Neuropeptides Regulate Expression of Angiogenic Growth Factors in Human Dental Pulp Fibroblasts

Abstract<br/>INTRODUCTION:<br/>Neuropeptides play an important role in inflammation and repair and have been implicated in mediating angiogenesis. Pulp fibroblasts express neuropeptide receptors, and the aim of this research was to investigate whether neuropeptides could regulate angiogenic growth factor expression in vitro<br/>METHODS:<br/>An angiogenic array was used to determine the levels of 10 angiogenic growth factors expressed by human pulp fibroblasts.<br/>RESULTS:<br/>Pulp fibroblasts were shown to express angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin-binding epidermal growth factor, hepatocyte growth factor, leptin, platelet-derived growth factor, placental growth factor, and vascular endothelial growth factor. Furthermore, the neuropeptides substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and neuropeptide Y altered angiogenic growth factor expression in vitro.<br/>CONCLUSIONS:<br/>The regulation of angiogenic growth factor expression by neuropeptides suggests a novel role for neuropeptides in pulpal inflammation and repair.

Inhibition of platelet-derived growth factor promotes pericyte loss and angiogenesis in ischemic retinopathy

We investigated whether inhibition of platelet-derived growth factor (PDGF) receptor tyrosine kinase activity would affect pericyte viability, vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor-2 (VEGFR-2) expression and angiogenesis in a model of retinopathy of prematurity (ROP). ROP was induced in Sprague Dawley rats by exposure to 80% oxygen from postnatal (P) days 0 to 11 (with 3 hours/day in room air), and then room air from P12-18 (angiogenesis period). Shams were neonatal rats in room air from P0-18. STI571, a potent inhibitor of PDGF receptor tyrosine kinase, was administered from P12-18 at 50 or 100 mg/kg/day intraperitoneal (i.p.). Electron microscopy revealed that pericytes in the inner retina of both sham and ROP rats appeared normal; however STI571 induced a selective pericyte and vascular smooth muscle degeneration. Immunolabeling for caspase-3 and a-smooth muscle cell actin in consecutive paraffin sections of retinas confirmed that these degenerating cells were apoptotic pericytes. In all groups, VEGF and VEGFR-2 gene expression was located in ganglion cells, the inner nuclear layer, and retinal pigment epithelium. ROP was associated with an increase in both VEGF and VEGFR-2 gene expression and blood vessel profiles in the inner retina compared to sham rats. STI571 at both doses increased VEGF and VEGFR-2 mRNA and exacerbated angiogenesis in ROP rats, and in sham rats at 100 mg/kg/day. In conclusion, PDGF is required for pericyte viability and the subsequent prevention of VEGF/VEGFR-2 overexpression and angiogenesis in ROP.

Histone deacetylase 7 controls endothelial cell growth through modulation of beta-catenin

Rationale: Histone deacetylase (HDAC)7 is expressed in the early stages of embryonic development and may play a role in endothelial function.<br/><br/>Objective: This study aimed to investigate the role of HDAC7 in endothelial cell (EC) proliferation and growth and the underlying mechanism.<br/><br/>Methods and Results: Overexpression of HDAC7 by adenoviral gene transfer suppressed human umbilical vein endothelial cell (HUVEC) proliferation by preventing nuclear translocation of ß-catenin and downregulation of T-cell factor-1/Id2 (inhibitor of DNA binding 2) and cyclin D1, leading to G1 phase elongation. Further assays with the TOPFLASH reporter and quantitative RT-PCR for other ß-catenin target genes such as Axin2 confirmed that overexpression of HDAC7 decreased ß-catenin activity. Knockdown of HDAC7 by lentiviral short hairpin RNA transfer induced ß-catenin nuclear translocation but downregulated cyclin D1, cyclin E1 and E2F2, causing HUVEC hypertrophy. Immunoprecipitation assay and mass spectrometry analysis revealed that HDAC7 directly binds to ß-catenin and forms a complex with 14-3-3 e, ?, and ? proteins. Vascular endothelial growth factor treatment induced HDAC7 degradation via PLC?-IP3K (phospholipase C?–inositol-1,4,5-trisphosphate kinase) signal pathway and partially rescued HDAC7-mediated suppression of proliferation. Moreover, vascular endothelial growth factor stimulation suppressed the binding of HDAC7 with ß-catenin, disrupting the complex and releasing ß-catenin to translocate into the nucleus.<br/><br/>Conclusions: These findings demonstrate that HDAC7 interacts with ß-catenin keeping ECs in a low proliferation stage and provides a novel insight into the mechanism of HDAC7-mediated signal pathways leading to endothelial growth

