Vascular Endothelial Growth Factor A Secretion Research Articles

Neuropilin-1 expressing melanoma cells as a model to study the aggressiveness of metastatic melanoma

Background The molecular mechanisms associated with the acquisition of a metastatic phenotype by melanoma cells are not very well understood. Therefore, the identification of molecular determinants involved in the metastatic switch that may either cause or contribute to the aggressiveness of melanoma is of primary relevance. We had previously identified neuropilin-1 (NRP-1), a co-receptor of the vascular endothelial growth factor-A (VEGF-A), as an important determinant of melanoma aggressiveness, in clones of the human melanoma cell line M14, expressing or not NRP-1 [1,2]. We demonstrated that even though the simultaneous presence of both VEGFR-2 and NRP-1 potentiates VEGF-A secretion and the aggressiveness of melanoma cells, NRP-1 is by itself able to promote cell invasion [1]. During melanoma progression, tumour cells show increased adhesiveness to the vascular wall, invade the extracellular matrix (ECM) and frequently form functional channels similar to vascular vessels (vasculogenic mimicry) [3]. In the present study we analysed the mechanisms responsible for the aggressive phenotype of NRP-1 expressing melanoma cells. Materials and methods Melanoma aggressiveness was evaluated in vitro as cell ability to migrate through an ECM layer in Boyden chambers and to form tubule-like structures on matrigel gels. Pre-incubation of the cells with specific blocking antibodies allowed the identification of specific integrins and other molecules relevant to these processes. The results obtained by anti-integrin antibodies, showing the involvement of avb 5i ntegrin in the aggressiveness of melanoma cells expressing NRP-1, were confirmed by ITGB5 gene silencing and by the use of cilengitide, a potent inhibitor of aν integrins activation. Results

Cilengitide downmodulates invasiveness and vasculogenic mimicry of neuropilin 1 expressing melanoma cells through the inhibition of αvβ5 integrin.

During melanoma progression, tumour cells show increased adhesiveness to the vascular wall, invade the extracellular matrix (ECM) and frequently form functional channels similar to vascular vessels (vasculogenic mimicry). These properties are mainly mediated by the interaction of integrins with ECM components. Since we had previously identified neuropilin 1 (NRP-1), a coreceptor of vascular endothelial growth factor A (VEGF-A), as an important determinant of melanoma aggressiveness, aims of this study were to identify the specific integrins involved in the highly invasive phenotype of NRP-1 expressing cells and to investigate their role as targets to counteract melanoma progression. Melanoma aggressiveness was evaluated in vitro as cell ability to migrate through an ECM layer and to form tubule-like structures using transfected cells. Integrins relevant to these processes were identified using specific blocking antibodies. The αvβ5 integrin was found to be responsible for about 80% of the capability of NRP-1 expressing cells to adhere on vitronectin. In these cells αvβ5 expression level was twice higher than in low-invasive control cells and contributed to the ability of melanoma cells to form tubule-like structures on matrigel. Cilengitide, a potent inhibitor of αν integrins activation, reduced ECM invasion, vasculogenic mimicry and secretion of VEGF-A and metalloproteinase 9 by melanoma cells. In conclusion, we demonstrated that ανβ5 integrin is involved in the highly aggressive phenotype of melanoma cells expressing NRP-1. Moreover, we identified a novel mechanism that contributes to the antimelanoma activity of the αv integrin inhibitor cilengitide based on the inhibition of vasculogenic mimicry.

Targeting filamin B induces tumor growth and metastasis via enhanced activity of matrix metalloproteinase-9 and secretion of VEGF-A.

