Stimulates Vascular Endothelial Growth Factor Production Research Articles

Prognostic Role of IL-6 in POEMS Syndrome

Prognostic Role of IL-6 in POEMS Syndrome

S-layer protein 2 of vaginal Lactobacillus crispatus 2029 enhances growth, differentiation, VEGF production and barrier functions in intestinal epithelial cell line Caco-2

We have previously demonstrated the ability of the human vaginal strain Lactobacillus crispatus 2029 (LC2029) for strong adhesion to cervicovaginal epithelial cells, expression of the surface layer protein 2 (Slp2), and antagonistic activity against urogenital pathogens. Slp2 forms regular two-dimensional structure around the LC2029 cells,which is secreted into the medium and inhibits intestinal pathogen-induced activation of caspase-9 and caspase-3 in the human intestinal Caco-2 cells. Here, we elucidated the effects of soluble Slp2 on adhesion of proteobacteria pathogens inducing necrotizing enterocolitis (NEC), such as Escherichia coli ATCC E 2348/69, E. coli ATCC 31705, Salmonella Enteritidis ATCC 13076, Campylobacter jejuni ATCC 29428, and Pseudomonas aeruginosa ATCC 27853 to Caco-2 cells, as well as on growth promotion, differentiation, vascular endothelial growth factor (VEGF) production, and intestinal barrier function of Caco-2 cell monolayers. Slp2 acts as anti-adhesion agent for NEC-inducing proteobacteria, promotes growth of immature Caco-2 cells and their differentiation, and enhances expression and functional activity of sucrase, lactase, and alkaline phosphatase. Slp2 stimulates VEGF production, decreases paracellular permeability, and increases transepithelial electrical resistance, strengthening barrier function of Caco-2 cell monolayers. These data support the important role of Slp2 in the early postnatal development of the human small intestine enterocytes.

Abstract PS3-22: A new computational model for visualization and prediction of breast cancer growth based on reinforcement learning

Abstract Introduction: Breast cancer is a complex disease with no simple solution, which is why researchers are tackling it from every angle. A relatively new effort of predictive oncology is to develop a systematic method for predicting the future status of an individual breast tumor given representation of the initial conditions and an appropriate mathematical model. Using a medical imaging-based approach, the goal of this preclinical research project was to obtain high resolution images of the tumor angiogenic network and predict cancer growth using a novel mathematical model that we recently develop. Methodology: In the proposed model, we consider each cell and vessel as an agent and decisions are made based on the state-action-reward-state-action (SARSA) concept, which is an incremental reinforcement learning algorithm. Two types of cells (i.e. cancer and normal) and vessels (i.e. tip and stalk) are used in the environment. This spatial collection of cells and vessels then have six (i.e. apoptosis, hypoxia, necrosis, migration, proliferation and quiescence) and three (i.e. branch, expansion and sprout) allowable actions. The model agent has a high degree of autonomy and performs actions based on the knowledge it receives from the surrounding environment. One of the more important parts of our model is the release and diffusion of select nutrients into the tumor environment, e.g. oxygen, vascular endothelial growth factor (VEGF), etc. Using a breast cancer xenograft mouse model, animals were administrated an intravascular contrast agent (200 microliters, ExiTron Nano 12000, Miltenyi Biotec) and tumors were scanned using an ultrahigh resolution computed tomography (CT) system (OI/CT, MILabs). The tumor microvascular network was segmented and used as an input and initial state of our model that then simulated cancer growth. Subsequent CT scans (on the order of weeks) were performed to validate model accuracy. Results: Initial results demonstrate that the SARSA model can simulate tumor growth. As the concentration of available nutrients around the tumor decreases, normal cells began to decline sharply and were eliminated by selecting the apoptotic phenotype. Cancer cells were more resistant to nutrient deficiencies during simulation. Consequently, these cancer cells proliferated, and tumor volume increased. Our findings also revealed that decreased oxygen availability (i.e. hypoxia) stimulated VEGF production and growth of the microvascular network, which agreed with repeat in vivo CT imaging results. Conclusion: A predictive model of breast cancer growth was developed and preliminary preclinical results using in vivo medical images of breast cancer-bearing animals highlighted the utility of this new oncological tool. Citation Format: M. Hossein Zangooei, Ryan Margolis, Kenneeth Hoyt. A new computational model for visualization and prediction of breast cancer growth based on reinforcement learning [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS3-22.