Tumor necrosis factor receptor expression and signaling in renal cell carcinoma

<p>Clear cell renal cell carcinoma (ccRCC), a tubular epithelial cell (TEC) malignancy, frequently secretes tumor necrosis factor (TNF). TNF signals via two distinct receptors (TNFRs). TNFR1, expressed in normal kidney primarily on endothelial cells, activates apoptotic signaling kinase 1 and nuclear factor-kappaB (NF-kappaB) and induces cell death, whereas TNFR2, inducibly expressed on endothelial cells and on TECs by injury, activates endothelial/epithelial tyrosine kinase (Etk), which trans-activates vascular endothelial growth factor receptor 2 (VEGFR2) to promote cell proliferation. We investigated TNFR expression in clinical samples and function in short-term organ cultures of ccRCC tissue treated with wild-type TNF or specific muteins selective for TNFR1 (R1-TNF) or TNFR2 (R2-TNF). There is a significant increase in TNFR2 but not TNFR1 expression on malignant TECs that correlates with increasing malignant grade. In ccRCC organ cultures, R1-TNF increases TNFR1, activates apoptotic signaling kinase and NF-kappaB, and promotes apoptosis in malignant TECs. R2-TNF increases TNFR2, activates NF-kappaB, Etk, and VEGFR2 and increases entry into the cell cycle. Wild-type TNF induces both sets of responses. R2-TNF actions are blocked by pretreatment with a VEGFR2 kinase inhibitor. We conclude that TNF, acting through TNFR2, is an autocrine growth factor for ccRCC acting via Etk-VEGFR2 cross-talk, insights that may provide a more effective therapeutic approach to this disease.</p>

Vascular Endothelial Cell Growth–Activated XBP1 Splicing in Endothelial Cells Is Crucial for Angiogenesis

<p>BACKGROUND - : Vascular endothelial cell growth factor plays a pivotal role in angiogenesis via regulating endothelial cell proliferation. The X-box binding protein 1 (XBP1) is believed to be a signal transducer in the endoplasmic reticulum stress response. It is unknown whether there is crosstalk between vascular endothelial cell growth factor signaling and XBP1 pathway. </p><p>METHODS AND RESULTS - : We found that vascular endothelial cell growth factor induced the kinase insert domain receptor internalization and interaction through C-terminal domain with the unspliced XBP1 and the inositol requiring enzyme 1 α in the endoplasmic reticulum, leading to inositol requiring enzyme 1 α phosphorylation and XBP1 mRNA splicing, which was abolished by siRNA-mediated knockdown of kinase insert domain receptor. Spliced XBP1 regulated endothelial cell proliferation in a PI3K/Akt/GSK3β/β- catenin/E2F2-dependent manner and modulated the cell size increase in a PI3K/Akt/GSK3β/β-catenin/E2F2-independent manner. Knockdown of XBP1 or inositol requiring enzyme 1 α decreased endothelial cell proliferation via suppression of Akt/GSK3β phosphorylation, β-catenin nuclear translocation, and E2F2 expression. Endothelial cell-specific knockout of XBP1 (XBP1ecko) in mice retarded the retinal vasculogenesis in the first 2 postnatal weeks and impaired the angiogenesis triggered by ischemia. Reconstitution of XBP1 by Ad-XBP1s gene transfer significantly improved angiogenesis in ischemic tissue in XBP1ecko mice. Transplantation of bone marrow from wild-type o XBP1ecko mice could also slightly improve the foot blood reperfusion in ischemic XBP1ecko mice. </p><p>CONCLUSIONS - : These results suggest that XBP1 can function via growth factor signaling pathways to regulate endothelial proliferation and angiogenesis. </p>

Clinical and testing protocols for the analysis of epidermal growth factor receptor mutations in East Asian patients with non-small cell lung cancer:a combined clinical-molecular pathological approach

Several randomized phase III studies in advanced stage non-small cell lung cancer (NSCLC) confirmed the superior response rate and progression-free survival of using epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor as first-line therapy compared with chemotherapy in patients with activating EGFR mutations. Despite the need for EGFR mutation tests to guide first-line therapy in East Asian NSCLC, there are no current standard clinical and testing protocols.