Filamins regulate cell locomotion and associate with diverse signaling molecules. We have recently found that targeting filamin A (FLNA) reduces RAS-induced lung adenocarcinomas. In this study, we explored the role of another major filamin isoform, filamin B (FLNB), in tumor development. In contrast to FLNA, we report that targeting FLNB enhances RAS-induced tumor growth and metastasis which is associated with higher matrix metallopeptidase-9 (MMP-9) and extracellular signal-regulated kinase (ERK) activity. Flnb deficiency in mouse embryonic fibroblasts results in increased proteolytic activity of MMP-9 and cell invasion mediated by the RAS/ERK pathway. Similarly, silencing FLNB in multiple human cancer cells increases the proteolytic activity of MMP-9 and tumor cell invasion. Furthermore, we observed that Flnb-deficient RAS-induced tumors display more capillary structures that is correlated with increased vascular endothelial growth factor-A (VEGF-A) secretion. Inhibition of ERK activation blocks phorbol myristate acetate-induced MMP-9 activity and VEGF-A secretion in vitro. In addition, silencing FLNB in human ovarian cancer cells increases secretion of VEGF-A that induces endothelial cells to form more vascular structures in vitro. We conclude that FLNB suppresses tumor growth and metastasis by regulating the activity of MMP-9 and secretion of VEGF-A which is mediated by the RAS/ERK pathway.

Visualising dual downregulation of insulin-like growth factor receptor-1 and vascular endothelial growth factor-A by heat shock protein 90 inhibition effect in triple negative breast cancer

PurposeTriple negative breast cancer (TNBC) is biologically characterised by heterogeneous presence of molecular pathways underlying it. Insulin-like growth factor receptor-1 (IGF-1R) expression and vascular endothelial growth factor-A (VEGF-A) have been identified as key factors in these pathways in TNBC. In this study, we aimed at in vivo PET imaging the effect of heat shock protein (Hsp) 90 inhibition by means of NVP-AUY922 on these pathways, with zirconium-89 (89Zr) labelled antibodies targeting IGF-1R and VEGF-A. Materials and methodsIn vitro NVP-AUY922 effects on cellular IGF-1R expression and VEGF-A secretion were determined in MCF-7 and MDA-MB-231 cell lines. Moreover human TNBC bearing MDA-MB-231 mice received 50mg/kg NVP-AUY922 or vehicle q3d intraperitoneally for 21days. PET scans with 89Zr-MAB391 and 89Zr-bevacizumab for visualisation of IGF-1R and VEGF-A were performed before and during treatment. Ex vivo biodistribution and correlative tissue analyses were performed. ResultsNVP-AUY922 treatment reduced IGF-1R expression and VEGF-A excretion in both cell lines. Hsp90 inhibition lowered tumour uptake on 89Zr-MAB391-PET by 37.3% (P<0.01) and on 89Zr-bevacizumab-PET by 44.4% (P<0.01). This was confirmed by ex vivo biodistribution with a reduction of 41.3% injected dose (ID)/g for 89Zr-MAB391 and 37.8% ID/g for 89Zr-bevacizumab, while no differences were observed for other tissues. This coincided with reduced IGF-1R expression and mean vessel density in the NVP-AUY922 treated tumours. Conclusion89Zr-MAB391 and 89Zr-bevacizumab PET reflect effect of Hsp90 inhibitors and can therefore potentially be used to monitor therapeutic effects of Hsp90 inhibitor therapy in TNBC.

Hypoxia inducible factor-1 is involved in growth factor, glucocorticoid and hypoxia mediated regulation of vascular endothelial growth factor-A in human meningiomas

In meningiomas, neovascularization through angiogenesis is essential for tumor expansion. As the vascular endothelial growth factor-A (VEGF-A) plays an outstanding role in this process, we have studied basal VEGF-A release and some aspects of its regulation in 46 meningiomas and in Ben-Men-1 cells in vitro. Among two putative VEGF-A stimulating growth factors tested, TGF-1β was more potent than TGF-α in enhancing VEGF-A secretion. Hypoxia-mimicking conditions induced by CoCl2 treatment also strongly increased VEGF-A secretion. The synthetic glucocorticoid dexamethasone (DEX) potently suppressed both basal and growth factor or CoCl2-induced VEGF-A release. All these effects were also seen in the Ben-Men-1 cell line in which studies on the role of HIF-1 in the regulation of VEGF-A showed that not only hypoxia but also the growth factors induced HIF-1α and DEX suppressed HIF-1α induction. Therefore, in Ben-Men-1 cells with HIF-1α knock-down the effects of hypoxia, growth factors and DEX on VEGF-A production were strongly impaired. This clearly indicates that HIF-1 not only regulates hypoxia-induced VEGF-A production but also mediates at least in part the effects of growth factors and DEX on VEGF-A synthesis and release. Our findings show the complexity of VEGF-A regulation in meningiomas and point to new options for the pharmacological treatment of these tumors.