Nasal expression of the vascular endothelial growth factor and its receptors is reduced by mepolizumab in chronic rhinosinusitis with nasal polyposis

Chronic rhinosinusitis with nasal polyposis (CRSwNP) is a phenotype of chronic rhinosinusitis characterized by the presence of nasal polyps. To date, medical therapy and surgery can manage the disease in most cases. 1 Fokkens W.J. Lund V.J. Hopkins C. et al. European Position Paper on Rhinosinusitis and Nasal Polyps 2020. Rhinology. 2020; 58: 1-464 Google Scholar However, despite improvements in surgical techniques, CRSwNP is characterized by relapses in severe cases, thus leading to the study of biologic drugs such as mepolizumab, antagonizing interleukin (IL)-5. Experimental and clinical evidence demonstrates that CRSwNP is driven by a type 2 immune response and displays eosinophil infiltration, mucosal inflammation, and hyperplasia of the epithelium. 2 Bachert C. Marple B. Hosemann W. Cavaliere C. Wen W. Zhang N. Endotypes of chronic rhinosinusitis with nasal polyps: pathology and possible therapeutic implications. J Allergy Clin Immunol Pract. 2020; 8: 1514-1519 Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Furthermore, it has been described that vascular endothelial growth factor (VEGF) is strongly up-regulated in the mucosa of patients with CRSwNP, promoting edema and epithelial cell growth in nasal polyps when compared with normal mucosa. 3 Wittekindt C. Hess A. Bloch W. Sultanie S. Michel O. Immunohistochemical expression of VEGF and VEGF receptors in nasal polyps as compared to normal turbinate mucosa. Eur Arch Otorhinolaryngol. 2002; 259: 294-298 Crossref PubMed Scopus (56) Google Scholar Notably, other reports found that IL-5 stimulates VEGF production, which may influence immune cells’ activity in inflammatory conditions. 4 Puxeddu I. Ribatti D. Crivellato E. Levi-Schaffer F. Mast cells and eosinophils: a novel link between inflammation and angiogenesis in allergic diseases. J Allergy Clin Immunol. 2005; 116: 531-536 Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar

Triiodothyronine stimulates steroid and VEGF production in murine Leydig cells via cAMP-PKA pathway.

Thyroid hormones affect testicular development as well as functions like spermatogenesis and steroidogenesis, thereby influencing male fertility. Our group earlier showed that the stimulatory role of the thyroid hormone, T3 , on the production of vascular endothelial growth factor (VEGF) by murine Leydig cells is mediated by steroids and hypoxia-inducible factor-1 (HIF-1α). The current study further defines the signalling pathway(s) utilised by T3 to stimulate the production of steroids, VEGF and HIF-1α in mouse Leydig tumour cell line (MLTC-1). Specific inhibitors for different signalling molecules were used to study the role of cyclic AMP (cAMP), and its downstream mediators. Expression of VEGF and HIF-1α mRNA were measured by quantitative RT-PCR; VEGF secretion by ELISA; steroid secretion by radioimmunoassay and HIF-1α protein levels by western blotting. Inhibitors of adenylate cyclase (AC), protein kinase A (PKA), sarcoma kinase (SrcK), phosphoinositide 3-kinase (PI3K) and MAP kinase kinase (MEK1/2) abolished the T3 -induced increase in VEGF mRNA and protein levels. The same signalling molecules also mediated the increased production of steroids and HIF-1α protein in response to T3 . Therefore, it was concluded that T3 stimulates steroid secretion and HIF-1α protein in MLTC-1 cells through the AC-cAMP-PKA-PI3K-MEK pathway, which in turn stimulate VEGF production.

Lipopolysaccharide-Induced VEGF Production and Ambient Oxidative Stress in Type 2 Diabetes.