Dual targeting of tumour cells and host endothelial cells by novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines

<p>PURPOSE: Some members of a novel series of pyrrolo-1,5-benzoxazepines (PBOXs) are microtubule-targeting agents capable of inducing apoptosis in a variety of human cancerous cells, hence, they are currently being developed as potential anti-cancer agents. The purpose of this study was to first characterise the activities of a novel PBOX analogue, PBOX-16 and then investigate the anti-angiogenic potential of both PBOX-16 and its prototype PBOX-6.</p><p>METHODS: The effects of PBOX-6 and -16 on cancerous cells (chronic myeloid leukaemia K562 cells and ovarian carcinoma A2780 cells) and primary cultured human umbilical vein endothelial cells (HUVECs) were examined by assessing cell proliferation, microtubular organisation, DNA analysis of cell cycle progression and caspase-3/7 activity. Their anti-angiogenic properties were then investigated by examining their ability to interfere with HUVEC differentiation into capillary-like structures and vascular endothelial growth factor (VEGF)-stimulated HUVEC migration.</p><p>RESULTS: PBOX-6 and -16 inhibited proliferation of K562, A2780 and HUVEC cells in a concentration-dependent manner. PBOX-16, confirmed as a novel depolymerising agent, was approximately tenfold more potent than PBOX-6. Inhibition of cell proliferation was mediated by G(2)/M arrest followed by varying degrees of apoptosis depending on the cell type; endothelial cells underwent less apoptosis than either of the cancer cell lines. In addition to the antitumourigenic properties, we also describe a novel antiangiogenic function for PBOXs: treatment with PBOXs inhibited the spontaneous differentiation of HUVECs into capillary-like structures when grown on a basement membrane matrix preparation (Matrigelâ„¢) and also significantly reduced VEGF-stimulated HUVEC migration.</p><p>CONCLUSION: Dual targeting of both the tumour cells and the host endothelial cells by PBOX compounds might enhance the anti-cancer efficacy of these drugs.</p>

Epidermal Growth Factor Receptor Activity Determines Response of Colorectal Cancer Cells to Gefitinib Alone and in Combination with Chemotherapy.

Purpose: Up to now, there have been no established predictive markers for response to epidermal growth factor receptor (EGFR/HER1/erbB1) inhibitors alone and in combination with chemotherapy in colorectal cancer. To identify markers that predict response to EGFR-based chemotherapy regimens, we analyzed the response of human colorectal cancer cell lines to the EGFR-tyrosine kinase inhibitor, gefitinib (Iressa, AstraZeneca, Wilmington, DE), as a single agent and in combination with oxaliplatin and 5-fluorouracil (5-FU). Experimental Design: Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and crystal violet cell viability assays and analyzed by ANOVA. Apoptosis was measured by flow cytometry, poly(ADP-ribose) polymerase, and caspase 3 cleavage. EGFR protein phosphorylation was detected by Western blotting. Results: Cell lines displaying high constitutive EGFR phosphorylation (a surrogate marker for EGFR activity) were more sensitive to gefitinib. Furthermore, in cell lines exhibiting low constitutive EGFR phosphorylation, an antagonistic interaction between gefitinib and oxaliplatin was observed, whereas in cell lines with high basal EGFR phosphorylation, the interaction was synergistic. In addition, oxaliplatin treatment increased EGFR phosphorylation in those cell lines in which oxaliplatin and gefitinib were synergistic but down-regulated EGFR phosphorylation in those lines in which oxaliplatin and gefitinib were antagonistic. In contrast to oxaliplatin, 5-FU treatment increased EGFR phosphorylation in all cell lines and this correlated with synergistic decreases in cell viability when 5-FU was combined with gefitinib. Conclusions: These results suggest that phospho-EGFR levels determine the sensitivity of colorectal cancer cells to gefitinib alone and that chemotherapy-mediated changes in phospho-EGFR levels determine the nature of interaction between gefitinib and chemotherapy.

