Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids

Background

Roundabouts are axon guidance molecules that have recently been identified to play a role in vascular guidance as well. In this study, we have investigated gene knockdown analysis of endothelial Robos, in particular roundabout 4 (robo4), the predominant Robo in endothelial cells using small interfering RNA technology in vitro.

Results

Robo1 and Robo4 knockdown cells display distinct activity in endothelial cell migration assay. The knockdown of robo4 abrogated the chemotactic response of endothelial cells to serum but enhanced a chemokinetic response to Slit2, while robo1 knockdown cells do not display chemotactic response to serum or VEGF. Robo4 knockdown endothelial cells unexpectedly show up regulation of Rho GTPases. Zebrafish Robo4 rescues both Rho GTPase homeostasis and serum reduced chemotaxis in robo4 knockdown cells. Robo1 and Robo4 interact and share molecules such as Slit2, Mena and Vilse, a Cdc42-GAP. In addition, this study mechanistically implicates IRSp53 in the signaling nexus between activated Cdc42 and Mena, both of which have previously been shown to be involved with Robo4 signaling in endothelial cells.

Conclusion

This study identifies specific components of the Robo signaling apparatus that work together to guide directional migration of endothelial cells.     

Polysialic acid is released by human umbilical vein endothelial cells (HUVEC) in vitro

Background

Sialic acids represent common terminal residues on numerous mammalian glycoconjugates, thereby influencing e.g. lumen formation in developing blood vessels. Interestingly, besides monosialylated also polysialylated glycoconjugates are produced by endothelial cells. Polysialic acid (polySia) is formed in several organs during embryonal and postnatal development influencing, for instance, cell migration processes. Furthermore, the function of cytokines like basic fibroblast growth factor (bFGF) is modulated by polySia.

Results

In this study, we demonstrated that human umbilical vein endothelial cells (HUVEC) also secrete polysialylated glycoconjugates. Furthermore, an interaction between polySia and vascular endothelial growth factor (VEGF) was observed. VEGF modulates like bFGF the migration of HUVEC. Since both growth factors interact with polySia, we examined, if polySia modulates the migration of HUVEC. To this end scratch assays were performed showing that the migration of HUVEC is stimulated, when polySia was degraded.

Conclusions

Since polySia can interact with bFGF as well as VEGF and the degradation of polySia resulted in an increased cell migration capacity in the applied scratch assay, we propose that polySia may trap these growth factors influencing their biological activity. Thus, polySia might also contribute to the fine regulation of physiological processes in endothelial cells.

     

Nitric Oxide Donors Augment Interleukin-1β-Induced Vascular Endothelial Growth Factor in Airway Smooth Muscle Cells

Angiogenesis and microvascular leakage are features of chronic inflammatory diseases of which molecular mechanisms are poorly understood. We investigated the effects of interleukin-1β (IL-1β) on the expression and secretion of vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) in porcine airway smooth muscle cells (PASMC) in relation to a nitric oxide (NO) pathway. Serum-deprived (48 h) PASMC were stimulated with IL-1β alone or with NO donor, l-arginine and/or NO synthase inhibitor l-NAME for 4 and 24 h. IL-1β did not affect PlGF release, but augmented VEGF release (2.4-fold) after 24 h. VEGF release was inhibited by l-NAME (531.8 ± 52 pg/ml), but restored and further elevated by l-arginine (1,529 ± 287 pg/ml). IL-1β up-regulated VEGF mRNA (1.8-fold) and this response was attenuated by l-NAME (1.1-fold) and augmented by l-arginine (3.8-fold) at 4 h. Restoration of a NO pathway by l-arginine in l-NAME-treated cells resulted in elevated VEGF mRNA levels (2.2-fold). [³H]Thymidine incorporation assay revealed enhanced porcine pulmonary artery endothelial cell proliferation in response to IL-1β, VEGF and PlGF, and this mitogenic effect was not influenced via the NO pathway. Our results suggest that a NO pathway modulates VEGF synthesis during inflammation contributing to bronchial angiogenesis and vascular leakage.     

Requirement of vasculogenesis and blood circulation in late stages of liver growth in zebrafish

Background

In the vascular system, Notch receptors and ligands are expressed mainly on arteries, with Delta-like 4 (Dll4) being the only ligand known to be expressed early during the development of arterial endothelial cells and capillaries. Dll4 null embryos die very early in development with severely reduced arterial calibre and lumen and loss of arterial cell identity.

