VEGF Release Research Articles

Picea mariana polyphenolic extract inhibits phlogogenic mediators produced by TNF-α-activated psoriatic keratinocytes: Impact on NF-κB pathway

Ethnopharmacological relevancePicea mariana ((Miller) Britton, Sterns, and Poggenburg; Pinaceae) bark has been traditionally used by North American natives for treating topical inflammations. It has been also suggested to improve various inflammatory skin disorders like Psoriasis vulgaris. Extracts from this bark storage protein contain polyphenolic compounds which have well-known antiinflammatory activities. Based on the capacity of polyphenolic compounds to modulate functions of normal human keratinocytes, this study was set up to decipher the mechanisms of action of a chemically characterized polyphenolic extract from Picea mariana bark (BS-EAcf) on lesional keratinocytes of skin with psoriasis vulgaris, a disease driven by the immune system in which TNF-α plays a significant role. Materials and methodsBS-EAcf corresponds to the ethyl acetate soluble fraction from the hot water extract of Picea mariana bark. BS-EAcf effects were evaluated in normal human (NHK) and psoriatic (PK) keratinocytes stimulated by TNF-α. Cell viability was assessed by lactate deshydrogenase release and propidium iodide (PI) staining. The mechanisms of action of BS-EAcf in keratinocytes were investigated by flow cytometry, ELISAs, RT-PCR and western blot analyses. ResultsPK exhibited a higher response to TNF-α than NHK regarding the ICAM-1 expression and the production of NO, IL-6, IL-8, fractalkine and PGE2, whereas BS-EAcf significantly inhibited this TNF-α-induced increase at concentrations without causing keratinocyte toxicity. Additionally, this extract significantly inhibited the TNF-α-induced release of elafin and VEGF by PK and NHK. Since TNF-α activation of most of these factors is dependent on the NF-κB pathway, this latter was studied in TNF-α-activated PK. BS-EAcf inhibited the TNF-α-induced phosphorylation and degradation of total IκBα as well as phosphorylation of NF-κB p65. ConclusionsThe ethyl acetate fraction from Picea mariana bark extract showed inhibitory effects of cytokines, chemokines, adhesion molecules, nitric oxide and prostaglandins produced by keratinocytes under TNF-α activation through down-regulating the NF-κB pathway. This study demontrated that this extract could be a potential antiinflammatory agent capable of improving psoriatic skin.

Fumaric Acid Esters Stimulate Astrocytic VEGF Expression through HIF-1α and Nrf2

Fumaric acid esters (FAE) are oral analogs of fumarate that have recently been shown to decrease relapse rate and disease progression in multiple sclerosis (MS), prompting to investigate their protective potential in other neurological diseases such as amyotrophic lateral sclerosis (ALS). Despite efficacy in MS, mechanisms of action of FAEs are still largely unknown. FAEs are known to activate the transcription factor Nrf2 and downstream anti-oxidant responses through the succination of Nrf2 inhibitor KEAP1. However, fumarate is also a known inhibitor of prolyl-hydroxylases domain enzymes (PhD), and PhD inhibition might lead to stabilization of the HIF-1α transcription factor under normoxic conditions and subsequent activation of a pseudo hypoxic response. Whether Nrf2 activation is associated with HIF-1α stabilization in response to FAEs in cell types relevant to MS or ALS remains unknown. Here, we show that FAEs elicit HIF-1α accumulation, and VEGF release as its expected consequence, in astrocytes but not in other cell types of the central nervous system. Reporter assays demonstrated that increased astrocytic VEGF release in response to FAEs was dependent upon both HIF-1α and Nrf2 activation. Last, astrocytes of transgenic mice expressing SOD1(G93A), an animal model of ALS, displayed reduced VEGF release in response to FAEs. These studies show that FAEs elicit different signaling pathways in cell types from the central nervous system, in particular a pseudo-hypoxic response in astrocytes. Disease relevant mutations might affect this response.