Src and ADAM-17-Mediated Shedding of Transforming Growth Factor-alpha Is a Mechanism of Acute Resistance to TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L) has emerged as a promising anticancer agent. However, resistance to TRAIL is likely to be a major problem, and sensitization of cancer cells to TRAIL may therefore be an important anticancer strategy. In this study, we examined the effect of the epidermal growth factor receptor (EGFR)tyrosine kinase inhibitor (TKI) gefitinib and a human epidermal receptor 2 (HER2)-TKI (M578440) on the sensitivity of human colorectal cancer (CRC) cell lines to recombinant human TRAIL (rhTRAIL). A synergistic interaction between rhTRAIL and gefitinib and rhTRAIL and M578440 was observed in both rhTRAIL-sensitive and resistant CRC cells. This synergy correlated with an increase in EGFR and HER2 activation after rhTRAIL treatment. Furthermore, treatment of CRC cells with rhTRAIL resulted in activation of the Src family kinases (SFK). Importantly, we found that rhTRAIL treatment induced shedding of transforming growth factor-alpha (TGF-alpha) that was dependent on SFK activity and the protease ADAM-17. Moreover, this shedding of TGF-alpha was critical for rhTRAIL-induced activation of EGFR. In support of this, SFK inhibitors and small interfering RNAs targeting ADAM-17 and TGF-alpha also sensitized CRC cells to rhTRAIL-mediated apoptosis. Taken together, our findings indicate that both rhTRAIL-sensitive and resistant CRC cells respond to rhTRAIL treatment by activating an EGFR/HER2-mediated survival response and that these cells can be sensitized to rhTRAIL using EGFR/HER2-targeted therapies. Furthermore, this acute response to rhTRAIL is regulated by SFK-mediated and ADAM-17-mediated shedding of TGF-alpha, such that targeting SFKs or inhibiting ADAM-17, in combination with rhTRAIL, may enhance the response of CRC tumors to rhTRAIL. [Cancer Res 2008;68(20):8312-21]

eNOS overexpression exacerbates vascular closure in the obliterative phase of OIR and increases angiogenic drive in the subsequent proliferative stage

Purpose: In ischemic retinopathies, the misdirection of reparative angiogenesis away from the hypoxic retina leads to pathologic neovascularization. Thus, therapeutic strategies that reverse this trend would be extremely beneficial. Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is an important mediator of vascular endothelial growth factor (VEGF) function facilitating vascular growth and maturation. However, in addition to NO, eNOS can also produce superoxide (O), exacerbating pathology. Here, our aim was to investigate the effect of eNOS overexpression on vascular closure and subsequent recovery of the ischemic retina. <br/><br/>Methods: Mice overexpressing eNOS-GFP were subjected to oxygen-induced retinopathy (OIR) and changes in retinal vascularization quantified. Background angiogenic drive was assessed during vascular development and in aortic rings. NOS activity was measured by Griess assay or conversion of radiolabeled arginine to citrulline, nitrotyrosine (NT), and superoxide by immunolabeling and dihydroethidium fluorescence and VEGF by ELISA. <br/><br/>Results: In response to hyperoxia, enhanced eNOS expression led to increased NOS-derived superoxide and dysfunctional NO production, NT accumulation, and exacerbated vessel closure associated with tetrahydrobiopterin (BH) insufficiency. Despite worse vaso-obliteration, eNOS overexpression resulted in elevated hypoxia-induced angiogenic drive, independent of VEGF production. This correlated with increased vascular branching similar to that observed in isolated aortas and during development. Enhanced recovery was also associated with neovascular tuft formation, which showed defective NO production and increased eNOS-derived superoxide and NT levels. <br/><br/>Conclusions: In hyperoxia, reduced BH bioavailability causes overexpressed eNOS to become dysfunctional, exacerbating vaso-obliteration. In the proliferative phase, however, eNOS has important prorepair functions enhancing angiogenic growth potential and recovery in ischemia. © 2012 The Association for Research in Vision and Ophthalmology, Inc.

HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells

Reendothelialization involves endothelial progenitor cell (EPC) homing, proliferation, and differentiation, which may be influenced by fluid shear stress and local flow pattern. This study aims to elucidate the role of laminar flow on embryonic stem (ES) cell differentiation and the underlying mechanism. We demonstrated that laminar flow enhanced ES cell-derived progenitor cell proliferation and differentiation into endothelial cells (ECs). Laminar flow stabilized and activated histone deacetylase 3 (HDAC3) through the Flk-1-PI3K-Akt pathway, which in turn deacetylated p53, leading to p21 activation. A similar signal pathway was detected in vascular endothelial growth factor-induced EC differentiation. HDAC3 and p21 were detected in blood vessels during embryogenesis. Local transfer of ES cell-derived EPC incorporated into injured femoral artery and reduced neointima formation in a mouse model. These data suggest that shear stress is a key regulator for stem cell differentiation into EC, especially in EPC differentiation, which can be used for vascular repair, and that the Flk-1-PI3K-Akt-HDAC3-p53-p21 pathway is crucial in such a process.