SUMO-Conjugating Enzyme UBC9 Promotes Proliferation and Migration of Fibroblast-like Synoviocytes in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease with high morbidity and mortality. Fibroblast-like synoviocytes (FLS) in the synovial tissues play critical roles in joint destruction. Recent studies implicate the sumoylation in the regulation of the inflammation and arthritis. Thus, we explored whether SUMO-conjugating enzyme UBC9 is involved in the progression of RA using a mouse collagen-induced arthritis (CIA) model. The effects of UBC9 siRNA on cell invasion and migration in human RA-FLS were also assessed in vitro. Treatment with siRNA against UBC9 for 3weeks reduced the arthritis score and joint destruction. The expression of SUMO-1 and UBC9 protein in CIA joints was inhibited by UBC9 knockdown. Serum levels of anti-collagen (CII) antibodies, vascular endothelial growth factor A (VEGF-A), matrix metalloproteinases (MMP)-3, and MMP-9 were also decreased in CIA mice. In vitro, UBC9 silencing inhibited the secretion of VEGF-A, MMP-3, and MMP-9 from TNF-α-stimulated human RA-FLS. TNF-α-induced RA-FLS proliferation and migration were significantly attenuated by UBC9 knockdown. These findings indicate that SUMO-conjugating enzyme UBC9 promotes proliferation and migration of fibroblast-like synoviocytes in rheumatoid arthritis. Inhibition of UBC9 activity may be a viable therapeutic target in amelioration of disease progression in RA by attenuating FLS proliferation, migration, and invasion.

Aryl Hydrocarbon Receptor Antagonism Attenuates Growth Factor Expression, Proliferation, and Migration in Fibroblast-Like Synoviocytes from Patients with Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease with high morbidity and mortality. Within the inflammatory milieu, resident fibroblast-like synoviocytes (FLS) in the synovial tissue undergo hyperplasia, which leads to joint destruction. Epidemiologic studies and our previous research suggest that activation of the aryl hydrocarbon receptor (AHR) pathway plays an instrumental role in the inflammatory and destructive RA phenotype. In addition, our recent studies implicate the AHR in the regulation of the expression of several growth factors in established tumor cell lines. Thus, under inflammatory conditions, we hypothesized that the AHR is involved in the constitutive and inducible expression of several growth factors, FLS proliferation and migration, along with protease-dependent invasion in FLS from patients with RA (RA-FLS). Treatment with the AHR antagonist GNF351 inhibits cytokine-induced expression of vascular endothelial growth factor-A (VEGF-A), epiregulin, amphiregulin, and basic fibroblast growth factor mRNA through an AHR-dependent mechanism in both RA-FLS and FLS. Secretion of VEGF-A and epiregulin from RA-FLS was also inhibited upon GNF351 treatment. RA-FLS cell migration, along with cytokine-induced RA-FLS cell proliferation, was significantly attenuated by GNF351 exposure. Treatment of RA-FLS with GNF351 mitigated cytokine-mediated expression of matrix metalloproteinase-2 and -9 mRNA and diminished the RA-FLS invasive phenotype. These findings indicate that inhibition of AHR activity may be a viable therapeutic target in amelioration of disease progression in RA by attenuating growth factor release; FLS proliferation, migration, and invasion; and inflammatory activity.

Angiogenesis stimulated by human kallikrein‐related peptidase 12 actingviaa platelet‐derived growth factor B‐dependent paracrine pathway

KLK12, a kallikrein peptidase, is thought to take part in the control of angiogenesis. Our analysis of the secretome of endothelial cells (ECs) that had been treated with KLK12 showed that KLK12 converts the extracellular matrix- or membrane-bound precursor of platelet-derived growth factor B (PDGF-B) into a soluble form. Both PDGF-B and vascular endothelial growth factor A (VEGF-A) take part in the induction of angiogenesis by KLK12 in a coculture model of angiogenesis that mimics endothelial tubule formation. We used a cellular approach to analyze the interplay between KLK12, PDGF-B, and VEGF-A and showed that release of PDGF-B by KLK12 leads to the fibroblast-mediated secretion of VEGF-A. This then stimulates EC differentiation and the formation of capillary tube-like structures. Thus, KLK12 favors the interaction of ECs and stromal cells. The released PDGF-B acts as a paracrine factor that modulates VEGF-A secretion by stromal cells, which ultimately leads to angiogenesis. Moreover, the genes encoding KLK12 and PDGFB are both expressed in ECs and up-regulated in tumor cells kept under hypoxic conditions, which is consistent with the physiological involvement of KLK12 in PDGF-B maturation.

