cells was detected by Western blot upon transfection with EGFL7 overexpression vectors and vehicle control for 0, 10, 30 and 60 min. RESULTS: Migration and angiogenesis of endothelial cells were notably enhanced by EGFL7 overexpression. ERK pathway was strongly activated by EGFL7, whereas AKT remained constant in endothelial cells. Inhibition of ERK impaired EGFL7 induced ERK activation and endothelial cell migration and angiogenesis. CONCLUSION: EGFL7 effectively promotes migration and angiogenesis through ERK signaling pathway in endothelial cells.

MicroRNA-223 antagonizes angiogenesis by targeting ß1 integrin and preventing growth factor signaling in endothelial cells.

RATIONALE: Endothelial cells in situ are largely quiescent, and their isolation and culture are associated with the switch to a proliferative phenotype. OBJECTIVE: To identify antiangiogenic microRNAs expressed by native endothelial cells that are altered after isolation and culture, as well as the protein targets that regulate responses to growth factors. METHODS AND RESULTS: Profiling studies revealed that miR-223 was highly expressed in freshly isolated human, murine, and porcine endothelial cells, but those levels decreased in culture. In primary cultures of endothelial cells, vascular endothelial cell growth factor and basic fibroblast growth factor further decreased miR-223 expression. The overexpression of precursor-miR-223 did not affect basal endothelial cell proliferation but abrogated vascular endothelial cell growth factor-induced and basic fibroblast growth factor-induced proliferation, as well as migration and sprouting. Inhibition of miR-223 in vivo using specific antagomirs potentiated postnatal retinal angiogenesis in wild-type mice, whereas recovery of perfusion after femoral artery ligation and endothelial sprouting from aortic rings from adult miR-223(-/y) animals were enhanced. MiR-223 overexpression had no effect on the growth factor-induced activation of ERK1/2 but inhibited the vascular endothelial cell growth factor-induced and basic fibroblast growth factor-induced phosphorylation of their receptors and activation of Akt. ß1 integrin was identified as a target of miR-223 and its downregulation reproduced the defects in growth factor receptor phosphorylation and Akt signaling seen after miR-223 overexpression. Reintroduction of ß1 integrin into miR-223-ovexpressing cells was sufficient to rescue growth factor signaling and angiogenesis. CONCLUSIONS: These results indicate that miR-223 is an antiangiogenic microRNA that prevents endothelial cell proliferation at least partly by targeting ß1 integrin.

Recombinant human endostatin improves anti-tumor efficacy of paclitaxel by normalizing tumor vasculature in Lewis lung carcinoma.

PURPOSE: Normalization of the tumor vasculature and microenvironment by several angiogenesis inhibitors has been reported. Given that recombinant human endostatin (rh-endostatin) is also an endogenous angiogenesis inhibitor, a comprehensive evaluation of the effects of rh-endostatin on tumor vasculature and microenvironment and chemotherapy sensitivity would be favorable. METHODS: Multiple treatment schedules of the combination of rh-endostatin and paclitaxel were tested in Lewis lung carcinoma. Further, we monitored microvascular density, tumor hypoxic fraction, and collagen covered tumor vessels at three different time points following the treatment of rh-endostatin, as well as the transcription of angiogenesis related factors (vascular endothelial growth factor-A and thrombospondin-1) and vasculature markers (regulator of G-protein signaling 5 and platelet/endothelial cell adhesion molecule-1). RESULTS: The anti-tumor efficacy of paclitaxel was significantly improved 7 days after the treatment of rh-endostatin. Tumor microvascular density was decreased by rh-endostatin, although it became even higher 7 days after termination of rh-endostatin. Non-necrotic hypoxic fraction was significantly reduced 7 days after treatment of rh-endostatin, accompanied with increased collagen covered tumor vessels and coverage of pericytes around endothelial cells. Rh-endostatin could transiently upregulate the transcription of thrombospondin-1 and modulate the imbalance between vascular endothelial growth factor-A and thrombospondin-1. CONCLUSION: Rh-endostatin could normalize the tumor vasculature and microenvironment in Lewis lung carcinoma probably via modulation of the balance between vascular endothelial growth factor-A and thrombospondin-1. During the time of vascular normalization, paclitaxel treatment was found to have maximal effect on tumor growth delay.

Novel role for epidermal growth factor-like domain 7 in metastasis of human hepatocellular carcinoma.