Pre-transplant signal induction for vascularisation in human islets

Human islet transplant success is partially impaired by slow revascularisation. Our study investigated the potential for rotational cell culture (RC) of human islets combined with thiazolidinedione (TZD) stimulation of peroxisome proliferator-activated receptor gamma (PPARγ) to upregulate vascular endothelial growth factor (VEGF) expression in the islets. Four groups of human islets were studied: static culture (SC) with and without 25 mmol/L TZD and RC with and without 25 mmol/L TZD. These were assessed for insulin secretion and soluble VEGF-A release. Both proteins were quantified by enzyme-linked immunosorbent assay (ELISA), supported with qualitative immunofluorescence staining. RC + TZD increased insulin secretion by >20% (p < 0.05-0.001) in response to 16.7 mmol/L glucose and 16.7 mmol/L glucose + 10 mmol/L theophylline (G + T). This effect was seen at all time intervals compared with SC and without addition of TZD. Soluble VEGF-A release was significantly augmented by RC and TZD exposure with an increased effect of >30% (p < 0.001) at 72 h under both SC and RC conditions. RC supplemented with a TZD enhances and prolongs the release of insulin and soluble VEGF-A by isolated human islets.

De novo hem- and lymphangiogenesis by endothelial progenitor and mesenchymal stem cells in immunocompetent mice.

Cellular pro-angiogenic therapies may be applicable for the treatment of peripheral vascular diseases. Interactions between mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) may provide such a treatment option. With the exception of some studies in man, experiments have only been performed in immunodeficient mice and rats. We studied an immunocompetent syngeneic mouse model. We isolated MSCs from bone marrow and EPCs from the lung of adult C57/Bl.6 mice and co-injected them in Matrigel subcutaneously in adult C57/Bl.6 mice. We demonstrate development of both blood vessels and lymphatics. Grafted EPCs integrated into the lining of the two vessel types, whereas MSCs usually did not incorporate into the vessel wall. Injections of each separate cell type did not, or hardly, reveal de novo angiogenesis. The release of VEGF-A by MSCs has been shown before, but its inhibitors, e.g., soluble VEGF receptors, have not been studied. We performed qualitative and quantitative studies of the proteins released by EPCs, MSCs, and cocultures of the cells. Despite the secretion of VEGF inhibitors (sVEGFR-1, sVEGFR-2) by EPCs, VEGF-A was secreted by MSCs at bioavailable amounts (350pg/ml). We confirm the secretion of PlGF, FGF-1, MCP-1, and PDGFs by EPCs/MSCs and suggest functions for VEGF-B, amphiregulin, fractalkine, CXCL10, and CXCL16 during MSC-induced hem- and lymphangiogenesis. We assume that lymphangiogenesis is induced indirectly by growth factors from immigrating leukocytes, which we found in close association with the lymphatic networks. Inflammatory responses to the cellular markers GFP and cell-tracker red (CMPTX) used for tracing of EPCs or MSCs were not observed. Our studies demonstrate the feasibility of pro-angiogenic/lymphangiogenic therapies in immunocompetent animals and indicate new MSC/EPC-derived angiogenic factors.

Platelet‐mediated angiogenesis is independent of VEGF and fully inhibited by aspirin

Platelets are major players in every step of vessel development through the local delivery of angiogenesis-modulating factors, including the pro-angiogenic protein VEGF and the anti-angiogenic endostatin. Although thrombin is a potent agonist and is highly elevated in angiogenesis-related diseases, studies regarding its action on the release of platelet angiogenic factors are scarce and controversial. Herein, we have investigated the role of thrombin not only in VEGF and endostatin release but also in net platelet angiogenic activity. Human platelets were stimulated with thrombin in the presence of the various inhibitors of the signalling pathways involved in platelet activation. Supernatants/releasates were used to determine the levels of angiogenic molecules and to induce angiogenic responses. We found that thrombin induced the secretion of both VEGF and endostatin; however, the overall effect of the releasates was pro-angiogenic as they promoted tubule-like formation and increased the proliferation of endothelial cells. Both responses were only slightly suppressed in the presence of a VEGF receptor-neutralizing antibody. Pharmacological studies revealed that while inhibitors of PKC, p38, ERK1/2, Src kinases or PI3K/Akt exerted only partial inhibitory effects, aspirin fully blocked the pro-angiogenic activity of the releasate. In contrast to current belief, platelet pro-angiogenic responses are independent of VEGF and appear to be the result of the combined action of several molecules. Moreover, our data reinforce the notion that aspirin is a good candidate for a therapeutic agent to treat angiogenesis-related diseases.