Results

The current detailed analysis of these mutants shows that the arterial defect precedes the initiation of blood flow and that the arterial Dll4 ^-/- endothelial cells proliferate and migrate more actively. Dll4 ^-/- mutants reveal a defective basement membrane around the forming aorta and increased endothelial cell migration from the dorsal aorta to peripheral regions, which constitute the main causes of arterial lumen reduction in these embryos. The increased proliferation and migration of Dll4 ^-/- endothelial cells was found to coincide with increased expression of the receptors VEGFR-2 and Robo4 and with downregulation of the TGF-β accessory receptor Endoglin.

Conclusion

Together, these results strongly suggest that Notch signalling can increase arterial stability and calibre by decreasing the response of arterial endothelial cells to local gradients of pro-angiogenic factors like VEGF.     

The antiangiogenic 16K prolactin impairs functional tumor neovascularization by inhibiting vessel maturation

Background

Angiogenesis, the formation of new blood vessels from existing vasculature, plays an essential role in tumor growth, invasion, and metastasis. 16K hPRL, the antiangiogenic 16-kDa N-terminal fragment of human prolactin was shown to prevent tumor growth and metastasis by modifying tumor vessel morphology.

Methodology/Principal Findings

Here we investigated the effect of 16K hPRL on tumor vessel maturation and on the related signaling pathways. We show that 16K hPRL treatment leads, in a murine B16-F10 tumor model, to a dysfunctional tumor vasculature with reduced pericyte coverage, and disruption of the PDGF-B/PDGFR-B, Ang/Tie2, and Delta/Notch pathways. In an aortic ring assay, 16K hPRL impairs endothelial cell and pericyte outgrowth from the vascular ring. In addition, 16K hPRL prevents pericyte migration to endothelial cells. This event was independent of a direct inhibitory effect of 16K hPRL on pericyte viability, proliferation, or migration. In endothelial cell-pericyte cocultures, we found 16K hPRL to disturb Notch signaling.

Conclusions/Significance

Taken together, our data show that 16K hPRL impairs functional tumor neovascularization by inhibiting vessel maturation and for the first time that an endogenous antiangiogenic agent disturbs Notch signaling. These findings provide new insights into the mechanisms of 16K hPRL action and highlight its potential for use in anticancer therapy.     

Approach to diagnosis and pathological examination in bronchial Dieulafoy disease: a case series

Background

We previously observed that allergen-exposed mice exhibit remodeling of large bronchial-associated blood vessels. The aim of the study was to examine whether vascular remodeling occurs also in vessels where a spill-over effect of bronchial remodeling molecules is less likely.

Methods

We used an established mouse model of allergic airway inflammation, where an allergic airway inflammation is triggered by inhalations of OVA. Remodeling of bronchial un-associated vessels was determined histologically by staining for α-smooth muscle actin, procollagen I, Ki67 and von Willebrand-factor. Myofibroblasts were defined as and visualized by double staining for α-smooth muscle actin and procollagen I. For quantification the blood vessels were divided, based on length of basement membrane, into groups; small (≤250 μm) and mid-sized (250–500 μm).

Results

We discovered marked remodeling in solitary small and mid-sized blood vessels. Smooth muscle mass increased significantly as did the number of proliferating smooth muscle and endothelial cells. The changes were similar to those previously seen in large bronchial-associated vessels. Additionally, normally poorly muscularized blood vessels changed phenotype to a more muscularized type and the number of myofibroblasts around the small and mid-sized vessels increased following allergen challenge.

Conclusion

We demonstrate that allergic airway inflammation in mice is accompanied by remodeling of small and mid-sized pulmonary blood vessels some distance away (at least 150 μm) from the allergen-exposed bronchi. The present findings suggest the possibility that allergic airway inflammation may cause such vascular remodeling as previously associated with lung inflammatory conditions involving a risk for development of pulmonary hypertension.     

Influence of oxygen tension on myocardial performance. Evaluation by tissue Doppler imaging

Background

Endothelial function in hypercholesterolemic rabbits is usually evaluated ex vivo on isolated aortic rings. In vivo evaluation requires invasive imaging procedures that cannot be repeated serially.

Aim

We evaluated a non-invasive ultrasound technique to assess early endothelial function in rabbits and compare data with ex vivo measurements.

Methods

Twenty-four rabbits (fed with a cholesterol diet (0.5%) for 2 to 8 weeks) were given progressive infusions of acetylcholine (0.05–0.5 μg/kg/min) and their endothelial function was assessed in vivo by transcutaneous vascular ultrasound of the abdominal aorta. Ex vivo endothelial function was evaluated on isolated aortic rings and compared to in vivo data.