UNLABELLED: Epidermal growth factor-like domain 7 (Egfl7) is a recently identified secreted protein that is believed to be primarily expressed in endothelial cells (ECs). Although its expression was reported elevated during tumorigenesis, whether and how Egfl7 contributes to human malignancies remains unknown. In the present study overexpression of Egfl7 was found predominantly in hepatocellular carcinoma (HCC) cells in HCC tissues and closely correlated with poor prognosis of HCC. The expression of Egfl7 in cancer cells was further verified with HCC cell lines including HepG2, MHCC97-L, and HCCLM3, and the Egfl7 expression levels positively correlated with metastatic potential of HCC cell lines was tested. To functionally characterize Egfl7 in HCC, we depleted its expression in HCCLM3 cells by using small interfering RNA. Interestingly, reduction of Egfl7 expression resulted in significant inhibition of migration but not growth of HCCLM3 cells. Biochemical analysis indicated that Egfl7 could facilitate the phosphorylation of focal adhesion kinase (FAK) and therefore promote the migration of HCCLM3 cells. In addition, this effect was almost completely blocked by inhibition of epidermal growth factor receptor (EGFR), indicating that the activation of FAK by Egfl7 is mediated through EGFR. Finally, we used a mouse model to demonstrate that down-regulation of Egfl7 was associated with suppression of intrahepatic and pulmonary metastases of HCC. Collectively, our study shows for the first time that overexpression of Egfl7 in HCC and Egfl7 promotes metastasis of HCC by enhancing cell motility through EGFR-dependent FAK phosphorylation. CONCLUSION: Our study suggests Egfl7 as a novel prognostic marker for metastasis of HCC and a potential therapeutic target.

Epidermal growth factor-like domain 7 (EGFL7) modulates Notch signalling and affects neural stem cell renewal.

Epidermal growth factor-like domain 7 (EGFL7) is a secreted factor implicated in cellular responses such as cell migration and blood vessel formation; however the molecular mechanisms underlying the effects of EGFL7 are largely unknown. Here we have identified transmembrane receptors of the Notch family as EGFL7-binding molecules. Secreted EGFL7 binds to a region in Notch involved in ligand-mediated receptor activation, thus acting as an antagonist of Notch signalling. Expression of EGFL7 in neural stem cells (NSCs) in vitro decreased Notch-specific signalling and consequently, reduced proliferation and self-renewal of NSCs. Such altered Notch signalling caused a shift in the differentiation pattern of cultured NSCs towards an excess of neurons and oligodendrocytes. We identified neurons as a source of EGFL7 in the brain, suggesting that brain-derived EGFL7 acts as an endogenous antagonist of Notch signalling that regulates proliferation and differentiation of subventricular zone-derived adult NSCs.

Cyclic GMP signaling is involved in the luteinizing hormone-dependent meiotic maturation of mouse oocytes.

It is well established that cAMP signaling is an important regulator of the oocyte meiotic cell cycle. Conversely, the function of cGMP during oocyte maturation is less clear. Herein, we evaluated the expression of cGMP-hydrolyzing phosphodiesterases (PDEs) in the somatic and germ cell compartments of the mouse ovarian follicle and demonstrate that PDE5 is preferentially expressed in somatic cells. Cyclic GMP is a potent inhibitor of cAMP hydrolysis from oocyte extracts, with a 50% inhibitory concentration of 97 nM. Luteinizing hormone (LH) stimulation of cultured preovulatory follicles results in a marked decrease in cGMP content, and a nadir is reached in 1.5 h; similarly, oocyte cGMP levels decrease after gonadotropin stimulation in vivo. The LH-dependent decrease in cGMP requires activation of the epidermal growth factor network. Treatment of follicles with a PDE5 inhibitor increases cGMP in the follicle well above unstimulated levels. Although LH causes a decrease in cGMP in follicles preincubated with PDE5 inhibitors, the levels of this nucleotide remain above unstimulated levels. Under these conditions of elevated cGMP, LH stimulation does not cause oocyte maturation after 5 h of incubation. Microinjection of a cGMP-specific PDE into oocytes causes meiotic maturation of wild-type oocytes, suggesting that an intraoocyte pool of cGMP is involved in the maintenance of meiotic arrest. This effect is absent in PDE3A-deficient oocytes. Taken together, these findings provide evidence that cGMP and cAMP signaling cooperate in maintaining meiotic arrest via regulation of PDE3A and that a decrease in cGMP in the somatic compartment is one of the signals contributing to meiotic maturation.

[Perspectives of regenerative mechanisms in cardiovascular disease spotlighting endothelial progenitor cells]. / Perspektiven regenerativer Mechanismen bei Herz-Kreislauf-Erkrankungen am Beispiel endothelialer Progenitorzellen.