Oxidative stress is implicated in the development of vascular disease and is associated with an upregulation of vascular endothelial growth factor (VEGF), which is pathogenetically linked to the microvascular complications of diabetes. Patients of African origin have an increased susceptibility to microvascular kidney disease compared with white patients; the reasons and mechanisms that contribute to this vulnerability are unclear. To investigate whether there are ethnic differences in lipopolysaccharide-induced monocyte VEGF production in whole blood cell cultures. In addition, to assess whether stimulated VEGF production is related to prevailing oxidative stress assessed by plasma lipid hydroperoxides (LOOHs) and α-tocopherol. Cross-sectional study at a secondary care center in North London, United Kingdom, serving an inner-city community of 154,000 adults. African-Caribbean and white patients with type 2 diabetes mellitus [(T2DM); n = 52]. Lipopolysaccharide-induced production of VEGF in whole blood cultures [61.8 (31.9) pg/mL to 78.4 (6.0) pg/mL; P < 0.001] correlated positively with LOOH levels (r = 0.3, P = 0.04) and was significantly higher in African-Caribbean than white patients with T2DM [404 (207.5) vs 268.8 (137.0)] pg/mL ×109/L monocytes; P = 0.018]. Plasma α-tocopherol concentration was higher in Caucasian (white) patients [40.3 (18.3) vs 30.0 (9.6)] µmol/L; P = 0.04] compared with African-Caribbean patients. This study suggests that the redox environment influences VEGF production in response to proinflammatory stimuli in T2DM. The differential responsiveness by ethnic origin may be of relevance in the variations in susceptibility to the long-term microvascular complications.

Synthetic Clay–based Hypoxia Mimetic Hydrogel for Pulp Regeneration: The Impact on Cell Activity and Release Kinetics Based on Dental Pulp–derived Cells In Vitro

IntroductionThixotropic synthetic clays have been successfully used for tissue engineering in regenerative medicine. The impact of these clays on the dental pulp, in particular in combination with hypoxia-based approaches using hypoxia mimetic agents (HMAs), is unknown. Our aim was to reveal the response of dental pulp–derived cells (DPCs) to a synthetic clay–based hydrogel and evaluate the release of HMAs. MethodsUsing resazurin-based toxicity assays, live-dead staining, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining, the viability of human DPCs seeded onto a synthetic clay–based hydrogel of 5%–0.15% as well as onto the hydrogels loaded with the HMAs dimethyloxalylglycine (DMOG), desferrioxamine, L-mimosine, and CoCl2 was evaluated. Furthermore, supernatant of the hydrogels loaded with HMAs were generated. Vascular endothelial growth factor (VEGF) production of DPCs in response to the supernatant was measured to reveal the cellular response to the HMAs. ResultsWe found that the synthetic clay–based hydrogel did not impair the viability of DPCs. Cell monolayer and cell cluster formations were observed on the hydrogel. No significant increase of VEGF levels was observed in the supernatant when DPCs were cultured on hydrogels loaded with HMAs. Supernatant of DMOG-loaded hydrogels stimulated VEGF production in DPCs in the first hour, whereas the effect of desferrioxamine, L-mimosine, and CoCl2 did not reach a level of significance. ConclusionsThe synthetic clay–based hydrogel represents a promising biomaterial that does not induce prominent toxic effects in DPCs. It can be loaded with DMOG to induce hypoxia mimetic activity. Overall, we provided first insights into the impact of synthetic clays on DPCs for tissue engineering purposes in regenerative endodontics.

Naturally Occurring Hair Growth Peptide: Water-Soluble Chicken Egg Yolk Peptides Stimulate Hair Growth Through Induction of Vascular Endothelial Growth Factor Production.