Phyllostachys edulis leaf extract reduces TNFα-induced release of VEGF and IL-8 in immortalized HaCaT cells

Phyllostachys edulis Carriere (Poaceae) has received considerable attention in pharmacological research due to their potent antitumor, anti-inflammatory, antimicrobial, and antiulcerogenic activities. In this study, we investigated the antiinflammatory effects of two leaf extracts (young versus old harvested leaves) prepared from Phyllostachys edulis on TNFα-induced overproduction of IL-8, VEGF and IL-6 in immortalized human keratinocytes (HaCaT). These cytokines play important roles in various inflammatory skin diseases, such as psoriasis. Bamboo leaves of different maturity and age levels were used since we expected a different phytochemical profile and therefore a different pharmacological activity. Both leaf extracts were prepared by Soxhlet extraction using water as extraction solvent. The amounts of major flavonoids were quantified using a LC-MS/MS method. Isoorientin was detected as the main flavonoid in both extracts with amounts of 1590 mg/kg (old leaves) and 1440 mg/kg (young leaves). The Soxhlet extract prepared from the young bamboo leaves (SEYL) dose dependently (25 – 250 µg/ml) inhibited the release of TNFα-induced IL-8, and VEGF, but not IL-6, in HaCaT cells while the extract prepared from the old leaves (SEOL) had no effect. In addition, isoorientin (ISO; 10 – 100µM) dose dependently reduced the levels of VEGF, IL-8 and IL-6 in TNFα-treated HaCaT cells, comparable to the positive control hydrocortisone (HC; 10µM). Cell viability was determined by the MTT (3-[4,5-dimethylthiazol-2yl]-diphenyl tetrazolium bromide) colorimetric assay. SEYL and SEOL up to a concentration of 250 µg/ml as well as ISO and HC (10 – 100µM, respectively) did not have any toxic effects on HaCaT cells. Taken together, an extract prepared from the young leaves of Phyllostachys edulis as well as isoorientin exerted anti-inflammatory effects in TNFα-treated HaCaT cells, suggesting interesting cosmetical and pharmacological applications.

Vascular Endothelial Growth Factor and Physiological Compressive Loading Synergistically Promote Bone Formation of Tissue-Engineered Bone

Angiogenesis and osteogenesis are essential for healing bone injuries, especially those related to critically sized bone defect (CSBD). Bone tissue engineering is a promising method for repairing bone defect, but the inherently poor nutrient supply and the slow osteogenesis of large size defect are two obstacles. This leads to an urgent need to understand how to orchestrate angiogenesis and osteogenesis for promoting bone defect healing. In this article, poly-L-lysine-coated vascular endothelial growth factor/alginate controlled releasing microspheres (VEGF-AG-PLL) were fabricated in tissue-engineered bone (TEB), and then the composite was implanted into the CSBD of goat femurs. Dynamic intramedullary rod was utilized to fixate the femur and produce an analogous physiological axial compression. We found that VEGF released in early stage not only promoted angiogenesis, but also brought benefit to osteogenesis. Meanwhile, in early stage physiological axial compression could regulate angiogenesis and facilitate VEGF release from the composite. In later stage, compression could accelerate reconstruction of TEB. In conclusion, this study demonstrated that the collaborative application of VEGF and compression could accelerate the reconstruction of TEB; thus, providing a new strategy for clinically repairing large bone defect in future.

PKC activation affects, via ELAVL1/HuR protein, VEGF expression in pericytic/endothelial coculture

AbstractPurpose To explore whether, following direct contact, there is a mutual influence between pericytes (PC) and endothelial cells (EC), and to establish whether PKC activation, a condition associated with hyperglycaemia in diabetic retinopathy (DR), can affect, via the mRNA‐binding ELAVL1/HuR protein, VEGF expression.Methods Retinal PC and EC were cultured separately (MONO) or in direct contact (1:1 ratio; coculture; COCU), and exposed or not to PKC activator phorbol esthers (100 nM PMA, 15 min.). PKCβI/βII, HuR, VEGF proteins were analysed by Western blotting; VEGF secretion in cell culture medium was evaluated by ELISA. VEGF mRNA analysis was performed by real‐time qPCR. Immunocytochemistry was performed to evaluate PMA‐induced HuR translocation.Results In MONO, VEGF mRNA/protein basal levels are higher in PC than EC. In COCU, VEGF protein amount is up‐regulated in EC and down‐regulated in PC, respectively. VEGF release is favoured by physical contact between PC and EC and further increased by PMA exposure. PKCβ activation induces HuR translocation from nucleus to cytoplasm, and it increases the protein levels of the kinase itself, HuR and VEGF in PC and EC in both culture conditions.Conclusion VEGF expression varies in function of the cell culture conditions. In all cultures, PKC activation increases PKCβI/βII, HuR and VEGF protein levels, changes which may occur also at very early stages of DR. The COCU model may be useful to study PC/EC interactions in both physiological and pathological conditions.