Cardiovascular diseases are the most common cause of death in the Western world. In general, the underlying disease is atherosclerosis, which is hallmarked by deterioration of the endothelial monolayer. Restoration of an intact endothelial monolayer for prevention and therapy of cardiovascular diseases is one key concept of regenerative medicine. This article offers a review of state-of-the-art regenerative mechanisms in cardiovascular disease spotlighting endothelial progenitor cells, and further features the perspectives of regenerative medicine in vascular biology.

New insights into bone morphogenetic protein signaling: focus on angiogenesis.

PURPOSE OF REVIEW: The role of bone morphogenetic proteins (BMPs) in vasculogenesis is still not well understood, despite many recent developments in this area of research. In this review, we discuss the most recent studies that identify new critical mechanisms through which BMP signaling acts with a focus on angiogenesis. RECENT FINDINGS: New evidence brought to light over the last few years suggests that BMP-binding proteins, formerly thought of as antagonists, can also increase BMP activity under certain conditions. It has also recently been determined that components of the extracellular matrix are involved in the BMP signaling pathways that regulate angiogenesis. Through the BMP pathway, myosin-X and cyclooxygenase 2 serve as target genes that have been determined to play a role in blood vessel formation. BMPs also conduct Smad-independent signaling and crosstalk with other pathways. Finally, BMPs have been shown to play an antiangiogenic role in specific settings. SUMMARY: Better understanding of the BMP signaling pathway and its regulators can have potentially great effects on therapeutic strategies from cardiovascular disease to cancer.

Epidermal growth factor-like domain 7 protects endothelial cells from hyperoxia-induced cell death.

Hyperoxia is one of the major contributors to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants. Emerging evidence suggests that the arrested lung development of BPD is associated with pulmonary endothelial cell death and vascular dysfunction resulting from hyperoxia-induced lung injury. A better understanding of the mechanism of hyperoxia-induced endothelial cell death will provide critical information for the pathogenesis and therapeutic development of BPD. Epidermal growth factor-like domain 7 (EGFL7) is a protein secreted from endothelial cells. It plays an important role in vascular tubulogenesis. In the present study, we found that Egfl7 gene expression was significantly decreased in the neonatal rat lungs after hyperoxic exposure. The Egfl7 expression was returned to near normal level 2 wk after discounting oxygen exposure during recovery period. In cultured human endothelial cells, hyperoxia also significantly reduced Egfl7 expression. These observations suggest that diminished levels of Egfl7 expression might be associated with hyperoxia-induced endothelial cell death and lung injury. When we overexpressed human Egfl7 (hEgfl7) in EA.hy926 human endothelial cell line, we found that hEgfl7 overexpression could partially block cytochrome c release from mitochondria and decrease caspase-3 activation. Further Western blotting analyses showed that hEgfl7 overexpression could reduce expression of a proapoptotic protein, Bax, and increase expression of an antiapoptotic protein, Bcl-xL. Theses findings indicate that hEGFL7 may protect endothelial cell from hyperoxia-induced apoptosis by inhibition of mitochondria-dependent apoptosis pathway.

The role of heat shock protein 60, vascular endothelial growth factor and antiphospholipid antibodies in Behçet disease.

BACKGROUND: Behçet disease is a systemic inflammatory disease of unknown aetiology. T cells in this disease proliferate vigorously in response to a specific peptide of heat shock protein (HSP) 60 in an antigen-specific fashion. Vascular endothelial cell growth factor (VEGF) is a cytokine participating in the inflammatory process. One of the prominent features of Behçet disease is vasculitis as a result of endothelial dysfunction. Antiphospholipid antibodies (APA) may play a role in the development of thrombosis by inhibiting production of prostacyclin by endothelial cells. OBJECTIVES: To investigate the role of HSP60, VEGF and APA in Behçet disease and their relation to clinical manifestations and disease activity. METHODS: Thirty patients with Behçet disease were included; 17 were in the active stage and 13 were in the inactive. Fifteen age- and sex-matched healthy subjects served as controls. Complete clinical examination and Doppler examination were done. Serum levels of HSP60, VEGF and APA were performed. RESULTS: Serum levels of HSP60, VEGF and APA were significantly higher in patients than in controls; however, their level did not correlate with disease activity. The serum level of VEGF correlated significantly with the presence of vascular manifestations and ocular involvement. The serum level of APA was greater in patients with thrombosis. HSP60 has an important role in aetiopathogenesis of Behçet disease, which sheds new light on its autoimmune nature. CONCLUSIONS: An elevated serum level of VEGF may be a risk factor for the development of ocular disease contributing to poor visual outcome.