Results

Significant endothelial dysfunction was demonstrated in hypercholesterolemic animals as early as 2 weeks after beginning the cholesterol diet (aortic cross-sectional area variation: -2.9% vs. +4% for controls, p < 0.05). Unexpectedly, response to acetylcholine at 8 weeks was more variable. Endothelial function improved in 5 rabbits while 2 rabbits regained a normal endothelial function. These data corroborated well with ex vivo results.

Conclusion

Endothelial function can be evaluated non-invasively in vivo by transcutaneous vascular ultrasound of the abdominal aorta in the rabbit and results correlate well with ex vivo data.     

Robust simplifications of multiscale biochemical networks

Background

Angiogenesis is a process by which new capillaries are formed from pre-existing blood vessels in physiological (e.g., exercise, wound healing) or pathological (e.g., ischemic limb as in peripheral arterial disease, cancer) contexts. This neovascular mechanism is mediated by the vascular endothelial growth factor (VEGF) family of cytokines. Although VEGF is often targeted in anti-angiogenic therapies, there is little knowledge about how its concentration may vary between tissues and the vascular system. A compartment model is constructed to study the VEGF distribution in the tissue (including matrix-bound, cell surface receptor-bound and free VEGF isoforms) and in the blood. We analyze the sensitivity of this distribution to the secretion rate, clearance rate and vascular permeability of VEGF.

Results

We find that, in a physiological context, VEGF concentration varies approximately linearly with the VEGF secretion rate. VEGF concentration in blood but not in tissue is dependent on the vascular permeability of healthy tissue. Model simulations suggest that relative VEGF increases are similar in blood and tissue during exercise and return to baseline within several hours. In a pathological context (tumor), we find that blood VEGF concentration is relatively insensitive to increased vascular permeability in tumors, to the secretion rate of VEGF by tumors and to the clearance. However, it is sensitive to the vascular permeability in the healthy tissue. Finally, the VEGF distribution profile in healthy tissue reveals that about half of the VEGF is complexed with the receptor tyrosine kinase VEGFR2 and the co-receptor Neuropilin-1. In diseased tissues, this binding can be reduced to 15% while VEGF bound to the extracellular matrix and basement membranes increases.

Conclusion

The results are of importance for physiological conditions (e.g., exercise) and pathological conditions (e.g., peripheral arterial disease, coronary artery disease, cancer). This mathematical model can serve as a tool for understanding the VEGF distribution in physiological and pathological contexts as well as a foundation to investigate pro- or anti-angiogenic strategies.     

Differential effects of Th1 versus Th2 cytokines in combination with hypoxia on HIFs and angiogenesis in RA

Introduction

Dysregulated angiogenesis is implicated in the pathogenesis of rheumatoid arthritis (RA). To provide a more profound understanding of arthritis-associated angiogenesis, we evaluated the expression of angiogenesis-modulating genes at onset, peak and declining phases of collagen-induced arthritis (CIA), a well-established mouse model for RA.

Methods

CIA was induced in DBA/1 mice with type II collagen. Functional capillary density in synovial tissue of knee joints was determined by intravital fluorescence microscopy. To assess the ability of arthritic joint homogenates to induce angiogenesis, an endothelial chemotaxis assay and an in vivo matrigel plug assay were employed. The temporal expression profile of angiogenesis-related genes in arthritic paws was analysed by quantitative real-time RT-PCR using an angiogenesis focused array as well as gene specific PCR. Finally, we investigated the therapeutic effect of a monoclonal antibody specifically blocking the binding of VEGF to neuropilin (NRP)-1.

Results

Although arthritic paw homogenates displayed angiogenic activity in vitro and in vivo, and synovia of arthritic paws appeared highly vascularised on histological examination, the functional capillary density in arthritic knee synovia was significantly decreased, whereas capillary diameter was increased. Of the 84 genes analysed, 41 displayed a differential expression in arthritic paws as compared to control paws. Most significant alterations were seen at the peak of clinical arthritis. Increased mRNA expression could be observed for VEGF receptors (Flt-1, Flk-1, Nrp-1, Nrp-2), as well as for midkine, hepatocyte growth factor, insulin-like growth factor-1 and angiopoietin-1. Signalling through NRP-1 accounted in part for the chemotactic activity for endothelial cells observed in arthritic paw homogenates. Importantly, therapeutic administration of anti-NRP1^B antibody significantly reduced disease severity and progression in CIA mice.

Conclusions

Our findings confirm that the arthritic synovium in murine CIA is a site of active angiogenesis, but an altered balance in the expression of angiogenic factors seems to favour the formation of non-functional and dilated capillaries. Furthermore, our results validate NRP-1 as a key player in the pathogenesis of CIA, and support the VEGF/VEGF receptor pathway as a potential therapeutic target in RA.     

