VEGF Synthesis Research Articles

CD147 expression was positively linked to aggressiveness and worse prognosis of gastric cancer: a meta and bioinformatics analysis

CD147 (also named as Basigin or EMMPRIN) might promote cancer invasion and metastasis by inducing MMP and VEGF synthesis in tumor microenvironment. We performed a systematic meta and bioinformatics analysis through multiple online databases up to March 14, 2017. Up-regulated CD147 expression was found in gastric cancer, compared with normal mucosa (p < 0.05). The male patients with gastric cancer showed higher CD147 expression than the female ones (p < 0.0001). CD147 expression was positively correlated with tumor size, depth of invasion, lymph node metastasis, TNM staging and unfavorable prognosis of gastric cancer (p < 0.05). At mRNA level, CD147 expression was higher in intestinal-type and mixed-type gastric carcinomas than normal tissues (p < 0.05). CD147 mRNA expression was negatively associated with histological grading and dedifferentiation of gastric cancer (p < 0.05). A higher CD147 mRNA expression was negatively correlated with overall and progression-free survival rates of all cancer patients, even stratified by clinicopathological features (p < 0.05). These findings indicated that CD147 expression might be employed as a potential marker to indicate gastric carcinogenesis and subsequent progression, even prognosis.

Hybrid hydrogels with high strength and biocompatibility for bone regeneration

The development of hydrogels for bone regeneration has highlighted the challenge that load-bearing hydrogels need to be biocompatible while achieving high strength. Several approaches have been reported to improve the toughness of hydrogels, but achieving high toughness and biocompatibility simultaneously remains a challenge. Here we report a polyacrylic acid/alginate/demineralized bone matrix (PAA/Alg/DBM) hybrid double network hydrogel, which was synthesized by a two-step sequential polymerization with embedded DBM, possessing both high strength and biocompatibility. With the persistence of DBM, it can promote the synthesis of VEGF and bFGF and the ALP activity of MG63 cells on hydrogel. All the results suggest that this hybrid double network hydrogel have potential for future application in bone regeneration.

Oxidative stress affects processing of amyloid precursor protein in vascular endothelial cells.

BackgroundOxidative stress is thought to be a key player in the pathogenesis of neurodegenerative dementia, including Alzheimer’s disease (AD). It has been assumed that oxidative stress contributes to the ß-amyloid deposition in cerebral blood vessels.MethodsIn order to prove this hypothesis, we examined the effect of oxidative stress on the processing of amyloid precursor protein (APP) in primary endothelial cells (EC) derived from cerebral cortical tissue of transgenic Tg2576 mice. Following exposure of EC by 1 μM hydrogen peroxide for up to 48 hours, formation and secretion of APP cleavage products sAPPα and sAPPß into the culture medium as well as the expression of endothelial APP were assessed.ResultsOxidative stress resulted in enhanced secretion of sAPPß into the culture medium as compared to controls (absence of hydrogen peroxide), which was accompanied by an increased APP expression, induction of VEGF synthesis, nitric oxide and oxygen free radicals productions, and differential changes of endothelial phospo-p42/44 MAPK expression.ConclusionThe data suggest that oxidative stress may represent a major risk factor in causing Aß deposition in the brain vascular system by initiating the amyloidogenic route of endothelial APP processing. The enhanced β-secretase activity following oxidative stress exposure, possibly promoted by phosphorylation of p42/44 MAPK.

Abstract P3-07-09: Synergistic effect of combinatorial treatment with maraviroc and tocilizumab on TNBC tumor growth and metastasis in mouse xenograft model