Abstract 4886: Caffeine normalizes breast stromal fibroblasts and suppresses their pro-carcinogenic effects.

Abstract Breast cancer is the leading cause of morbidity and second-leading cause of death in women worldwide. Breast stromal fibroblasts, the most abundant and the most reactive cells of the tumor stroma, actively promote tumor growth, invasion, migration, angiogenesis and metastasis through elevated secretion of several factors including SDF1, MMP-2 and vascular endothelial growth factor A (VEGF-A). These secretions are under the control of several tumor suppressor genes such as p16, p53 and CAV-1, which were fond to be down-regulated in most active fibroblasts. Therefore, our major aim in this study was to study the potential use of caffeine to up-regulate these tumor suppressor genes and hence reduces the secretion of these cancer-promoting factors. We have shown that low doses of caffeine up-regulate the tumor suppressor proteins p16, p21, p53 and CAV-1 in breast stromal fibroblasts. Consequently, the expression and secretion of MMP-2 and VEGF-A was reduced following caffeine treatment. Moreover, caffeine reduced the expression of SDF-1, TGF-β and α-SMA, three important markers of active fibroblast, suggesting that caffeine has the ability of “normalizing” stromal fibroblasts. To further confirm this, we have shown that caffeine suppresses stromal fibroblast-related enhancement of invasion/migration of breast cancer cells. Importantly, this effect was mediated through caffeine-dependent inactivation of the two major invasion/migration protein kinases Akt and Erk, through up-regulating PTEN in breast stromal fibroblasts. Furthermore, caffeine suppressed the expression/secretion of VEGF-A in breast stromal fibroblasts and their paracrine enhancement of angiogenesis in vitro. This effect was mediated through inhibition of the VEGF-A activator HIF-1α. Therefore, we have clearly shown here that caffeine, a natural and pharmacologically safe product, has the potential to normalize breast stromal fibroblasts and consequently suppresses their paracrine procarcinogenic effects. Citation Format: Mysoon M. Al Ansari, Abdelilah Aboussekhra. Caffeine normalizes breast stromal fibroblasts and suppresses their pro-carcinogenic effects. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4886. doi:10.1158/1538-7445.AM2013-4886

Time-Dependent Stabilization of Hypoxia Inducible Factor-1α by Different Intracellular Sources of Reactive Oxygen Species

Intratumoral hypoxia is a major obstacle in the development of effective cancer chemotherapy, decreasing the efficacy of anti-neoplastic drugs in several solid tumours. The hypoxic environment, through its master regulator hypoxia inducible factor-1 (HIF-1), is able to maintain an anti-apoptotic potential through activation of critical genes associated with drug resistance. Besides affecting metabolism and motility of tumour cells, hypoxia also paradoxically increases production of reactive oxygen species (ROS), which contribute to stabilize HIF-1 through a redox-mediated inhibition of its proteolysis. Here we reported that 1% O2 hypoxia increases the resistance of human metastatic melanoma cells to conventional chemotherapy with etoposide, and that the increase in chemoresistance strongly depends on ROS delivery due to hypoxia. We reported a biphasic redox-dependent role of HIF-1, involving mitochondrial complex III and NADPH oxidase as oxidants sources, synergising in enhancing survival to chemotherapy. The feed-forward loop engaged by hypoxia involves first an HIF-1-dependent vascular endothelial growth factor-A (VEGF-A) autocrine production and, in the later phase, activation of NADPH oxidase from VEGF/VEGFR2 interaction, finally leading to a further redox-dependent long lasting stabilization of HIF-1. We therefore identified a redox-dependent circuitry linking hypoxia-driven ROS to VEGF-A secretion and to enhanced melanoma cell survival to etoposide chemotherapy.

