Vascular endothelial growth factor-induced endothelial cell proliferation is regulated by interaction between VEGFR-2, SH-PTP1 and eNOS

VEGF receptor-2 plays a critical role in endothelial cell proliferation during angiogenesis. However, regulation of receptor activity remains incompletely explained. Here, we demonstrate that VEGF stimulates microvascular endothelial cell proliferation in a dose-dependent manner with VEGF-induced proliferation being greatest at 5 and 100 ng/ml and significantly reduced at intermediate concentrations (>50% at 20 ng/ml). Neutralization studies confirmed that signaling occurs via VEGFR-2. In a similar fashion, ERK/MAPK is strongly activated in response to VEGF stimulation as demonstrated by its phosphorylation, but with a decrease in phosphoryation at 20 ng/ml VEGF. Immunoblotting analysis revealed that VEGF did not cause a dose-dependent change in expression of VEGFR-2 but instead resulted in reduced phosphorylation of VEGFR-2 when cells were exposed to 10 and 20 ng/ml of VEGF. VEGFR-2 dephosphorylation was associated with an increase in the protein tyrosine phosphatase, SH-PTP1, and endothelial nitric oxide synthase (eNOS). Immunoprecipitation and selective immunoblotting confirmed the association between VEGFR-2 dephosphorylation and the upregulation of SH-PTP1 and eNOS. Transfection of endothelial cells with antisense oligonucleotide against VEGFR-2 completely abolished VEGF-induced proliferation, whereas anti SH-PTP1 dramatically increased VEGF-induced proliferation by 1 and 5-fold at 10 and 200 ng/ml VEGF, respectively. Suppression of eNOS expression only abolished endothelial cell proliferation at VEGF concentrations above 20 ng/ml. Taken together, these results indicate that activation of VEGFR-2 by VEGF enhances SH-PTP1 activity and eNOS expression, which in turn lead to two diverse events: one is that SH-PTP1 dephosphorylates VEGFR-2 and ERK/MAPK, which weaken VEGF mitogenic activity, and the other is that eNOS increases nitric oxide production which in turn lowers SH-PTP1 activity via S-nitrosylation.

Activation of vascular endothelial growth factor receptor-1 sustains angiogenesis and Bcl-2 expression via the phosphatidylinositol 3-kinase pathway in endothelial cells

Vascular insufficiency and retinal ischemia precede many proliferative retinopathies and stimulate secretion of various vasoactive growth factors, including vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). It is unclear, however, how PlGF, which is elevated in proliferative diabetic retinopathy and is a VEGF homolog that binds only to VEGF receptor (VEGFR)-1, promotes pathological angiogenesis. When primary microvascular endothelial cells were grown on collagen gels, PlGF-containing ligands upregulated Bcl-2 expression and stimulated the formation of capillary-like tube networks that were retained for up to 14 days in culture. The inhibition of VEGFR-1 results in a dramatic decrease in the number of capillary connections, indicating that VEGFR-1 ligands promote branching angiogenesis. In contrast, VEGF-induced tube formations and Bcl-2 expression were significantly decreased at the end of this period. Flow cytometry analysis of annexin-V/propidium iodide-stained cells revealed that PlGF and PlGF/VEGF heterodimer inhibited apoptosis in serum-deprived endothelial cells. These two growth factors stimulated a survival signaling pathway phosphatidylinositol 3-kinase (PI3K), as identified by increased Akt phosphorylation and because blocking PI3K signalling by adenovirus-mediated overexpression of wild-type phosphatase and tensin homolog on chromosome 10 (PTEN) disrupted angiogenesis and decreased Bcl-2 expression by PlGF and PlGF/VEGF heterodimer, whereas a dominant-negative PTEN mutant enhanced endothelial sprout formation and Bcl-2 expression. Together, these findings indicate that PlGF-containing ligands contribute to pathological angiogenesis by prolonging cell survival signals and maintaining vascular networks.

Activation of proteinase-activated receptor 2 stimulates soluble vascular endothelial growth factor receptor 1 release via epidermal growth factor receptor transactivation in endothelial cells

The proteinase-activated receptor 2 (PAR-2) expression is increased in endothelial cells derived from women with preeclampsia, characterized by widespread maternal endothelial damage, which occurs as a consequence of elevated soluble vascular endothelial growth factor receptor-1 (sVEGFR-1; commonly known as sFlt-1) in the maternal circulation. Because PAR-2 is upregulated by proinflammatory cytokines and activated by blood coagulation serine proteinases, we investigated whether activation of PAR-2 contributed to sVEGFR-1 release. PAR-2–activating peptides (SLIGRL-NH2 and 2-furoyl-LIGRLO-NH2) and factor Xa increased the expression and release of sVEGFR-1 from human umbilical vein endothelial cells. Enzyme-specific, dominant-negative mutants and small interfering RNA were used to demonstrate that PAR-2–mediated sVEGFR-1 release depended on protein kinase C-ß1 and protein kinase C-e, which required intracellular transactivation of epidermal growth factor receptor 1, leading to mitogen-activated protein kinase activation. Overexpression of heme oxygenase 1 and its gaseous product, carbon monoxide, decreased PAR-2–stimulated sVEGFR-1 release from human umbilical vein endothelial cells. Simvastatin, which upregulates heme oxygenase 1, also suppressed PAR-2–mediated sVEGFR-1 release. These results show that endothelial PAR-2 activation leading to increased sVEGFR-1 release may contribute to the maternal vascular dysfunction observed in preeclampsia and highlights the PAR-2 pathway as a potential therapeutic target for the treatment of preeclampsia.