Abstract Triple negative breast cancer (TNBC) as a metastatic disease is currently incurable. Unfortunately reliable and reproducible methods for testing drugs against metastasis are not available. We have previously developed a robust metastatic model in which mice are pretreated with tumor cell-conditioned media (TCM) from human TNBC cells (MDA-MB-231 and SUM149) for 2 weeks prior to tumor cell inoculation. In this model we found reproducible metastases in lymph nodes (LN) and lungs within 4-5 weeks after orthotopic tumor inoculation [1]. We have discovered that the TNBC cells secrete large amounts of interleukin-6 (IL-6) that “educates” lymphatic endothelial cells (LEC) in the LN and lungs. Stat3, a transcription factor, gets activated and induces the synthesis of CCL5 and VEGF among other factors. CCL5 recruits the tumor cells to the LN and lungs; VEGF helps build blood vessels in the LN to facilitate tumor cell survival; VEGF produced in the lung helps the tumor cells extravasate into the lung. We have confirmed the importance of these factors by showing that inhibitors of these factors significantly inhibit metastasis. In this report, using Maraviroc (CCR5 inhibitor) and cMR16-1 Ab (murine surrogate of the anti-IL-6R antibody), we investigated the effect of the combination treatment on the tumor growth and metastasis of orthotopic tumor xenografts generated from MDA-MB-231-Luc-D3H2LN cells. 2x106 TNBC cells tagged with luciferase were suspended in 100 μl PBS/Matrigel (1:1) and injected s.c. into female 3- to 4-week-old BALB/c nu/nu athymic mice pretreated with tumor cell-conditioned media (TCM) from TNBC cells for 2 weeks. We administered the Maraviroc (8 mg/kg Maraviroc, orally daily) and cMR16-1 Ab (i.p. 3-days per week) for 5 weeks. Our data show that tumor growth was dramatically inhibited by cMR16-1 Ab. Further, the drug combination of Maraviroc with cMR16-1 Ab caused significant reduction of TNBC tumor growth in mice compared to single agents. In addition, we measured thoracic metastases by adding luciferin to mice and measuring luminescence ex-vivo in the IVIS imager. Significantly, both single treatment of Maraviroc and the combination of Maraviroc with cMR16-1 abrogated the thoracic metastasis compared to control and single treatment of cMR16-1. These findings implicate IL-6 and CCL5 signaling as a critical event in TNBC tumor growth and metastasis via crosstalk between cancer cells and stromal components. Further, these studies suggest that IL-6 and CCL5 act as key regulators orchestrating TNBC metastatic breast cancer. Therefore, we have provided evidence that supports the hypothesis that functional inhibition of the IL-6 and CCL-5 signaling pathway has the potential to circumvent TNBC growth and metastasis. [1] E Lee et al. Breast cancer cells condition lymphatic endothelial cells within pre-metastatic niches to promote metastasis. Nat Commun. 2014 5:4715. Citation Format: Jin K, Popel AS. Synergistic effect of combinatorial treatment with maraviroc and tocilizumab on TNBC tumor growth and metastasis in mouse xenograft model [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-09.

Immunohistochemical Markers of Angiogenesis and Apoptosis in Chorionic Villi during Early Pregnancy Miscarriage

Objective: Aim of this study was to investigate changes in the expression of immunohistochemical markers of angiogenesis (VEGF) and apoptosis (Caspase 3) in chorionic villi of human embryos, obtained after sporadic, recurrent pregnancy miscarriage and compare them with such expression in normal pregnancy Chorionic villie. Methods: Histological material included 63 recurrent, sporadic miscarriage and 32 artificial abortions chorionic villi samples. Immunohistochemical technique was applied to study expression levels of VEGF and Caspase 3. Results: VEGF and Caspase 3 expression intensity and diversity in different Chorionic villie (CV) structural components was observed in different investigated groups. Conclusion: Our research has shown that an important role is played not only by a general decrease in VEGF expression but also a change in the allocation of this cytokine in the structural components of early pregnancy miscarriage CV. The processes of apoptosis were more active in the groups of early miscarriage; however, they did not block the synthesis of VEGF. These results may indicate the role of factors of angiogenesis and apoptosis in the development of early miscarriage of pregnancy.