Curcumin suppresses HIF1A synthesis and VEGFA release in pituitary adenomas

Curcumin (diferuloylmethane), a polyphenolic compound derived from the spice plant Curcuma longa, displays multiple actions on solid tumours including anti-angiogenic effects. Here we have studied in rodent and human pituitary tumour cells the influence of curcumin on the production of hypoxia inducible factor 1α (HIF1A) and vascular endothelial growth factor A (VEGFA), two key components involved in tumour neovascularisation through angiogenesis. Curcumin dose-dependently inhibited basal VEGFA secretion in corticotroph AtT20 mouse and lactosomatotroph GH3 rat pituitary tumour cells as well as in all human pituitary adenoma cell cultures (n=32) studied. Under hypoxia-mimicking conditions (CoCl(2) treatment) in AtT20 and GH3 cells as well as in all human pituitary adenoma cell cultures (n=8) studied, curcumin strongly suppressed the induction of mRNA synthesis and protein production of HIF1A, the regulated subunit of the hypoxia-induced transcription factor HIF1. Curcumin also blocked hypoxia-induced mRNA synthesis and secretion of VEGFA in GH3 cells and in all human pituitary adenoma cell cultures investigated (n=18). Thus, curcumin may inhibit pituitary adenoma progression not only through previously demonstrated anti-proliferative and pro-apoptotic actions but also by its suppressive effects on pituitary tumour neovascularisation.

Vascular endothelial growth factor A, secreted in response to transforming growth factor-β1 under hypoxic conditions, induces autocrine effects on migration of prostate cancer cells

Hypoxia and transforming growth factor-β1 (TGF-β1) increase vascular endothelial growth factor A (VEGFA) expression in a number of malignancies. This effect of hypoxia and TGF-β1 might be responsible for tumor progression and metastasis of advanced prostate cancer. In the present study, TGF-β1 was shown to induce VEGFA(165) secretion from both normal cell lines (HPV7 and RWPE1) and prostate cancer cell lines (DU145 and PC3). Conversely, hypoxia-stimulated VEGFA(165) secretion was observed only in prostate cancer cell lines. Hypoxia induced TGF-β1 expression in PC3 prostate cancer cells, and the TGF-β type I receptor (ALK5) kinase inhibitor partially blocked hypoxia-mediated VEGFA(165) secretion. This effect of hypoxia provides a novel mechanism to increase VEGFA expression in prostate cancer cells. Although autocrine signaling of VEGFA has been implicated in prostate cancer progression and metastasis, the associated mechanism is poorly characterized. VEGFA activity is mediated via VEGF receptor (VEGFR) 1 (Flt-1) and 2 (Flk-1/KDR). Whereas VEGFR-1 mRNA was detected in normal prostate epithelial cells, VEGFR-2 mRNA and VEGFR protein were expressed only in PC3 cells. VEGFA(165) treatment induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in PC3 cells but not in HPV7 cells, suggesting that the autocrine function of VEGFA may be uniquely associated with prostate cancer. Activation of VEGFR-2 by VEGFA(165) was shown to enhance migration of PC3 cells. A similar effect was also observed with endogenous VEGFA induced by TGF-β1 and hypoxia. These findings illustrate that an autocrine loop of VEGFA via VEGFR-2 is critical for the tumorigenic effects of TGF-β1 and hypoxia on metastatic prostate cancers.

Highly Invasive Melanoma Cells Activate the Vascular Endothelium via an MMP-2/Integrin αvβ5–Induced Secretion of VEGF-A