Alopecia is divided into two categories: androgenic alopecia and nonandrogenic alopecia. An androgen-dependent abnormality of biological functions causes alopecia in males, but the role of androgens is not yet elucidated in female pattern hair loss (FPHL). Modulation of androgenic activity is not effective in certain kinds of androgenic alopecia in females, as well as in cases of nonandrogenic alopecia in males and females. The hair growth drug, minoxidil, stimulates vascular endothelial growth factor (VEGF) production as well as vascularization and hair growth in females. Yet, because minoxidil has side effects with long-term use, a safe alternative hair growth agent is needed. Whereas hair develops after birth in mammalian species, hair mostly grows in a precocial bird, in the chicken, between hatching days 14 and 15. Therefore, we hypothesized that the chicken egg contains a key hair growth factor. In this study, we demonstrated that water-soluble peptides derived from the egg yolk stimulate VEGF production and human hair follicle dermal papilla cell growth. We also found that these peptides enhance murine hair growth and improve hair growth in FPHL. Finally, we characterized that water-soluble egg yolk peptides induce VEGF expression through insulin growth factor-1 receptor activation-induced hypoxia-inducible factor-1α transcription pathway. We have given the name "hair growth peptide (HGP)" to this water-soluble egg yolk peptide.

Triiodothyronine stimulates VEGF expression and secretion via steroids and HIF-1α in murine Leydig cells

ABSTRACTLeydig cells are the principal steroidogenic cells of the testis. Leydig cells also secrete a number of growth factors including vascular endothelial growth factor (VEGF) which has been shown to regulate both testicular steroidogenesis and spermatogenesis. The thyroid hormone, T3, is known to stimulate steroidogenesis in Leydig cells. T3 has also been shown to stimulate VEGF production in a variety of cell lines. However, studies regarding the effect of T3 on VEGF synthesis and secretion by the Leydig cells were lacking. Therefore, we investigated the effect of T3 on VEGF synthesis and secretion in a mouse Leydig tumour cell line, MLTC-1. The effect of T3 was compared with that of LH/cAMP and hypoxia, two known stimulators of Leydig cell functions. The cells were treated with T3, 8-Br-cAMP (a cAMP analogue), or CoCl2 (a hypoxia mimetic) and VEGF secreted in the cell supernatant was measured using ELISA. The mRNA levels of VEGF were measured by quantitative RT-PCR. In the MLTC-1 cells, T3, 8-Br-cAMP, and CoCl2 stimulated VEGF mRNA levels and the protein secretion. T3 also increased steroid secretion as well as HIF-1α protein levels, two well-established upstream regulators of VEGF. Inhibitors of steroidogenesis as well as HIF-1α resulted in inhibition of T3-stimulated VEGF secretion by the MLTC-1 cells. This suggested a mediatory role of steroids and HIF-1α protein in T3-stimulated VEGF secretion by MLTC-1 cells. The mediation by steroids and HIF-1α were independent of each other.Abbreviations: 8-Br-cAMP: 8-bromo – 3ʹ, 5ʹ cyclic adenosine monophosphate; CoCl2: cobalt chloride; HIF-1α: hypoxia inducible factor −1α; LH: luteinizing hormone; T3: 3, 5, 3ʹ-L-triiodothyronine; VEGF: vascular endothelial growth factor

Effects of Vitreomacular Adhesion on Age-Related Macular Degeneration.

Herein, we review the association between vitreomacular adhesion (VMA) and neovascular age-related macular degeneration (AMD). Meta-analyses have shown that eyes with neovascular AMD are twice as likely to have VMA as normal eyes. VMA in neovascular AMD may induce inflammation, macular traction, decrease in oxygenation, sequestering of vascular endothelial growth factor (VEGF), and other cytokines or may directly stimulate VEGF production. VMA may also interfere with the treatment effects of anti-VEGF therapy, which is the standard treatment for neovascular AMD, and releasing VMA can improve the treatment response to anti-VEGF treatment in neovascular AMD. We also reviewed currently available methods of relieving VMA.

IL-8 enhances the angiogenic potential of human bone marrow mesenchymal stem cells by increasing vascular endothelial growth factor.