Hypericin targets multiple signaling mediators in cancer cells generating unique, diverse anti-tumoral, anti-metastatic, and anti-angiogenic activities with evidence for clinical applicability

The goals of this review are to comprehensively analyze the diverse biological activities displayed by a most potent photodynamic agent–hypericin. Hypericin is a lipophilic redox-reactive molecule possessing a redox potential low enough to act as electron acceptor, subsequently discharging these electrons to oxygen, generating ROS. This property enables intracellular hypericin to retain redox activities in the dark. In cells hypericin sequesters in endoplasmic reticulum and Golgi apparatus membranes and photo-oxidizes membranal lipoproteins. However, the most relevant cytosolic hypericin target is the Hsp90 chaperone. We have shown that hypericin selectively binds to and oxidizes Hsp90, inducing its forced polyubiquitiylation, functional inactivation and rapid degradation in a proteasome independent manner. Hsp90 physiological association with a myriad of client proteins is disrupted and several signaling mediators, cell cycling and proliferation regulators are destabilized and degraded. Secondarily affected cell cycle checkpoints cause uneven, premature mitosis, (karyokinesis with no cytokinesis), forming polykaryonic giant cells, hallmark of mitotic catastrophe also known as mitotic cell death. HIF-1a, the master regulator of VEGF synthesis and angiogenesis inducer is also an Hsp90 client protein. HIF-1a is physiologically degraded by oxygen but also by the hypericin-induced Hsp90 ablation, inducing potent tumor neoangiogenesis inhibition. Hsp90 is implicated in mediating inheritable epigenetic modifications, causing epigenetic signature changes in key developmentally regulated genes and tumor cell exit from proliferation cycles. Expression of EZH2, the Polycomb repressor complex-2 catalytic subunit, which trimethylates histone H3lys27 is suppressed, class-I HDACs expression downregulated and HDAC1-Dnmt1-EZH2 complex formations diminish. Deficiencies in HDACs cellular contents lead to histones H3 and H4 hyperacetylation, which together with diminished H3K27-trimethylation relax chromatin structure, activating transcription including of differentiation-promoting genes. In GBM cells neuroglial differentiation antigens are expressed, cytoarchitecture modulated and the cells undergo tumor cell differentiation. Indeed, clinically significant anti-GBM effects were obtained in a clinical trial in recurrent, progressive GBM patients.

Ginkgolide B enhances the differentiation of preosteoblastic MC3T3-E1 cells through VEGF: Involvement of the p38 MAPK signaling pathway.

Ginkgolide B (GB) is one of the ginkgolides isolated from the leaves of the Ginkgo biloba tree. Our previous study indicated that GB promotes the proliferation, migration and adhesion of endothelial progenitor cells, and the induction of angiogenesis through vascular endothelial factor (VEGF). In the present study, the effects of GB on the differentiation of MC3T3‑E1 cells and the signaling pathway involved were investigated invitro. The MC3T3‑E1 cell viability activities were assessed using an MTS assay. Measurements of alkaline phosphatase activity and Alizarin Red staining were used to identify osteoblastic differentiation of the MC3T3‑E1 cells. The mRNA and secretion levels of VEGF were detected using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis and enzyme-linked immunosorbent assays, respectively. The protein expression levels of phosphorylation‑associated markers were detected using western blot analysis and associated gene expression was determined using RT‑qPCR analysis. It was found that GB significantly promoted alkaline phosphatase activity and osteoblastic mineralization in the MC3T3‑E1 cells. In addition, the mRNA expression and secretion levels of VEGF in the MC3T3‑E1 cells were significantly increased in MC3T3‑E1 cells treated with GB. SB203580, a specific inhibitor of p38 mitogen‑activated protein (MAP) kinase, markedly suppressed the GB‑induced p38 kinase phosphorylation and GB‑induced synthesis of VEGF. PD98059, an inhibitor of the upstream kinase, which activates p44/p42 MAP kinase, had minimal effect on the GB‑induced phosphorylation of p44/p42 MAP kinase or the GB‑induced synthesis of VEGF. Taken together, these results indicated that GB promoted osteoblastic differentiation of the MC3T3‑E1 cells through VEGF, and that the p38, but not the p44/p42 MAP kinase signaling pathway, was involved in the GB‑induced synthesis of VEGF.