Tumor cell extravasation is a critical step in the metastatic cascade and requires interaction between the tumor cell and the endothelium. Although cancer progression depends on a complex network of mechanisms, including inflammation and coagulation, the involvement of tumor-induced endothelium activation and the subsequent release of procoagulatory factors in this process are not well understood. Using tissue sections from patients with malignant melanoma, immunofluorescence studies for the presence of von Willebrand factor (VWF) clearly demonstrated endothelium activation and the formation of ultra-large VWF fibers in these patients. In vitro analyses revealed that supernatants from highly invasive melanoma cells induced an acute endothelium activation measured by VWF, P-selectin, and angiopoietin-2 release. Proteome profiling identified vascular endothelial growth factor A (VEGF-A) as the main mediator of endothelium activation. Inhibition and knock-down of VEGF-A in melanoma cells led to a rigorous decrease in VWF exocytosis. Selective small-interfering RNA to matrix metalloproteinase-2 (MMP-2) inhibited endothelium activation, and this effect correlated with reduced VEGF-A content in the supernatants of melanoma cells. Further experiments showed that active MMP-2 regulates VEGF-A in melanoma cells on a transcriptional level via an integrin αvβ5/phosphoinositide-3-kinase-dependent pathway. In conclusion, these results indicate an important role of VEGF-A in acute endothelium activation and provide clear evidence that MMP-2 plays a pivotal role in the autocrine regulation of VEGF-A expression in melanoma cells.

Abstract 2431: 89Zr-bevacizumab PET as an early biomarker for response to everolimus treatment in an ovarian cancer xenograft model

Abstract The mammalian target of rapamycin (mTOR) pathway is activated in the majority of ovarian cancers and is involved in tumor angiogenesis. Inhibitors of mTOR, like everolimus, are potentially interesting drugs as they can exert antitumor activity in part through reducing downstream vascular endothelial growth factor-A (VEGF-A) production. We investigated whether early effects of everolimus treatment could be monitored with 89Zr-bevacizumab positron emission tomography (VEGF-PET). Methods: The effect of everolimus on VEGF-A secretion was determined in three human ovarian cancer cell lines and in A2780luc+ ovarian cancer cells xenografted subcutaneously in BALB/c mice. Mice received daily everolimus (10 mg/kg intraperitoneally) for 14 days. PET scans with the tracer 89Zr-labeled bevacizumab were performed to monitor tumor VEGF-A expression before (baseline) and after treatment. Images were obtained 6 days after tracer injection. Tracer uptake was quantified and expressed as mean standardized uptake values (SUVmean). For ex vivo 89Zr-bevacizumab biodistribution and correlative tissue analyses, control animals were sacrificed after the baseline scans. Tumor VEGF-A levels were measured with ELISA in tumor lysates and mean vascular density (MVD) was determined with immunohistochemistry. Results: Everolimus treatment lowered VEGF-A levels in the supernatant of all cell lines. Everolimus lowered 89Zr-bevacizumab tumor uptake by 21.7 ± 4.0% (SUVmean 2.26 ± 0.18 versus 2.89 ± 0.20, p &amp;lt; 0.01). Ex vivo 89Zr-bevacizumab biodistribution showed less tracer uptake in the tumors of treated compared to control animals (7.78 ± 0.84 %ID/g versus 14.02 ± 1.68 %ID/g, p &amp;lt; 0.01), while no differences were observed for other tissues. VEGF-A protein levels in tumor lysates were lower in treated versus untreated tumors (p = 0.04), as was the MVD (p &amp;lt; 0.01). Conclusion: 89Zr-bevacizumab PET showed reduced tumor VEGF-A levels in vivo in response to everolimus therapy, coinciding with inhibition of tumor angiogenesis. Currently there are 2 clinical trials ongoing to study the value of 89Zr-bevacizumab PET to monitor tumor VEGF-A levels as an early biomarker of response to mTOR inhibitor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2431. doi:1538-7445.AM2012-2431

Distribution and Expression of Non-Neuronal Transient Receptor Potential (TRPV) Ion Channels in Rosacea

Rosacea is a frequent chronic inflammatory skin disease of unknown etiology. Because early rosacea reveals all characteristics of neurogenic inflammation, a central role of sensory nerves in its pathophysiology has been discussed. Neuroinflammatory mediators and their receptors involved in rosacea are poorly defined. Good candidates may be transient receptor potential (TRP) ion channels of vanilloid type (TRPV), which can be activated by many trigger factors of rosacea. Interestingly, TRPV2, TRPV3, and TRPV4 are expressed by both neuronal and non-neuronal cells. Here, we analyzed the expression and distribution of TRPV receptors in the various subtypes of rosacea on non-neuronal cells using immunohistochemistry, morphometry, double immunoflourescence, and quantitative real-time PCR (qRT-PCR) as compared with healthy skin and lupus erythematosus. Our results show that dermal immunolabeling of TRPV2 and TRPV3 and gene expression of TRPV1 is significantly increased in erythematotelangiectatic rosacea (ETR). Papulopustular rosacea (PPR) displayed an enhanced immunoreactivity for TRPV2, TRPV4, and also of TRPV2 gene expression. In phymatous rosacea (PhR)-affected skin, dermal immunostaining of TRPV3 and TRPV4 and gene expression of TRPV1 and TRPV3 was enhanced, whereas epidermal TRPV2 staining was decreased. Thus, dysregulation of TRPV channels also expressed by non-neuronal cells may be critically involved in the initiation and/or development of rosacea. TRP ion channels may be targets for the treatment of rosacea.