ATF4 and HIF-1α in bone: An intriguing relationship

As occurs in any other vascularized organ, one of the essential functions of blood vessels in bone is to be a source of O2, nutrients, hormones and growth factors (1). However, additional roles for bone blood vessels are emerging. For example, osteoblast precursors have been recently identified within their wall (2,3), which suggests that blood vessels are a source of osteoprogenitors. Moreover, it has been shown that endothelial cells are specialized niches for hematopoietic stem cells (4,5). In light of these findings, identification of the molecular and cellular mechanisms controlling angiogenesis in bone is essential in order to reach a full understanding of how bone and bone marrow development and homeostasis are regulated.

Insulin directly stimulates VEGF-A production in the glomerular podocyte

Podocytes are critically important for maintaining the integrity of the glomerular filtration barrier and preventing albuminuria. Recently, it has become clear that to achieve this, they need to be insulin sensitive and produce an optimal amount of VEGF-A. In other tissues, insulin has been shown to regulate VEGF-A release, but this has not been previously examined in the podocyte. Using in vitro and in vivo approaches, in the present study, we now show that insulin regulates VEGF-A in the podocyte in both mice and humans via the insulin receptor (IR). Insulin directly increased VEGF-A mRNA levels and protein production in conditionally immortalized wild-type human and murine podocytes. Furthermore, when podocytes were rendered insulin resistant in vitro (using stable short hairpin RNA knockdown of the IR) or in vivo (using transgenic podocyte-specific IR knockout mice), podocyte VEGF-A production was impaired. Importantly, in vivo, this occurs before the development of any podocyte damage due to podocyte insulin resistance. Modulation of VEGF-A by insulin in the podocyte may be another important factor in the development of glomerular disease associated with conditions in which insulin signaling to the podocyte is deranged.

Initiation of Aspirin Therapy Modulates Angiogenic Protein Levels in Women with Breast Cancer Receiving Tamoxifen Therapy

Aspirin has a range of antineoplastic properties linked to inhibition of cyclooxygenase enzymes in tumor cells, platelet inhibition and to inhibition of angiogenesis. We undertook a prospective study to determine the influence of a 45‐day course of aspirin therapy on circulating and intraplatelet levels of selected proangiogenic (vascular endothelial growth factor [VEGF]) and antiangiogenic (thrombospondin‐1 [TSP‐1]) proteins, and platelet protein release in women diagnosed with breast cancer who were receiving tamoxifen therapy. Initiation of aspirin therapy increases serum and intraplatelet levels of TSP‐1 without a corresponding increase in VEGF levels. Following aspirin therapy, VEGF levels decreased (relative to pretreatment levels) while TSP‐1 returned to pretreatment levels. Plasma TSP‐1 and VEGF levels did not change on aspirin therapy. Aspirin use also decreased thrombin receptor mediated release of TSP‐1 and VEGF from platelets. The selective impact on platelet angiogenic protein content and release supports one mechanism by which aspirin can modify the angiogenic balance in women receiving tamoxifen therapy. Aspirin therapy appears to favor an overall antiangiogenic balance in women with breast cancer who are receiving tamoxifen therapy.