P062 &lt;break /&gt; VEGF synthesis in distal lung fibroblasts from COPD patients and healthy control subjects; implications for pulmonary vascular remodelling

P062 &lt;break /&gt; VEGF synthesis in distal lung fibroblasts from COPD patients and healthy control subjects; implications for pulmonary vascular remodelling

Abstract 4084: Crosstalk between TNBC and stromal components via secreted factors enhances cell motility that can be attenuated by a CXCR1 inhibitor

Abstract Triple negative breast cancer (TNBC) as a metastatic disease is currently incurable. Unfortunately reliable and reproducible methods for testing drugs against metastasis are not available. We have previously developed a robust metastatic model in which mice are pretreated with tumor cell-conditioned media (TCM) from human TNBC cells (MDA-MB-231 and SUM149) for 2 weeks prior to tumor cell inoculation. In this model we found reproducible metastases in lymph nodes (LN) and lungs within 4-5 weeks after orthotropic tumor inoculation. We have discovered that the TNBC tumor cells secrete large amounts of interleukin-6 (IL-6) that “educates” lymphatic endothelial cells (LEC) in the LN and lungs. Stat3, a transcription factor, gets activated and induces the synthesis of CCL5 and VEGF among other factors. CCL5 recruits the tumor cells to the LN and lungs; VEGF helps build blood vessels in the LN to facilitate tumor cell survival; VEGF produced in the lung helps the tumor cells extravasate into the lung. We have confirmed the importance of these factors by showing that inhibitors of these factors significantly inhibit metastasis. In this report, using a human antibody array, we identified factors secreted by fibroblasts (HGF, IL-6, IL-8, CCL7, MIF, GDF-15, EMMPRIN and VEGF) and by macrophages (CXCL5, IL-8 and uPAR) upon induction by MDA-MB-231 TCM. We ranked the expression level of each factor by real time qRT-PCR and determined that interleukin 8 (IL-8) was the top candidate. We confirmed by ELISA that IL-8 secreted from either fibroblasts or macrophages treated with MDA-MB-231 TCM was upregulated compared to treatment with serum free media (SFM). Our data showed that the proliferation of MDA-MB-231 cells incubated with conditioned media (CM) from TCM-induced fibroblasts or TCM-induced macrophages was enhanced compared to SFM treatment. Furthermore, MDA-MB-231 cell migration, a key step in tumor metastasis, was promoted by CM from TCM-induced fibroblasts or macrophages. Significantly, inhibition of the IL-8 signaling pathway by Reparixin, an inhibitor of the IL-8 receptor CXCR1, abrogated the upregulation of MDA-MB-231 cell proliferation and migration induced by TCM-induced fibroblasts or macrophages. These findings implicate IL-8 signaling as a critical event in TNBC cell proliferation and migration via crosstalk with stromal components. Further, these studies suggest that IL-8 acts as a key regulator orchestrating TNBC metastatic breast cancer. Therefore, we have provided evidence that supports the hypothesis that functional antagonism of the IL-8 signaling pathway has the potential to circumvent TNBC breast cancer growth and metastasis. Citation Format: Kideok Jin, Niranjan B. Pandey, Aleksander S. Popel. Crosstalk between TNBC and stromal components via secreted factors enhances cell motility that can be attenuated by a CXCR1 inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4084.

Effects of estradiol on VEGF and bFGF by Akt in endometrial cancer cells are mediated through the NF-κB pathway.

Endometrial carcinogenesis may be related to the long-term effects of estradiol with no antagonism. However, how estradiol regulates cell proliferation is unknown. In the present study, through investigating the molecular events involved in estradiol induced angiogenics factors VEGF and bFGF, we found that estradiol induced endometrial cancer cell division, proliferation, migratory and invasive capacity invitro and upregulated mRNA expression and protein synthesis of VEGF and bFGF. The estradiol-dependent induction of the expression of VEGF and bFGF was blocked by ER inhibitor, AKT inhibitor and NF-κB inhibitor (PDTC) in estrogen receptor positive Ishikawa cells and blocked by AKT inhibitor, NF-κB inhibitor (PDTC) in estrogen receptor negative HEC-1A cells. Moreover, estradiol activation of AKT was also blocked by AKT antagonist. NF-κB activation was restricted by estradiol concentration and time. Estradiol leading to VEGF and bFGF induction was also confirmed by the development of xenograft tumors invivo. Taken together, our data suggest that estradiol induces the production of angiogenic factors via a mechanism involving AKT-mediated NF-κB activation partly in non-genomic manner without the estrogen receptor.

Gonadotropin and tumorigenesis: Direct and indirect effects on inflammatory and immunosuppressive mediators and invasion.