The beneficial effects of mesenchymal stem cells (MSCs) are mediated partly by the paracrine production of cytoprotective and trophic factors. Vascular endothelial growth factor (VEGF) is released from MSCs as a paracrine trophic factor and contributes to the therapeutic effects of the stem cell by regulating angiogenesis and promoting revascularization in injured tissues. Interleukin-8 (IL-8), an inflammatory chemokine with potent proangiogenic properties, is upregulated in the ischemic brain and has been shown to promote homing of bone marrow-derived cells to injured sites. However, the effect of IL-8 on MSCs paracrine function remains unknown. We found that IL-8 induced VEGF production and phosphorylation of Akt and ERK. Both effects could be blocked by inhibitors (LY294002, PD098059) or siRNA-mediated silencing of Akt and ERK in human bone marrow MSCs (hBM-MSCs). IL-8-induced VEGF production in hBM-MSCs significantly increased tube formation on Matrigel compared with basal secreted VEGF. In a rat stroke model, administration of IL-8-treated hBM-MSCs decreased the infarction volume and increased angiogenesis in the ischemic boundary zone compared with hBM-MSC treatment alone. In conclusion, IL-8 stimulates VEGF production in hBM-MSCs in part via the PI3K/Akt and MAPK/ERK signal transduction pathways and that administration of IL-8-treated hBM-MSCs increases angiogenesis after stroke. This approach may be used to optimize MSC-based therapies for numerous diseases including stroke, myocardial ischemia, and spinal cord injury.

Luteinizing hormone, insulin like growth factor-1, and epidermal growth factor stimulate vascular endothelial growth factor production in cultured bubaline granulosa cells

The objective of this study was to characterize in vitro expression and secretion of vascular endothelial growth factor (VEGF) in bubaline granulosa cells (GC), grown in serum containing media supplemented with luteinizing hormone (LH), insulin like growth factor-1 (IGF-1), and epidermal growth factor (EGF) at three different doses and time durations. GCs were collected from ovarian follicles of varying diameters [Gp-I (small), 4–6mm; Gp-II (medium), 7–9mm; Gp-III (large), 10–13mm; Gp-IV (pre-ovulatory), >13mm]. In general, each of the three treatments resulted in a dose as well as time dependent increase in the mRNA expression and secretion of VEGF in the cultured GCs of Gp-IV follicles. These results were well supported by our observations on immunocytochemistry in Gp IV granulosa cell culture (GCC). We also looked into the expression dynamics of an anti-apoptotic factor – proliferating cellular antigen (PCNA) and a pro-apoptotic factor – Bcl-2-associated X protein (BAX) in GCs of Gp IV follicles on treatments with LH, IGF-1, and EGF to evaluate their cytoprotective/anti-apoptotic property. Relative expressions of PCNA and BAX showed a mutually opposite trend with the PCNA expression increasing and BAX expression decreasing with increase in dose and time to reach the zenith (P<0.05) and nadir (P<0.05) at the highest dose(s) at the maximum time duration (72h) for PCNA and BAX respectively on treatment with all the three factors. Thus, it can be concluded that LH, IGF-1, and EGF treatments have a cytoprotective/anti-apoptotic effect and stimulate VEGF production in granulosa cells of bubaline pre-ovulatory follicles.

Phosphodiesterase-4 Inhibition Augments Human Lung Fibroblast Vascular Endothelial Growth Factor Production Induced by Prostaglandin E2

Lung fibroblasts are believed to be a major source of vascular endothelial growth factor (VEGF), which supports the survival of lung endothelial cells and modulates the maintenance of the pulmonary microvasculature. VEGF has been related to the pathogenesis of lung diseases, including chronic obstructive pulmonary disease (COPD). Prostaglandin E2 (PGE2) stimulates VEGF production from lung fibroblasts via the E-prostanoid (EP)-2 receptor. The EP2 signaling pathway uses cyclic adenosine monophosphate (cAMP) as a second messenger, and cAMP is degraded by phosphodiesterases (PDEs). This study investigates whether phosphodiesterase inhibition modulates the human lung fibroblast VEGF production induced by PGE2. Human fetal lung fibroblasts were cultured with PGE2 and PDE inhibitors. The PDE4 inhibitors roflumilast, roflumilast N-oxide, and rolipram with PGE2 increased VEGF release, as quantified in supernatant media by ELISA. In contrast, PDE3, PDE5, and PDE7 inhibitors did not affect VEGF release. Roflumilast increased VEGF release with either an EP2 or an EP4 agonist. Roflumilast augmented the cytosolic cAMP levels induced by PGE2 and VEGF release with other agents that use the cAMP signaling pathway. Roflumilast-augmented VEGF release was completely inhibited by a protein kinase A (PKA) inhibitor. Roflumilast with PGE2 increased VEGF mRNA levels, and the blockade of mRNA synthesis inhibited the augmented VEGF release. The stimulatory effect of roflumilast on VEGF release was replicated using primary healthy and COPD lung fibroblasts. These findings demonstrate that PDE4 inhibition can modulate human lung fibroblast VEGF release by PGE2 acting through the EP2 and EP4 receptor-cAMP/PKA signaling pathway. Through this action, PDE4 inhibitors such as roflumilast could contribute to the survival of lung endothelial cells.