Carbon monoxide inhibits sprouting angiogenesis and vascular endothelial growth factor receptor-2 phosphorylation

Carbon monoxide (CO) is a gaseous autacoid known to positively regulate vascular tone; however, its role in angiogenesis is unknown. The aim of this study was to investigate the effect of CO on angiogenesis and vascular endothelial growth factor (VEGF) receptor-2 phosphorylation. Human umbilical vein endothelial cells (HUVECs) were cultured on growth factor- reduced Matrigel and treated with a CO-releasing molecule (CORM-2) or exposed to CO gas (250 ppm). Here, we report the surprising finding that exposure to CO inhibits vascular endothelial growth factor (VEGF)-induced endothelial cell actin reorganisation, cell proliferation, migration and capillary-like tube formation. Similarly, CO suppressed VEGF-mediated phosphorylation of VEGFR-2 at tyrosine residue 1175 and 1214 and basic fibroblast growth factor- (FGF-2) and VEGF-mediated Akt phosphorylation. Consistent with these data, mice exposed to 250 ppm CO (1h/day for 14 days) exhibited a marked decrease in FGF-2-induced Matrigel plug angiogenesis (p<0.05). These data establish a new biological function for CO in angiogenesis and point to a potential therapeutic use for CO as an anti-angiogenic agent in tumour suppression.

The expression and prognostic significance of bcl-2-associated transcription factor 1 in rectal cancer following neoadjuvant therapy

Date of Acceptance: 12/07/2015 © 2015 John Wiley & Sons Ltd. Acknowledgements This study was supported by funding from the Encompass kick start and SMART:Scotland award schemes of Scottish Enterprise and Friends of Anchor. The Grampian Biorepository assisted with the immunohistochemical investigations.

The expression and prognostic significance of bcl-2-associated transcription factor 1 in rectal cancer following neoadjuvant therapy

Date of Acceptance: 12/07/2015 © 2015 John Wiley & Sons Ltd. Acknowledgements This study was supported by funding from the Encompass kick start and SMART:Scotland award schemes of Scottish Enterprise and Friends of Anchor. The Grampian Biorepository assisted with the immunohistochemical investigations.

Elevated hepatocyte growth factor levels in osteoarthritis osteoblasts contribute to their altered response to bone morphogenetic protein-2 and reduced mineralization capacity

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Effect of the synthetic coumarin, ethyl 2-oxo-2H-chromene-3-carboxylate, on activity of Crotalus durissus ruruima sPLA2 as well as on edema and platelet aggregation induced by this factor

We show that ethyl 2-oxo-2H-chromene-3-carboxylate (EOCC), a synthetic coumarin, irreversibly inhibits phospholipase A(2) (sPLA2) from Crotalus durissus ruruima venom (sPLA2r) with an IC(50) of 3.1 +/- 0.06 nmol. EOCC strongly decreased the V(max) and K(m), and it virtually abolished the enzyme activity of sPLA2r as well as sPLA2s from other sources. The edema induced by 5PLA2r + EOCC was less than that induced by 5PLA2r treated with p-bromophenacyl bromide, which was more efficient at neutralizing the platelet aggregation activity of native 5PLA2r. Native 5PLA2r induced platelet aggregation of 91.54 +/- 9.3%, and sPLA2r +/- EOCC induced a platelet aggregation of 18.56 +/- 6.5%. EOCC treatment also decreased the myotoxic effect of sPLA2r. Mass spectrometry showed that EOCC formed a stable complex with sPLA2r, which increased the mass of native 5PLA2r from 14,299.34 da to 14,736.22 Da. Moreover, the formation of this complex appeared to be involved in the loss of 5PLA2r activity. Our results strongly suggest that EOCC can be used as a pharmacological agent against the 5PLA2 in Crotalus durissus sp. venom as well as other sPLA2s. (C) 2010 Elsevier Ltd. All rights reserved.