Molecular profile and partial functional analysis of novel endothelial cell-derived growth factors that regulate hematopoiesis.

Recent progress has been made in the identification of the osteoblastic cellular niche for hematopoietic stem cells (HSCs) within the bone marrow (BM). Attempts to identify the soluble factors that regulate HSC self-renewal have been less successful. We have demonstrated that primary human brain endothelial cells (HUBECs) support the ex vivo amplification of primitive human BM and cord blood cells capable of repopulating non-obese diabetic/severe combined immunodeficient repopulating (SCID) mice (SCID repopulating cells [SRCs]). In this study, we sought to characterize the soluble hematopoietic activity produced by HUBECs and to identify the growth factors secreted by HUBECs that contribute to this HSC-supportive effect. Extended noncontact HUBEC cultures supported an eight-fold increase in SRCs when combined with thrombopoietin, stem cell factor, and Flt-3 ligand compared with input CD34(+) cells or cytokines alone. Gene expression analysis of HUBEC biological replicates identified 65 differentially expressed, nonredundant transcripts without annotated hematopoietic activity. Gene ontology studies of the HUBEC transcriptome revealed a high concentration of genes encoding extracellular proteins with cell-cell signaling function. Functional analyses demonstrated that adrenomedullin, a vasodilatory hormone, synergized with stem cell factor and Flt-3 ligand to induce the proliferation of primitive human CD34(+)CD38(-)lin(-) cells and promoted the expansion of CD34(+) progenitors in culture. These data demonstrate the potential of primary HUBECs as a reservoir for the discovery of novel secreted proteins that regulate human hematopoiesis.

Thymidine phosphorylase inhibitors: recent developments and potential therapeutic applications.

Thymidine Phosphorylase (TPase) catalyses the reversible phosphorolysis of pyrimidine 2'-deoxynucleosides to 2-deoxyribose-1-phosphate and their respective pyrimidine bases, including the phosphorolysis of nucleoside analogues with important antiviral or anticancer properties. Moreover, TPase, identified also as the angiogenic platelet-derived endothelial cell growth factor (PD-ECGF), stimulates endothelial cell migration in vitro and angiogenesis in vivo and plays an important role in tumour progression and metastasis. Here we have summarized the most recent approaches in the search for novel TPase inhibitors together with the potential therapeutic applications of such inhibitors.

Emphysema: an autoimmune vascular disease?

We propose that an endogenous maintenance program controls lung cell turnover, apoptosis, and tissue repair, and that emphysema is a manifestation of the breakdown of the lung structure maintenance program. Emphysema can be induced experimentally in rats by three methods: blockade of vascular endothelial growth factor receptors using SU5416, a small molecule-tyrosine kinase inhibitor; methylprednisolone, which activates matrix metalloproteinase-9 and decreases Akt phosphorylation; and antibodies directed against endothelial cells (autoimmune emphysema). SU5416-induced emphysema is associated with lung induction of cytochrome P450 and oxidant stress, and a superoxide dismutase mimetic or N-acetylcysteine prevents this form of emphysema. A broad-spectrum metalloproteinase inhibitor prevents methylprednisolone-induced emphysema and, finally, autoimmune emphysema is associated with increased lung tissue metalloproteinase-9 expression and alveolar septal cell apoptosis. Athymic rats, which lack CD4+ T cells, are protected against autoimmune emphysema, whereas adoptive transfer of CD4+ T cells causes autoimmune emphysema in naive adult rats. It appears that vascular endothelial growth factor and signaling via its receptors plays a central role in the lung structural maintenance program, and oxidative stress, proteolysis, and apoptosis may coincide in the moment of lung cell destruction. Interestingly, the methylprednisolone model illustrates that inflammation is not necessary for the development of emphysema.

The role of growth factors in the pathogenesis of diabetic retinopathy.