Effect of Prostaglandin E2 on Vascular Endothelial Growth Factor Production in Nasal Polyp Fibroblasts

PurposeAngiogenesis is involved in the pathogenesis of chronic rhinosinusitis with nasal polyps. We aimed to investigate the effects of prostaglandin E2 (PGE2) on vascular endothelial growth factor (VEGF) production, the role of E-prostanoid (EP) 4 receptors, and the signal transduction pathway mediating VEGF production in nasal polyp-derived fibroblasts (NPDFs).MethodsEight primary NPDF cultures were established from nasal polyps, which were incubated with or without PGE2. Reverse transcription-polymerase chain reaction amplification of EP receptors (EP1, EP2, EP3, and EP4) and immunofluorescence staining for VEGF production were performed. VEGF production via the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K) pathways was evaluated by enzyme-linked immunosorbent assay.ResultsAll EP receptors were expressed in NPDFs. PGE2 significantly increased VEGF production concentration- and time dependently, and VEGF production was regulated by an EP4 receptor. Activation of intracellular cAMP regulated VEGF production. VEGF production was decreased by PKA and PI3K inhibitors via intracellular cAMP.ConclusionsPGE2 stimulates VEGF production via the EP4 receptor in NPDFs. These results indicate that PGE2-induced VEGF production is mediated, at least partially, through cAMP-dependent signaling pathways. Therapies targeting the EP4 receptor may be effective in inhibiting the development of nasal polyps.

Enamel matrix derivative induces production of vascular endothelial cell growth factor in human gingival fibroblasts

Enamel matrix derivative (EMD) may enhance periodontal wound healing by inducing angiogenesis. We sought to investigate the effect and the mechanism of action of EMD on vascular endothelial growth factor (VEGF) production by human gingival fibroblasts. Cells were stimulated with EMD, transforming growth factor-β1 (TGF-β1), or fibroblast growth factor 2 (FGF-2), with or without antibodies to TGF-β1 or FGF-2. The levels of VEGF in the culture media were measured using an ELISA. We examined the effects of SB203580 [a p38 mitogen-activated protein kinase (MAPK) inhibitor], U0126 [an extracellular signal-regulated kinase (ERK) inhibitor], SP600125 [a c-Jun N-terminal kinase (JNK) inhibitor], and LY294002 [a phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor] on EMD-induced VEGF production. Enamel matrix derivative stimulated the production of VEGF in a dose- and time-dependent manner. Treatment of human gingival fibroblasts with antibodies to TGF-β1 or FGF-2 significantly decreased EMD-induced VEGF production, whereas the addition of exogenous TGF-β1 and FGF-2 stimulated VEGF production. Enamel matrix derivative-induced VEGF production was significantly attenuated by SB203580, U0126, and LY294002. Our results suggest that EMD stimulates VEGF production partially via TGF-β1 and FGF-2 in human gingival fibroblasts and that EMD-induced VEGF production is regulated by ERK, p38 MAPK, and PI3K/Akt pathways. Enamel matrix derivative-induced production of VEGF by human gingival fibroblasts may be involved in the enhancement of periodontal wound healing by inducing angiogenesis.