Molecular analysis of exons 8, 9 and 10 of the fibroblast growth factor receptor 2 (FGFR2) gene in two families with index cases of Apert Syndrome

Introduction: Apert syndrome (AS) is a craniosynostosis condition caused by mutations in the Fibroblast Growth Factor Receptor 2 (FGFR2) gene. Clinical features include cutaneous and osseous symmetric syndactily in hands and feet, with variable presentations in bones, brain, skin and other internal organs. Methods: Members of two families with an index case of Apert Syndrome were assessed to describe relevant clinical features and molecular analysis (sequencing and amplification) of exons 8, 9 and 10 of FGFR2 gen. Results: Family 1 consists of the mother, the index case and half -brother who has a cleft lip and palate. In this family we found a single FGFR2 mutation, S252W, in the sequence of exon 8. Although mutations were not found in the study of the patient affected with cleft lip and palate, it is known that these diseases share signaling pathways, allowing suspected alterations in shared genes. In the patient of family 2, we found a sequence variant T78.501A located near the splicing site, which could interfere in this process, and consequently with the protein function.

Employing the five-factor mentoring instrument : analysing mentoring practices for teaching primary science.

Primary science education is a concern around the world and quality mentoring within schools can develop preservice teachers’ practices. A five-factor model for mentoring has been identified, namely, personal attributes, system requirements, pedagogical knowledge, modelling, and feedback. Final-year preservice teachers (mentees, n=211) from three Turkish universities were administered a previously validated instrument to gather perceptions of their mentoring in primary science teaching. ANOVA indicated that each of these five factors was statistically significant (p<.001) with mean scale scores ranging from 3.36 to 4.12. Although mentees perceived their mentors to provide evaluation feedback (95%), model classroom management (88%), guide their preparation (96%), and outline the science curriculum (92%), the majority of mentors were perceived not to assist their mentees in 10 of the 34 survey items. Professional development programmes that target the specific needs of these mentors may further enhance mentoring practices for advancing primary science teaching.

Confirmatory factor analysis of the Way of Coping Checklist-Revised (WCCL-R) in the Asian context

Industrial employment growth has been one of the most dynamic areas of expansion in Asia; however, current trends in industrialised working environments have resulted in greater employee stress. Despite research showing that cultural values affect the way people cope with stress, there is a dearth of psychometrically established tools for use in non-Western countries to measure these constructs. Studies of the "Way of Coping Checklist-Revised" (WCCL-R) in the West suggest that the WCCL-R has good psychometric properties, but its applicability in the East is still understudied. A confirmatory factor analysis (CFA) is used to validate the WCCL-R constructs in an Asian population. This study used 1,314 participants from Indonesia, Sri Lanka, Singapore, and Thailand. An initial exploratory factor analysis revealed that original structures were not confirmed; however, a subsequent EFA and CFA showed that a 38-item, five-factor structure model was confirmed. The revised WCCL-R in the Asian sample was also found to have good reliability and sound construct and concurrent validity. The 38-item structure of the WCCL-R has considerable potential in future occupational stress-related research in Asian countries.

Multi-factor password-authenticated key exchange

We consider a new form of authenticated key exchange which we call multi-factor password-authenticated key exchange, where session establishment depends on successful authentication of multiple short secrets that are complementary in nature, such as a long-term password and a one-time response, allowing the client and server to be mutually assured of each other's identity without directly disclosing private information to the other party. Multi-factor authentication can provide an enhanced level of assurance in higher-security scenarios such as online banking, virtual private network access, and physical access because a multi-factor protocol is designed to remain secure even if all but one of the factors has been compromised. We introduce a security model for multi-factor password-authenticated key exchange protocols, propose an efficient and secure protocol called MFPAK, and provide a security argument to show that our protocol is secure in this model. Our security model is an extension of the Bellare-Pointcheval-Rogaway security model for password-authenticated key exchange and accommodates an arbitrary number of symmetric and asymmetric authentication factors.

Vitronectin modulates human mesenchymal stem cell response to insulin-like growth factor-I and transforming growth factor beta 1 in a serum-free environment

The use of animal sera for the culture of therapeutically important cells impedes the clinical use of the cells. We sought to characterize the functional response of human mesenchymal stem cells (hMSCs) to specific proteins known to exist in bone tissue with a view to eliminating the requirement of animal sera. Insulin-like growth factor-I (IGF-I), via IGF binding protein-3 or -5 (IGFBP-3 or -5) and transforming growth factor-beta 1 (TGF-beta(1)) are known to associate with the extracellular matrix (ECM) protein vitronectin (VN) and elicit functional responses in a range of cell types in vitro. We found that specific combinations of VN, IGFBP-3 or -5, and IGF-I or TGF-beta(1) could stimulate initial functional responses in hMSCs and that IGF-I or TGF-beta(1) induced hMSC aggregation, but VN concentration modulated this effect. We speculated that the aggregation effect may be due to endogenous protease activity, although we found that neither IGF-I nor TGF-beta(1) affected the functional expression of matrix metalloprotease-2 or -9, two common proteases expressed by hMSCs. In summary, combinations of the ECM and growth factors described herein may form the basis of defined cell culture media supplements, although the effect of endogenous protease expression on the function of such proteins requires investigation.