Molecular mapping the presence of druggable targets in preinvasive and precursor breast lesions:a comprehensive review of biomarkers related to therapeutic interventions

The analysis of clinical breast samples using biomarkers is integral to current breast cancer management. Currently, a limited number of targeted therapies are standard of care in breast cancer treatment. However, these targeted therapies are only suitable for a subset of patients and resistance may occur. Strategies to prevent the occurrence of invasive lesions are required to reduce the morbidity and mortality associated with the development of cancer. In theory, application of targeted therapies to pre-invasive lesions will prevent their progression to invasive lesions with full malignant potential. The diagnostic challenge for pathologists is to make interpretative decisions on early detected pre-invasive lesions. Overall, only a small proportion of these pre-invasive lesions will progress to invasive carcinoma and morphological assessment is an imprecise and subjective means to differentiate histologically identical lesions with varying malignant potential. Therefore differential biomarker analysis in pre-invasive lesions may prevent overtreatment with surgery and provide a predictive indicator of response to therapy. There follows a review of established and emerging potential druggable targets in pre-invasive lesions and correlation with lesion morphology.

Endothelial progenitor cells in diabetic retinopathy

Diabetic retinopathy (DR) is a leading cause of visual impairment worldwide. Patients with DR may irreversibly lose sight as a result of the development of diabetic macular edema (DME) and/or proliferative diabetic retinopathy (PDR); retinal blood vessel dysfunction and degeneration plays an essential role in their pathogenesis. Although new treatments have been recently introduced for DME, including intravitreal vascular endothelial growth factor inhibitors (anti-VEGFs) and steroids, a high proportion of patients (~40-50%) do not respond to these therapies. Furthermore, for people with PDR, laser photocoagulation remains a mainstay therapy despite this being an inherently destructive procedure. Endothelial progenitor cells (EPCs) are a low-frequency population of circulating cells known to be recruited to sites of vessel damage and tissue ischemia where they promote vascular healing and re-perfusion. A growing body of evidence suggests that the number and function of EPCs are altered in patients with varying degrees of diabetes duration, metabolic control, and in the presence or absence of DR. Although there are no clear-cut outcomes from these clinical studies, there is mounting evidence that some EPC sub-types may be involved in the pathogenesis of DR and may also serve as biomarkers for disease progression and stratification. Moreover, some EPC sub-types have considerable potential as therapeutic modalities for DME and PDR in the context of cell therapy. This study presents basic clinical concepts of DR and combines this with a general insight on EPCs and their relation to future directions in understanding and treating this important diabetic complication.

Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3

It is well-known that atherosclerosis occurs geographically at branch points where disturbed flow predisposes to the development of plaque via triggering of oxidative stress and inflammatory reactions. In this study, we found that disturbed flow activated anti-oxidative reactions via up-regulating heme oxygenase 1 (HO-1) in an X-box binding protein 1 (XBP1) and histone deacetylase 3 (HDAC3)-dependent manner. Disturbed flow concomitantly up-regulated the unspliced XBP1 (XBP1u) and HDAC3 in a vascular endothelial growth factor receptor (VEGFR) and PI3K/Akt dependent manner. The presence of XBP1 was essential for the up-regulation of HDAC3 protein. Over-expression of XBP1u and/or HDAC3 activated Akt1 phosphorylation, Nrf2 protein stabilization and nuclear translocation, and HO-1 expression. Knockdown of XBP1u decreased the basal level and disturbed flow-induced Akt1 phosphorylation, Nrf2 stabilization and HO-1 expression. Knockdown of HDAC3 ablated XBP1u-mediated effects. The mammalian target of rapamycin complex 2 (mTORC2) inhibitor, AZD2014, ablated XBP1u or HDAC3 or disturbed flow-mediated Akt1 phosphorylation, Nrf2 nuclear translocation and HO-1 expression. Neither actinomycin D nor cycloheximide affected disturbed flow-induced up-regulation of Nrf2 Protein. Knockdown of Nrf2 abolished XBP1u or HDAC3 or disturbed flow-induced HO-1 up-regulation. Co-immunoprecipitation assays demonstrated that XBP1u physically bound to HDAC3 and Akt1. The region of amino acids 201 to 323 of the HDAC3 protein was responsible for the binding to XBP1u. Double immunofluorescence staining revealed that the interactions between Akt1 and mTORC2, Akt1 and HDAC3, Akt1 and XBP1u, HDAC3 and XBP1u occurred in the cytosol. Thus, we demonstrate that XBP1u and HDAC3 exert a protective effect on disturbed flow-induced oxidative stress via up-regulation of mTORC2-dependent Akt1 phosphorylation and Nrf2-mediated HO-1 expression.