Prolyl Hydroxylase Inhibitors Increase the Production of Vascular Endothelial Growth Factor in Dental Pulp–derived Cells

IntroductionProlyl hydroxylase (PHD) inhibitors can induce a proangiogenic response that stimulates regeneration in soft and hard tissues. However, the effect of PHD inhibitors on the dental pulp is unclear. The purpose of this study was to evaluate the effects of PHD inhibitors on the proangiogenic capacity of human dental pulp–derived cells. MethodsTo test the response of dental pulp–derived cells to PHD inhibitors, the cells were exposed to dimethyloxalylglycine, desferrioxamine, L-mimosine, and cobalt chloride. To assess the response of dental pulp cells to a capping material supplemented with PHD inhibitors, the cells were treated with supernatants from calcium hydroxide. Viability, proliferation, and protein synthesis were assessed by formazan formation, 3[H]thymidine, and 3[H]leucine incorporation assays. The effect on the proangiogenic capacity was measured by immunoassays for vascular endothelial growth factor (VEGF). ResultsWe found that all 4 PHD inhibitors can reduce viability, proliferation, and protein synthesis at high concentrations. At nontoxic concentrations and in the presence of supernatants from calcium hydroxide, PHD inhibitors stimulated the production of VEGF in dental pulp–derived cells. When calcium hydroxide was supplemented with the PHD inhibitors, the supernatants from these preparations did not significantly elevate VEGF levels. ConclusionsThese results show that PHD inhibitors can stimulate VEGF production of dental pulp–derived cells, suggesting a corresponding increase in their proangiogenic capacity. Further studies will be required to understand the impact that this might have on pulp regeneration.

Dual Inhibition of Met Kinase and Angiogenesis to Overcome HGF-Induced EGFR-TKI Resistance in EGFR Mutant Lung Cancer

Acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a serious problem in the management of EGFR mutant lung cancer. We recently reported that hepatocyte growth factor (HGF) induces resistance to EGFR-TKIs by activating the Met/PI3K pathway. HGF is also known to induce angiogenesis in cooperation with vascular endothelial growth factor (VEGF), which is an important therapeutic target in lung cancer. Therefore, we hypothesized that dual inhibition of HGF and VEGF may be therapeutically useful for controlling HGF-induced EGFR-TKI-resistant lung cancer. We found that a dual Met/VEGF receptor 2 kinase inhibitor, E7050, circumvented HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer cell lines by inhibiting the Met/Gab1/PI3K/Akt pathway in vitro. HGF stimulated VEGF production by activation of the Met/Gab1 signaling pathway in EGFR mutant lung cancer cell lines, and E7050 showed an inhibitory effect. In a xenograft model, tumors produced by HGF-transfected Ma-1 (Ma-1/HGF) cells were more angiogenic than vector control tumors and showed resistance to gefitinib. E7050 alone inhibited angiogenesis and retarded growth of Ma-1/HGF tumors. E7050 combined with gefitinib induced marked regression of tumor growth. Moreover, dual inhibition of HGF and VEGF by neutralizing antibodies combined with gefitinib also markedly regressed tumor growth. These results indicate the therapeutic rationale of dual targeting of HGF-Met and VEGF-VEGF receptor 2 for overcoming HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer.

Neuropeptide Y stimulates VEGF production and secretion and promotes angiogenesis in murine and human breast cancer

The functional impact of NPY on breast cancer progression has been a question of growing interest. Our group has reported augmented mitogenic and migratory effects of NPY on breast cancer cells, however the anigogenic potential of NPY remains to be examined in models of breast cancer. In the current study we hypothesized that NPY promotes angiogenesis by stimulating the production and secretion of vascular endothelial growth factor (VEGF) from cancer cells. Endothelial tube formation assays (human umbilical vein endothelial cells: HUVEC) were conducted using control and NPY treated (10−11M to 10−7M for 24‐hr) conditioned media (CM) from 4T1 and MDA‐MB‐231 breast cancer cells. Compared to control, there was a concentration‐dependent increase in the number of endothelial tube branch‐points (BP) and endothelial cell networks (EN) in HUVEC exposed to NPY CM. The CM from 10−8 M NPY treatment induced the greatest angiogenic response, which resulted in a 2‐fold &amp; 5‐fold increase in number of BP (4T1 and MDA‐MB‐231 resp.) and 3‐ fold &amp; 21‐fold increase in EN (4T1 and MDA‐MB‐231 resp.). Congruently, NPY treatment caused a 108% increase in VEGF expression in cancer cells and a 65% increase in VEGF secretion. These data support our ongoing in vivo studies showing a correlation between NPY availability and tumor angiogenesis; thus, highlighting an additional pathological implication of NPY in breast cancer. NSERC &amp; CBCF.