Human chorionic gonadotropin (hCG), a hormone essential for pregnancy, is also ectopically expressed by a variety of cancers and is associated with poor prognosis; molecular mechanisms which may contribute to tumor progression remain ill-defined. Exogenous hCG enhanced the viability of human colorectal and lung cancer cells and promoted the growth of syngeneic tumors in mice. It induced the synthesis of VEGF, IL-8, matrix metalloprotease (MMP)-2 and MMP-9, and increased invasiveness in an MMP-dependent manner. While inducing the secretion of the tumor-associated extra-cellular matrix proteoglycan versican from tumor cells, hCG consequently caused the TLR-2-mediated generation of the inflammatory, tumor-associated cytokines TNF-α and IL-6 from peripheral blood adherent cells. The molecule up-modulated the Treg-associated transcription factor FOXP3 in tumor cells and increased the secretion of TGFβ and IL-10, thereby inhibiting T cell proliferation and inducing the differentiation FOXP3- CD4+ CD25- cells into functional FOXP3+ CD4+ CD25+ suppressor cells. Co-culture of hCG-treated tumor cells with mature bone-marrow derived dendritic cells induced the generation of active indoleamine deoxygenase. While anti-hCG antibodies restricted the growth of implanted tumor cells in nude mice, immunization of immune competent mice with a βhCG-TT conjugate supplemented with Mycobacterium indicus pranii provided synergistic survival benefit in animals implanted with syngeneic, hCG-responsive tumor cells. These studies elucidate the pathways by which hCG can promote tumorigenesis, providing further rationale for anti-hCG vaccination in the treatment of gonadotropin-sensitive tumors. © 2016 Wiley Periodicals, Inc.

Delivery of mesenchymal stem cells in biomimetic engineered scaffolds promotes healing of diabetic ulcers.

We hypothesized that delivery of mesenchymal stem cells (MSCs) in a biomimetic collagen scaffold improves wound healing in a diabetic mouse model. Rolled collagen scaffolds containing MSCs were implanted or applied topically to diabetic C57BL/6 mice with excisional wounds. Rolled scaffolds were hypoxic, inducing MSC synthesis and secretion of VEGF. Diabetic mice with wounds treated with rolled scaffolds containing MSCs showed increased healing compared with controls. Histologic examination showed increased cellular proliferation, increased VEGF expression and capillary density, and increased numbers of macrophages, fibroblasts and smooth muscle cells. Addition of laminin to the collagen scaffold enhanced these effects. Activated MSCs delivered in a biomimetic-collagen scaffold enhanced wound healing in a translationally relevant diabetic mouse model.

Pursuing a novel bioactive dressing: stimulation of dermal fibroblasts with calcium-phosphate nanoparticles

Event Abstract Back to Event Pursuing a novel bioactive dressing: stimulation of dermal fibroblasts with calcium-phosphate nanoparticles Claudia Navarro-Requena1, 2, Soledad Pérez-Amodio1, 2, Óscar Castaño1, 2, 3 and Elisabeth Engel1, 2, 3 1 Institute for Bioengineering of Catalonia, Spain 2 CIBER de Bioingeniería, Biomateriales y Nanomedicina, Spain 3 Technical University of Catalonia, Materials Science and Metallurgical Engineering, Spain Introduction: Hard-to-heal wounds are a major socio-economic problem in developed countries[1]. In the recent years, bioactive dressings have been developed, mainly based in the addition of biological factors. However, these present significant limitations, such as high cost of production or regulatory challenges. Extracellular calcium could be considered as an alternative biostimulator, since it has been probed to affect the healing process of skin injuries[2],[3]. However, a better understanding of its effect is needed, together with a system to achieve a controlled release. In this study we tested whether the ionic dissolution of calcium-releasing nanoparticles achieved a similar stimulating effect as extracellular calcium on dermal fibroblast in vitro. Materials and Methods: Rat dermal fibroblasts were cultured in the presence of medium containing different calcium concentrations, normally ranging from 0.1 to 3.5mM Ca2+. Before adding it to the cells, media was incubated with calcium chloride(CaCl2) or with calcium-phosphate nanoparticles (CaP) for 24h at 37ºC and filter sterilized. Several relevant aspects in the wound healing process were assessed by different in vitro assays. Metabolic activity was determined with the Alamarblue® kit. Proliferation was measured by DNA quantification. Cell migration was quantified by a wound scratch assay. Total collagen synthesis was determined by means of a commercial kit. Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) levels of activity were analyzed by a zymography assay. Finally, VEGF synthesis was measured with an ELISA. Results and Discussion: The effects stimulated by calcium from media conditioned with CaP did not exactly correlate with the one from CaCl2. This means that the other factors that the CaP are releasing, such as phosphates, are also affecting the cells. Interestingly, 3.5mM from both CaCl2 and CaP enhanced metabolic activity, in vitro wound closure and collagen synthesis. However, CaP decreased MMP activity, proliferation and VEGF synthesis. Conclusions: Even though calcium coming from CaP did not have the same effect as CaCl2 on dermal fibroblasts, the concentration of 3.5mM Ca2+ from CaP was able to enhance metabolic activity, migration, collagen synthesis and to decrease MMP activity. All these effects might promote a faster healing. A better understanding of how other cell types involved in the healing process respond to a range of concentrations of calcium-phosphate particles may lead to a wiser design of novel bioactive dressings for skin wound healing. FPU grant ref. AP-2012-5310; MAT2012-38793