PEDF and VEGF-A Output from Human Retinal Pigment Epithelial Cells Grown on Novel Microcarriers

Human retinal pigment epithelial (hRPE) cells have been tested as a cell-based therapy for Parkinson's disease but will require additional study before further clinical trials can be planned. We now show that the long-term survival and neurotrophic potential of hRPE cells can be enhanced by the use of FDA-approved plastic-based microcarriers compared to a gelatin-based microcarrier as used in failed clinical trials. The hRPE cells grown on these plastic-based microcarriers display several important characteristics of hRPE found in vivo: (1) characteristic morphological features, (2) accumulation of melanin pigment, and (3) high levels of production of the neurotrophic factors pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor-A (VEGF-A). Growth of hRPE cells on plastic-based microcarriers led to sustained levels (>1 ng/ml) of PEDF and VEGF-A in conditioned media for two months. We also show that the expression of VEGF-A and PEDF is reciprocally regulated by activation of the GPR143 pathway. GPR143 is activated by L-DOPA (1 μM) which decreased VEGF-A secretion as opposed to the previously reported increase in PEDF secretion. The hRPE microcarriers are therefore novel candidate delivery systems for achieving long-term delivery of the neuroprotective factors PEDF and VEGF-A, which could have a value in neurodegenerative conditions such as Parkinson's disease.

Hypoxia Alters the Main Characteristics of the Hematopoietic Stem and Progenitor Cell Microenvironment in Vitro

Abstract 4803 Background:Hematopoietic stem and progenitor cells (HSPC) are located in a specialized microenvironment, called the stem cell niche, where their stem cell phenotype and differentiation are tightly regulated via interactions with the supporting mesenchymal stromal cells (MSC). These niches have been shown to be localized in regions with a lower oxygen tension which may also impact on the functional properties of MSC. For a better understanding to what extent hypoxia contributes to the establishment of an undifferentiated niche microenvironment that prevents inopportune differentiation of HSPC, we investigated MSC/HSPC co-cultures as well as MSC single cultures under low oxygen conditions. Design and Methods:Distribution, functional and phenotypical characteristics of CD34+ HSPC in hypoxic co-cultures (0.5% O2) were analyzed by flow cytometry. The effect of co-culture medium on the HSPC migration potential was tested in a transwell assay. The secretion of vascular endothelial growth factor A (VEGF-A), stromal-derived factor 1 (SDF-1), IL-6 and IL-8 by MSC was determined using ELISA whereas the expression of cell surface molecules was detected by flow cytometry. Moreover, the MSC proliferation as well as adipogenic and osteogenic differentiation was compared between hypoxic and normoxic culture conditions. Results:In the hypoxic co-culture, the adhesion of HSPC to the MSC layer was inhibited, whereas HSPC transmigration beneath the MSC layer was favoured. Increased VEGF-A secretion by MSC under hypoxic conditions, which enhanced the permeability of the MSC monolayer, was responsible for this effect. Furthermore, VEGF expression in hypoxic MSC was induced via hypoxia-inducible factor (HIF) signalling. Whereas IL-6 and IL-8 secretion were increased, SDF-1 expression by MSC was down-regulated under hypoxic conditions in a HIF-independent manner. The MSC immunophenotype which is characterized by expression of CD73, CD90, CD105, and CD166 was not significantly changed by hypoxia. Interestingly, a significant decrease of CD146 mRNA and protein expression levels was observed. The MSC proliferation was not significantly affected by lower oxygen tension. Culture of MSC in adipogenic induction medium for 14 days under hypoxia resulted in a reduced appearance of adipocyte-like cells containing lipid droplets and almost 50 % lower mRNA levels of fatty acid binding protein 2. The ALP activity as readout for osteogenic differentiation was decreased between 10% and 60% in hypoxic MSC. Conclusions:Low oxygen tension reduces the in vitro differentiation capacity and alters the cytokine secretion profile of primary human MSC. These functional changes may favour the homing and maintenance of quiescent HSC simulating the physiologically hypoxic niche conditions in vitro. Disclosures:No relevant conflicts of interest to declare.