Release kinetics of platelet-derived and plasma-derived growth factors from autologous plasma rich in growth factors

Many studies have evaluated the biological effects of platelet rich plasma reporting the final outcomes on cell and tissues. However, few studies have dealt with the kinetics of growth factor delivery by plasma rich in growth factors. Venous blood was obtained from three healthy volunteers and processed with PRGF-Endoret technology to prepare autologous plasma rich in growth factors. The gel-like fibrin scaffolds were then incubated in triplicate, in a cell culture medium to monitor the release of PDGF-AB, VEGF, HGF and IGF-I during 8 days of incubation. A leukocyte-platelet rich plasma was prepared employing the same technology and the concentrations of growth factors and interleukin-1β were determined after 24h of incubation. After each period, the medium was collected, fibrin clot was destroyed and the supernatants were stored at -80°C until analysis. The growth factor delivery is diffusion controlled with a rapid initial release by 30% of the bioactive content after 1h of incubation and a steady state release when almost 70% of the growth factor content has been delivered. Autologous fibrin matrix retained almost 30% of the amount of the growth factors after 8 days of incubation. The addition of leukocytes to the formula of platelet rich plasma did not increase the concentration of the growth factors, while it drastically increased the presence of pro-inflammatory IL-1β. Further studies employing an in vitro inflammatory model would be interesting to study the difference in growth factors and pro-inflammatory cytokines between leukocyte-free and leukocyte-rich platelet rich plasma.

Glycated collagen and altered glucose increase endothelial cell adhesion strength

Cell adhesion strength is important to cell survival, proliferation, migration, and mechanotransduction, yet changes in endothelial cell adhesion strength have not yet been examined in diseases such as diabetes with high rates of cardiovascular complications. We therefore investigated porcine aortic endothelial cell adhesion strength on native and glycated collagen-coated substrates and in low, normal, and high glucose culture using a spinning disc apparatus. Adhesion strength increased by 30 dynes/cm(2) in cells on glycated collagen as compared to native collagen. Attachment studies revealed that cells use higher adhesion strength αv β3 integrins to bind to glycated collagen instead of the typical α2 β1 integrins used to bind to native collagen. Similarly, endothelial cells cultured in low and high glucose had 15 dynes/cm(2) higher adhesion strength than cells in normal glucose after 2 days. Increased adhesion strength was due to elevated VEGF release and intracellular PKC in low and high glucose cells, respectively. Thus glucose increased endothelial cell adhesion strength via different underlying mechanisms. These adhesion strength changes could contribute to diabetic vascular disease, including accelerated atherosclerosis and disordered angiogenesis.

Modification of xenogenic bone substitute materials – effects on the early healing cascade in vitro

Initial platelet activation with subsequent cytokine release at the defect site plays a crucial role in tissue integration. The aim of this study was to evaluate the influence of topographic and biomimetic collagen modifications of a xenogenic bone substitute material (BSM) on in vitro platelet activation and cytokine release. Three types of xenogenic BSM were used. Two BSM with different levels of granularity (large granule BSM [XBSM/L], small granule BSM [XBSM/S]) and a BSM with collagen (XBSM/C). All three samples were incubated with platelet concentrate of four healthy volunteers at room temperature for 15min. For all groups, highly thrombogenic collagen type 1 served as a reference and an additional preparation with platelet concentrate only (without XBSM) served as control. Platelet count and cytokine release of VEGF, PDGF, TGF-β, and IGF into the supernatant were measured. Compared with the control group, XBSM/C showed an increase in platelets consumption (mean 41,000±26,000/ml vs. 471,000±38,000/ml), cytokine release of VEGF (mean 46.8±7.2pg/ml vs. 18.8±2.7pg/ml), and PDGF (mean 18,350±795pg/ml vs. 2726±410pg/ml) but not IGF (194,728±51,608pg/ml vs. 1,333,911±35,314pg/ml). There was also an increase in cytokine release of TGF-ß in XBSM/C compared with XBSM/S (77,188±27,413pg/ml vs. 38,648±13,191pg/ml), but no such difference when compared with XBSM/L (77,188±27,413pg/ml vs. 53,309±29,430pg/ml). XBSM/L showed higher platelets consumption (301,000±45,000 vs. 415,000±98,000) and a higher cytokine release of PDGF (3511±247pg/ml vs. 3165±78pg/ml) compared with XBSM/S. There was no distinct difference in the levels of VEGF, TGF-ß, and IGF between XBSM/L and XBSM/S. Topographic as well as biomimetic modifications of the xenogenic BSM showed an increased platelet activation and cytokine release in vitro. This effect on the intrinsic healing cascade could result in comparable enhanced soft- and hard-tissue regeneration in vivo.