The twisted survivin connection to angiogenesis.

Survivin, a member of the inhibitor of apoptosis family of proteins (IAPs) that controls cell division, apoptosis, metastasis and angiogenesis, is overexpressed in essentially all human cancers. As a consequence, the gene/protein is considered an attractive target for cancer treatment. Here, we discuss recent findings related to the regulation of survivin expression and its role in angiogenesis, particularly in the context of hypoxia. We propose a novel role for survivin in cancer, whereby expression of the protein in tumor cells promotes VEGF synthesis, secretion and angiogenesis. Mechanistically, we propose the existence of a positive feed-back loop involving PI3-kinase/Akt activation and enhanced β-Catenin-TCF/LEF-dependent VEGF expression followed by secretion. Finally, we elaborate on the possibility that this mechanism operating in cancer cells may contribute to enhanced tumor vascularization by vasculogenic mimicry together with conventional angiogenesis.

Effect of ovine luteinizing hormone (oLH) charge isoforms on VEGF and cAMP production

Although an increase in VEGF expression and synthesis in association with LH has been established; it is unknown if all LH isoforms act similarly. This study evaluated the production of cAMP and VEGF among LH isoforms in two in vitro bioassays. The LH was obtained from hypophyses and the group of isoforms was isolated by chromatofocusing. cAMP production was assessed using the in vitro bioassay of HEK-293 cells and VEGF production was evaluated in granulosa cells. Immunological activity was measured with a homologous RIA. Immunoactivity and bioactivity for each isoform were compared against a standard, by estimating the IC50 and the EC50. The basic isoforms were more immunoactive than the standard. The neutral and the moderately acidic had an immunological activity similar to the standard. The acidic isoform was the least immunoreactive. cAMP production at the EC50 dose was similar among the basic isoforms, the moderately acidic and the standard; for the neutral and the acidic, the EC50 dose was higher. It was observed that compared with the control, VEGF production at the lowest LH dose was no different in the standard and each isoform. In the intermediate dose, a positive response was caused in the standard and the neutral and basic isoforms. Although the acidic isoform showed a dose-dependent response, it was not significant relative to the control. In conclusion, the basic isoform generated the greatest cAMP and VEGF production, similar to the reference standard, and the acidic the smallest.

Leptin enhances ICAM-1 expression, induces migration and cytokine synthesis, and prolongs survival of human airway epithelial cells.