Diabetic retinopathy (DR) is the most severe of several ocular complications of diabetes. The earliest clinical signs of DR are microaneurysms and haemorrhages. Later signs include dilated, tortuous irregular veins and retinal non-profusion, leading to retinal ischaemia that ultimately results in neovascularisation. Diabetic macular oedema, which involves the breakdown of the blood-retinal barrier, also occurs and is responsible for a major part of vision loss, particularly in Type 2 diabetes. The pathogenesis of DR is very complex. Many biochemical mechanisms have been proposed as explanations for the development and progression of DR. Chronic hyperglycaemia leads to oxidative injury, microthrombi formation, cell adhesion molecule activation, leukostasis and cytokine activation. Next, ischaemia-mediated overexpression of growth factors and cytokines occurs. These factors include vascular endothelial growth factor, insulin-like growth factor-1, angiopoetin-1 and -2, stromal-derived factor-1, fibroblast growth factor-2 and tumour necrosis factor. Because of the complex interplay between these factors, targeting a single growth factor will be unlikely to result in therapeutic inhibition of angiogenesis. These growth factors no doubt act in synergy to mediate the steps of angiogenesis, including protease production, endothelial cell proliferation, migration and tube formation. This review attempts to provide an overview of perspectives regarding the pathogenesis of this disease. The focus, however, is on describing the unique features of selected relevant factors and how each growth factor may act in a synergistic manner with other factors.

[Pathogenic mechanisms of thrombosis in neoplasia: therapeutic implications]. / Patogenia de la trombosis asociada a enfermedades neoplásicas: implicaciones terapéuticas.

Malignancy is associated with a hypercoagulable state and a high risk for thrombohemorragic complications. Activation of blood coagulation in cancer is a complex phenomenon, involving many different pathways of the hemostatic system and numerous interactions of the tumour cell with other blood cells, including platelet, monocyte and endothelial cells. In addition, the involvement of fibrin formation in the processes of tumour spread and metastasis is important in this area. Experimental evidence suggest that TF expression in tumour cells is associated with enhanced procoagulant activity as well as increased tumour cell invasion, primary tumour growth and increased tumoral metastatic potential. Moreover, a close correlation exists between TF and the synthesis of the angiogenic cytokine VEGF in tumour cells and with angiogenesis in vivo. A number of agents designed specifically for targeting TF, VEGF and/or receptors are being evaluated in various clinical trials in cancer patients. This review discuss the current status in pharmacological interventions to block thrombogenicity and angiogenesis in the treatment of cancer.

VE-statin, an endothelial repressor of smooth muscle cell migration.

The recruitment and proliferation of smooth muscle cells and pericytes are two key events for the stabilization of newly formed capillaries during angiogenesis and, when out of control in the adult, are the main causes of arteriosclerosis. We have identified a novel gene, named VE-statin for vascular endothelial-statin, which is expressed specifically by endothelial cells of the developing mouse embryo and in the adult, and in early endothelial progenitors. The mouse and human VE-statin genes have been located on chromosome 2 and 9, respectively, they span >10 kbp and are transcribed in two major variants arising from independent initiation sites. The VE-statin transcripts code for a unique protein of 30 kDa that contains a signal peptide and two epidermal growth factor (EGF)-like modules. VE-statin is found in the cellular endoplasmic reticulum and secreted in the cell supernatant. Secreted VE-statin inhibits platelet-derived growth factor (PDGF)-BB-induced smooth muscle cell migration, but has no effects on endothelial cell migration. VE-statin is the first identified inhibitor of mural cell migration specifically produced by endothelial cells.

VEGF-A and alphaVbeta3 integrin synergistically rescue angiogenesis via N-Ras and PI3-K signaling in human microvascular endothelial cells.

We recently showed that normal fibroblasts mediate capillary-like differentiation of human microvascular endothelial cells (HMVEC) in a 3-D angiogenesis model. Here, we show that a collaborative effect of VEGF-A and alphaVbeta3 integrin is critical in fibroblast-mediated angiogenesis because enhancement of both VEGF production by fibroblasts and beta3 integrin expression in HMVEC can rescue capillary-like endothelial differentiation under reduced serum conditions. To investigate the downstream signaling mechanisms, we compared N-Ras and Rho/Rac/Cdc42, as well as phosphatidylinositol 3-kinase (PI3-K) and Akt, for their involvement in the capillary-like network formation. The dominant-negative mutant of N-Ras (N-RasN17), but not the mutants of Rho/Rac/Cdc42, suppressed network formation. Overexpression of a constitutively active form of PI3-K rescued the network formation, which was inhibited by a dominant-negative >beta3 integrin; however, an active form of Akt failed to rescue the inhibition but induced a phenotypic change in HMVEC. Moreover, PI3-K is a downstream target of N-Ras because it could be co-immunoprecipitated with N-Ras, and its active form could rescue the inhibitory effect of N-Ras N17. Thus, our data indicate the existence of N-Ras- and PI3-K-dependent but Rho/Rac/Cdc42- and Akt-independent signaling mechanisms for the synergistic effect of VEGF-A and alphaVbeta3 on fibroblast-mediated microvascular network formation.