Oxygen tension plays a critical role in the hematopoietic microenvironment in vitro

In the bone marrow mesenchymal stromal cells and osteoblasts form functional niches for hematopoietic stem and progenitor cells. This microenvironment can be partially mimicked using in vitro co-culture systems. In this study, we examined the oxygen tension in three distinct compartments in a co-culture system of purified CD34(+) cells and mesenchymal stromal cells with regard to different spatial localizations. Hypoxic cells in the co-culture were visualized by pimonidazole staining. Hematopoietic cell distribution, and functional and phenotypic characteristics were analyzed by flow cytometry. The secretion of vascular endothelial growth factor and stromal-derived factor-1 by mesenchymal stromal cells in low oxygen co-cultures was determined by an enzyme-linked immunosorbent assay. The effect of co-culture medium on the hematopoietic cell migration potential was tested in a transwell assay. In co-cultures under atmospheric oxygen tension, regions of low oxygen tension could be detected beneath the feeder layer in which a reservoir of phenotypically more primitive hematopoietic cells is located in vitro. In low oxygen co-culture, the adhesion of hematopoietic cells to the feeder layer was decreased, whereas hematopoietic cell transmigration beneath mesenchymal stromal cells was favored. Increased vascular endothelial growth factor-A secretion by mesenchymal stromal cells under low oxygen conditions, which increased the permeability of the monolayer, was responsible for this effect. Furthermore, vascular endothelial growth factor-A expression in low oxygen mesenchymal stromal cells was induced via hypoxia-inducible factor signaling. However, stromal cell-derived factor-1 secretion by mesenchymal stromal cells was down-regulated under low oxygen conditions in a hypoxia-inducible factor-independent manner. We demonstrate for the first time that differences in oxygen tension cause selective modification of hematopoietic cell and mesenchymal stromal cell interactions in a co-culture system, thus confirming that oxygen tension plays a critical role in the interaction between hematopoietic cells and the niche environment.

RSUME is implicated in HIF-1-induced VEGF-A production in pituitary tumour cells

The recently cloned small RWD-domain containing protein RSUME was shown to increase protein levels of hypoxia-inducible factor-1α (HIF-1α). The latter is the oxygen-regulated subunit of HIF-1, the most important transcription factor of the cellular adaptive processes to hypoxic conditions. It is also a major regulator of vascular endothelial growth factor-A (VEGF-A), which is critically involved in the complex process of tumour neovascularisation. In this study, the expression and role of RSUME in pituitary tumours was studied. We found that RSUME mRNA was up-regulated in pituitary adenomas and significantly correlated with HIF-1α mRNA levels. Hypoxia (1% O(2)) or treatment with hypoxia-mimicking CoCl(2) enhanced RSUME and HIF-1α expression, induced translocation of HIF-1α to the nuclei and stimulated VEGF-A production both in pituitary tumour cell lines and primary human pituitary adenoma cell cultures. When RSUME expression was specifically down-regulated by siRNA, the CoCl(2)-induced increase VEGF-A secretion was strongly reduced which was shown to be a consequence of the RSUME knockdown-associated reduction of HIF-1α synthesis. Thus, RSUME plays an important role in initiating pituitary tumour neovascularisation through regulating HIF-1α levels and subsequent VEGF-A production and may therefore be critically involved in pituitary adenoma progression.

Protein kinase D2 is a crucial regulator of tumour cell–endothelial cell communication in gastrointestinal tumours

BackgroundTumour angiogenesis is crucially dependent on the communication between the tumour and the associated endothelium. Protein kinase D (PKD) isoenzymes mediate vascular endothelial growth factor-A (VEGF-A) induced endothelial cell proliferation...