There is rising interest in how obesity affects respiratory diseases, since epidemiological findings indicate a strong relationship between the two conditions. Leptin is a potent adipokine produced mainly by adipocytes. It regulates energy storage and expenditure and also induces inflammation. Previous studies have shown that leptin is able to activate inflammatory cells such as lymphocytes and granulocytes, but little is known about its effect on lung structural cells. The present study investigated the effects of leptin on human airway epithelial cells by using human primary airway epithelial cells and a human airway epithelial cell line, BEAS-2B. Flow cytometry showed enhanced ICAM-1 expression by both of those cells in response to leptin, and that effect was abrogated by dexamethasone or NF-κB inhibitor. Flow cytometry and quantitative PCR showed that airway epithelial cells expressed leptin receptor (Ob-R), whose expression level was downregulated by leptin itself. Multiplex cytokine analysis demonstrated enhanced production of CCL11, G-CSF, VEGF, and IL-6 by BEAS-2B cells stimulated with leptin. Furthermore, transfection of Ob-R small interference RNA decreased the effect of leptin on CCL11 production as assessed by quantitative PCR. Finally, leptin induced migration of primary airway epithelial cells toward leptin, suppressed BEAS-2B apoptosis induced with TNF-α and IFN-γ, and enhanced proliferation of primary airway epithelial cells. In summary, leptin was able to directly activate human airway epithelial cells by binding to Ob-R and by NF-κB activation, resulting in upregulation of ICAM-1 expression, induction of CCL11, VEGF, G-CSF, and IL-6 synthesis, induction of migration, inhibition of apoptosis, and enhancement of proliferation.

Abstract 4166: Targeting NF-κB by the curcumin and its analogs EF-31 and UBS-109 decreases growth and angiogenesis of colon cancer

Abstract Purpose/Objective: NF-κB plays an essential role in cancer cell growth and metastasis by stimulating angiogenesis. Curcumin decreases the activity of several oncogenic transcriptional factors including NF-κB and HIF-1α, thus we investigated whether curcumin and its analogs EF-31 and UBS-109, could disrupt angiogenesis using colorectal cancer cells (CRC). Methods: HCT-116 and HT-29 cells were used in these experiments. HUVEC tube formation assay, Matrigel plug assay, Western blotting, and VEGF activity assay were carried out to determine the curcumin, EF-31 and UBS-109 role in angiogenesis. A subcutaneous xenograft mouse model was used to evaluate the in vivo effects of curcumin analogues. Results: Conditioned medium from HCT-116 or H-T29 cells exposed to curcumin, EF-31 and UBS-109 in vitro significantly blocked HUVEC tube assembly in comparison to control. In vivo, EF-31 and UBS-109 blocked the vascularization of subcutaneous matrigel plugs and the growth of CRC xenografts. We observed significant inhibition of VEGF synthesis and secretion in both colon cell lines treated with curcumin, EF-31 and UBS-109 in concert with the loss of HIF-1α expression, of which transcriptionally regulated by NF-κB. Similar effects were observed in tumors from animals treated with EF-31 and UBS-109. Further, EF-31 and UBS-109 treatment significantly decreased the tumor growth compared to untreated tumors. These results suggesting that curcumin, EF-31 and UBS-109 inhibits VEGF production in part through the degradation of HIF-1α and increased the cytoplasmic accumulation of NF-κB. Conclusion: Taken together, destabilization of HIF-1α and inhibition of NF-κB from cytoplasm to nuclear translocation may be important contributing factors to the antiangiogenic action of curcumin, EF-31 and UBS-109 in CRC. Note: This abstract was not presented at the meeting. Citation Format: Ganji Purnachandra Nagaraju, Mamoru Shoji, Bassel El Rayes. Targeting NF-κB by the curcumin and its analogs EF-31 and UBS-109 decreases growth and angiogenesis of colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4166. doi:10.1158/1538-7445.AM2015-4166

Crosstalk between platelets and PBMC: New evidence in wound healing

Platelet-derived products have proven useful in accelerating healing processes and tissue regeneration. However, despite their widespread use in clinical practice, the cellular and molecular mechanisms involved have not yet been completely clarified. Recent studies show that interaction between platelet gel (PG) and peripheral blood mononuclear cells (PBMC) can result in activation of PBMC and production of several cytokines involved in wound healing and tissue repair. The aim of our study was to analyze whether crosstalk between platelets and PBMC can influence wound healing by modulating release of VEGF, bFGF and IL-10 by PBMC. Cultures of PBMC alone and co-cultures with autologous PG of 24 healthy volunteers were incubated under normoxia for 24 h. VEGF, bFGF and IL-10 concentration and expression were then analyzed in supernatants by ELISA and by real-time RT-PCR. We observed a down-regulation of VEGF and bFGF release and an up-regulation of IL-10 release in co-cultures of PBMC and PG. Platelets are not only important in the early stages of the healing process (clot formation, direct release of growth factors), but also can influence the whole process of tissue regeneration by modulating synthesis and release of VEGF, bFGF and IL-10 by PBMC. These effects could give platelets a new key role in the control of healing processes and provide insights into the clinical success of platelet-derived products in many medical fields.