Hypertonic Stress Induces VEGF Production in Human Colon Cancer Cell Line Caco-2: Inhibitory Role of Autocrine PGE2

Vascular Endothelial Growth Factor (VEGF) is a major regulator of angiogenesis. VEGF expression is up regulated in response to micro-environmental cues related to poor blood supply such as hypoxia. However, regulation of VEGF expression in cancer cells is not limited to the stress response due to increased volume of the tumor mass. Lipid mediators in particular arachidonic acid-derived prostaglandin (PG)E2 are regulators of VEGF expression and angiogenesis in colon cancer. In addition, increased osmolarity that is generated during colonic water absorption and feces consolidation seems to activate colon cancer cells and promote PGE2 generation. Such physiological stimulation may provide signaling for cancer promotion. Here we investigated the effect of exposure to a hypertonic medium, to emulate colonic environment, on VEGF production by colon cancer cells. The role of concomitant PGE2 generation and MAPK activation was addressed by specific pharmacological inhibition. Human colon cancer cell line Caco-2 exposed to a hypertonic environment responded with marked VEGF and PGE2 production. VEGF production was inhibited by selective inhibitors of ERK 1/2 and p38 MAPK pathways. To address the regulatory role of PGE2 on VEGF production, Caco-2 cells were treated with cPLA2 (ATK) and COX-2 (NS-398) inhibitors, that completely block PGE2 generation. The Caco-2 cells were also treated with a non selective PGE2 receptor antagonist. Each treatment significantly increased the hypertonic stress-induced VEGF production. Moreover, addition of PGE2 or selective EP2 receptor agonist to activated Caco-2 cells inhibited VEGF production. The autocrine inhibitory role for PGE2 appears to be selective to hypertonic environment since VEGF production induced by exposure to CoCl2 was decreased by inhibition of concomitant PGE2 generation. Our results indicated that hypertonicity stimulates VEGF production in colon cancer cell lines. Also PGE2 plays an inhibitory role on VEGF production by Caco-2 cells exposed to hyperosmotic stress through EP2 activation.

Carbon Monoxide Promotes VEGF Expression by Increasing HIF-1α Protein Level via Two Distinct Mechanisms, Translational Activation and Stabilization of HIF-1α Protein

Carbon monoxide (CO) plays a significant role in vascular functions. We here examined the molecular mechanism by which CO regulates HIF-1 (hypoxia-inducible transcription factor-1)-dependent expression of vascular endothelial growth factor (VEGF), which is an important angiogenic factor. We found that astrocytes stimulated with CORM-2 (CO-releasing molecule) promoted angiogenesis by increasing VEGF expression and secretion. CORM-2 also induced HO-1 (hemeoxygenase-1) expression and increased nuclear HIF-1α protein level, without altering its promoter activity and mRNA level. VEGF expression was inhibited by treatment with HIF-1α siRNA and a hemeoxygenase inhibitor, indicating that CO stimulates VEGF expression via up-regulation of HIF-1α protein level, which is partially associated with HO-1 induction. CORM-2 activated the translational regulatory proteins p70(S6k) and eIF-4E as well as phosphorylating their upstream signal mediators Akt and ERK. These translational signal events and HIF-1α protein level were suppressed by inhibitors of phosphatidylinositol 3-kinase (PI3K), MEK, and mTOR, suggesting that the PI3K/Akt/mTOR and MEK/ERK pathways are involved in a translational increase in HIF-1α. In addition, CORM-2 also increased stability of the HIF-1α protein by suppressing its ubiquitination, without altering the proline hydroxylase-dependent HIF-1α degradation pathway. CORM-2 increased HIF-1α/HSP90α interaction, which is responsible for HIF-1α stabilization, and HSP90-specific inhibitors decreased this interaction, HIF-1α protein level, and VEGF expression. Furthermore, HSP90α knockdown suppressed CORM-2-induced increases in HIF-1α and VEGF protein levels. These results suggest that CO stimulates VEGF production by increasing HIF-1α protein level via two distinct mechanisms, translational stimulation and protein stabilization of HIF-1α.