C-X-C motif receptor 2, endostatin and proteinase-activated receptor 1 polymorphisms as prognostic factors in NSCLC

The progress of non-small cell lung cancer (NSCLC) is dependent on sufficient angiogenesis. Thrombin induced activation of proteinase-activated receptor 1 (PAR-1) on platelets leads to platelet secretion and aggregation. This influences cell survival, apoptosis and angiogenesis by the release of VEGF and Endostatin (ES), a potent angiogenesis inhibitor. Interleukin-8 (IL-8) induces tumor angiogenesis independent of the VEGF pathway through the chemokine C-X-C motif receptor 2 (CXCR-2). Our purpose was to evaluate germline polymorphisms of these potential therapy targets as prognostic markers for disease free survival (DFS) and overall survival (OS) in surgically treated NSCLC patients. In total 209 Caucasian patients, treated between 1996 and 2011, were included in this study. Genomic DNA was extracted from peripheral blood leucocytes. Genotyping of CXCR-2 +1208 C>T and +785 C>T, PAR-1 -506 Ins/del and -14 Ivs A>T and ES +4349 G>A was performed by TaqMan® genotyping assays or by polymerase chain reaction (PCR) followed by capillary electrophoresis. Chi-square test, Kaplan–Meier estimator and cox regression hazard model were used to assess the prognostic value of selected polymorphisms. The PAR-1 -14 Ivs A/A genotype was associated with advanced tumor stages (p=0.024) and, in univariate analysis, with shorter median OS in squamous cell lung carcinoma (SqCC, p=0.035). The CXCR-2 +1208T/T genotype was associated with aggressive tumor biology (p=0.038), and shorter DFS and OS (p=0.018, p=0.021) in NSCLC and especially in SqCC a negative predictor for DFS and OS (p=0.045, p=0.041). Genotyping of the CXCR-2 +1208 C>T polymorphism could be a useful tool to identify high-risk SqCC subgroups.

Engineered VEGF-releasing PEG-MAL hydrogel for pancreatic islet vascularization.

Biofunctionalized polyethylene glycol maleimide (PEG-MAL) hydrogels were engineered as a platform to deliver pancreatic islets to the small bowel mesentery and promote graft vascularization. VEGF, a potent stimulator of angiogenesis, was incorporated into the hydrogel to be released in an on-demand manner through enzymatic degradation. PEG-MAL hydrogel enabled extended in vivo release of VEGF. Isolated rat islets encapsulated in PEG-MAL hydrogels remained viable in culture and secreted insulin. Islets encapsulated in PEG-MAL matrix and transplanted to the small bowel mesentery of healthy rats grafted to the host tissue and revascularized by 4weeks. Addition of VEGF release to the PEG-MAL matrix greatly augmented the vascularization response. These results establish PEG-MAL engineered matrices as a vascular-inductive cell delivery vehicle and warrant their further investigation as islet transplantation vehicles in diabetic animal models.

VEGF receptor-1 involvement in pericyte loss induced byEscherichia coliin anin vitromodel of blood brain barrier

The key aspect of neonatal meningitis is related to the ability of pathogens to invade the blood-brain barrier (BBB) and to penetrate the central nervous system. In the present study we show that, in an in vitro model of BBB, on the basis of co-culturing primary bovine brain endothelial cells (BBEC) and primary bovine retinal pericytes (BRPC), Escherichia coli infection determines changes of transendothelial electrical resistance (TEER) and permeability (Pe) to sodium fluorescein. In the co-culture model, within BBEC, bacteria are able to stimulate cytosolic and Ca(2+)-independent phospholipase A2 (cPLA2 and iPLA2 ) enzyme activities. In supernatants of E. coli-stimulated co-cultures, an increase in prostaglandins (PGE2) and VEGF production in comparison with untreated co-cultures were found. Incubation with E. coli in presence of AACOCF3 or BEL caused a decrease of PGE2 and VEGF release. SEM and TEM images of BBEC and BRPC showed E. coli adhesion to BBEC and BRPC but only in BBEC the invasion occurs. VEGFR-1 but not VEGFR-2 blockade by the specific antibody reduced E. coli invasion in BBEC. In our model of BBB infection, a significant loss of BRPC was observed. Following VEGFR-1, but not VEGFR-2 blockade, or in presence of AACOCF3 or BEL, elevated TEER values, reduced permeability and BRPC loss were found. These data suggest that VEGFR-1 negatively regulates BRPC survival and its blockade protects the barrier integrity. PGs and VEGF could exert a biological effect on BBB, probably by BRPC coverage ablation, thus increasing BBB permeability. Our results show the role played by the BBEC as well as BRPC during a bacterial attack on BBB. A better understanding of the mechanisms by which E. coli enter the nervous system and how bacteria alter the communication between endothelial cells and pericytes may provide exciting new insight for clinical intervention.