Recombinant Human Keratinocyte Growth Factor Induces Akt Mediated Cell Survival Progression in Emphysematous Mice

IntroductionEmphysema has been associated with decreased VEGF and VEGFR-2 expression and the presence of high numbers of apoptotic alveolar cells. Keratinocyte growth factor stimulates VEGF synthesis which in turn confers normal lung structure maintenance via the Akt pathway. In this study the potential role of rHuKGF in the improvement of deregulated Akt mediated cell survival pathway in emphysematous mice was investigated. MethodsThree experimental groups, i.e., emphysema, treatment and control groups, were prepared. Lungs of mice were treated on 3 occasions by oropharyngeal instillation of 10mg rHuKGF per kg body weight after induction of emphysema with porcine pancreatic elastase. Subsequently, lung tissues from mice were collected for histopathology and molecular biology studies. Results and discussionHistopathology photomicrographs and destructive index analysis have shown that elastase-induced airspace enlargement and loss of alveoli recovered in the treatment group. rHuKGF stimulates VEGF production which in turn induces the Akt mediated cell survival pathway in emphysematous lungs. mRNA expression of VEGF, VEGFR, PI3K and Akt was significantly increased while Pten, Caspase-9 and Bad was notably decreased in treatment group when compared with emphysema group, being comparable with the control group. Moreover, VEGF protein expression was in accordance with that found for mRNA. ConclusionTherapeutic rHuKGF supplementation improves the deregulated Akt pathway in emphysema, resulting in alveolar cell survival through activation of the endogenous VEGF-dependent cell survival pathway. Hence rHuKGF may prove to be a potential drug in the treatment of emphysema.

The tyrosine phosphatase SHP-1 regulates hypoxia inducible factor-1α (HIF-1α) protein levels in endothelial cells under hypoxia.

IntroductionThe tyrosine phosphatase SHP-1 negatively influences endothelial function, such as VEGF signaling and reactive oxygen species (ROS) formation, and has been shown to influence angiogenesis during tissue ischemia. In ischemic tissues, hypoxia induced angiogenesis is crucial for restoring oxygen supply. However, the exact mechanism how SHP-1 affects endothelial function during ischemia or hypoxia remains unclear. We performed in vitro endothelial cell culture experiments to characterize the role of SHP-1 during hypoxia.ResultsSHP-1 knock-down by specific antisense oligodesoxynucleotides (AS-Odn) increased cell growth as well as VEGF synthesis and secretion during 24 hours of hypoxia compared to control AS-Odn. This was prevented by HIF-1α inhibition (echinomycin and apigenin). SHP-1 knock-down as well as overexpression of a catalytically inactive SHP-1 (SHP-1 CS) further enhanced HIF-1α protein levels, whereas overexpression of a constitutively active SHP-1 (SHP-1 E74A) resulted in decreased HIF-1α levels during hypoxia, compared to wildtype SHP-1. Proteasome inhibition (MG132) returned HIF-1α levels to control or wildtype levels respectively in these cells. SHP-1 silencing did not alter HIF-1α mRNA levels. Finally, under hypoxic conditions SHP-1 knock-down enhanced intracellular endothelial reactive oxygen species (ROS) formation, as measured by oxidation of H2-DCF and DHE fluorescence.ConclusionsSHP-1 decreases half-life of HIF-1α under hypoxic conditions resulting in decreased cell growth due to diminished VEGF synthesis and secretion. The regulatory effect of SHP-1 on HIF-1α stability may be mediated by inhibition of endothelial ROS formation stabilizing HIF-1α protein. These findings highlight the importance of SHP-1 in hypoxic signaling and its potential as therapeutic target in ischemic diseases.