RSUME is involved in the regulation of angiogenic factor production and growth of human neuroendocrine tumors

RSUME is a small RWD-containing sumoylation enhancing protein that shows maximum expression in pituitary and pancreas. RSUME is up regulated under hypoxic conditions and is critically involved in the production of angiogenic factors in pituitary adenoma cell cultures. We have therefore tested whether also in a neuroendocrine pancreatic cell line (Bon-1 cells) RSUME is involved in tumorigenic processes. RSUME was found to be expressed in Bon-1 cells under basal cell culture conditions and both RSUME mRNA synthesis and protein expression was stimulated under hypoxia-mimicking conditions (CoCl2 treatment). Subsequently HIF-1a expression as well as the release of VEGF-A into the cell culture supernatant was strongly enhanced. Knock-down of RSUME by siRNA strongly impaired CoCl2-induced HIF-1a production and VEGF-A secretion indicating a critical role of RSUME in angiogenic factor production by Bon-1 cells. Moreover, RSUME seems to play a role in Bon-1 cell proliferation as moderate but significant growth suppression was observed in Bon-1 cells in which RSUME was knocked down. These results suggest a role of RSUME in the progression of human neuroendocrine pancreatic tumors, which has to be studied in tissue and primary cell cultures of these tumors.

Hypoglycemia Reduces Vascular Endothelial Growth Factor A Production by Pancreatic Beta Cells as a Regulator of Beta Cell Mass

VEGF-A expression in beta cells is critical for pancreatic development, formation of islet-specific vasculature, and Insulin secretion. However, two key questions remain. First, is VEGF-A release from beta cells coupled to VEGF-A production in beta cells? Second, how is the VEGF-A response by beta cells affected by metabolic signals? Here, we show that VEGF-A secretion, but not gene transcription, in either cultured islets or purified pancreatic beta cells, was significantly reduced early on during low glucose conditions. In vivo, a sustained hypoglycemia in mice was induced with Insulin pellets, resulting in a significant reduction in beta cell mass. This loss of beta cell mass could be significantly rescued with continuous delivery of exogenous VEGF-A, which had no effect on beta cell mass in normoglycemic mice. In addition, an increase in apoptotic endothelial cells during hypoglycemia preceded an increase in apoptotic beta cells. Both endothelial and beta cell apoptosis were prevented by exogenous VEGF-A, suggesting a possible causative relationship between reduced VEGF-A and the loss of islet vasculature and beta cells. Furthermore, in none of these experimental groups did beta cell proliferation and islet vessel density change, suggesting a tightly regulated balance between these two cellular compartments. The average islet size decreased in hypoglycemia, which was also prevented by exogenous VEGF-A. Taken together, our data suggest that VEGF-A release in beta cells is independent of VEGF-A synthesis. Beta cell mass can be regulated through modulated release of VEGF-A from beta cells based on physiological need.

Natural killer cells in human pregnancy

While conventional NK cells play an important role in early defenses against pathogens thanks to their cytolytic activity and production of pro-inflammatory chemokines and cytokines, those present in decidua (dNK cells), during early pregnancy, are primarily involved in tissue building and remodeling and in the formation of new blood vessels. This occurs mainly via the release of IL-8, VEGF, SDF-1 and IP-10. In addition, we show that by interacting with particular myelomonocytic cells (dCD14+) they contribute to the induction of regulatory T cells (Tregs). In turn, Tregs are thought to play a pivotal role in immunosuppression and induction of tolerance toward the fetal allograft. We recently demonstrated that CD34+ hematopoietic precursors (dCD34+) are present in decidual tissues, thus suggesting that dNK cells might derive from such precursors. Indeed, this was confined by in vitro experiments in which dCD34+ cells differentiated into dNK cells upon culture with appropriate cytokine combinations or even in co-culture with decidua-derived stromal cells (dSC). It is possible to speculate that inappropriate cellular interactions in the decidual microenvironment or defects of dNK (or dCD14+) cell generation might negatively influence pregnancy success.