Expression,purification and characterization of a vascular endothelial growth factor fusion protein

Objectives

To prepare recombinant tPep-(vascular endothelial growth factor) VEGF-B and assess its biological activity.

Results

This new VEGF fusion protein was constructed using a targeting peptide and prepared using E.coli. The tPep-VEGF-B was refolded from inclusion bodies and purified using affinity chromatography. Its bioactivity was determined in vitro using proliferation assay and wounding healing assay, and in vivo in zebrafish. By using the optimized downstream process, recombinant tPep-VEGF-B can be obtained with a purity of >90 % and a yield of 80 mg protein/l culture medium. The refolded protein is highly effective in promoting cell migration in vitro and in enhancing angiogenesis in vivo.

Conclusion

We have constructed a new VEGF fusion protein with potential therapeutic application in treating metabolic diseases.

     

Smad gene expression in pulmonary fibroblasts: indications for defective ECM repair in COPD

Background

Endothelial dysfunction is a major complication of pulmonary endarterectomy (PTE) that can lead to pulmonary edema and persistent pulmonary hypertension. We hypothesized that endothelial dysfunction is related to increased endothelial-cell (EC) death.

Methods

In piglets, the left pulmonary artery (PA) was ligated to induce lung ischemia then reimplanted into the main PA to reperfuse the lung. Animals sacrificed 5 weeks after ligation (n = 5), 2 days after reperfusion (n = 5), or 5 weeks after reperfusion (n = 5) were compared to a sham-operated group (n = 5). PA vasoreactivity was studied and eNOS assayed. EC apoptosis was assessed by TUNEL in the proximal and distal PA and by caspase-3 activity assay in the proximal PA. Gene expression of pro-apoptotic factors (thrombospondin-1 (Thsp-1) and plasminogen activator inhibitor 1 (PAI-1)) and anti-apoptotic factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) was investigated by QRT-PCR.

Results

Endothelium-dependent relaxation was altered 5 weeks after ligation (p = 0.04). The alterations were exacerbated 2 days after reperfusion (p = 0.002) but recovered within 5 weeks after reperfusion. EC apoptosis was increased 5 weeks after PA ligation (p = 0.02), increased further within 2 days after reperfusion (p < 0.0001), and returned to normal within 5 weeks after reperfusion. Whereas VEGF and bFGF expressions remained unchanged, TSP and PAI-1 expressions peaked 5 weeks after ligation (p = 0.001) and returned to normal within 2 days after reperfusion.

Conclusion

Chronic lung ischemia induces over-expression of pro-apoptotic factors. Lung reperfusion is followed by a dramatic transient increase in EC death that may explain the development of endothelial dysfunction after PE. Anti-apoptotic agents may hold considerable potential for preventing postoperative complications.     

The chemopreventive effect of Ginkgo biloba and Silybum marianum extracts on hepatocarcinogenesis in rats

Background/objective

This study was designed to evaluate the potential chemopreventive activities of Ginkgo biloba extract (EGb) and Silybum marianum extract (silymarin) against hepatocarcinogenesis induced by N-nitrosodiethylamine (NDEA) in rats.

Methods

Rats were divided into 6 groups. Group 1 served as normal control rats. Group 2 animals were intragastrically administrated NDEA at a dose of 10 mg/kg five times a week for 12 weeks to induce hepatocellular carcinoma (HCC). Groups 3 and 4 animals were pretreated with silymarin and EGb respectively. Groups 5 and 6 animals were posttreated with silymarin and EGb respectively. The investigated parameters in serum are alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltransferase (GGT) and vascular endothelial growth factor (VEGF). The investigated parameters in liver tissue are malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and comet assay parameters.

Results

In NDEA group, MDA level was elevated with subsequent decrease in GSH level and SOD, GPx and GR activities. In addition, NDEA group revealed a significant increase in serum ALT, AST and GGT activities and VEGF level. Furthermore, NDEA administrated animals showed a marked increase in comet assay parameters. These biochemical alterations induced by NDEA were confirmed by the histopathological examination of rat livers intoxicated with NDEA that showed an obvious cellular damage and well differentiated HCC. In contrast, silymarin+NDEA treated groups (3&;5) and EGb+NDEA treated groups (4&;6) showed a significant decrease in MDA level and a significant increase in GSH content and SOD, GPx and GR activities compared to NDEA group. Silymarin and EGb also beneficially down-regulated the increase in serum ALT, AST, GGT activities and VEGF level induced by NDEA. In addition, silymarin and EGb significantly decreased comet assay parameters. Histopathological examination of rat livers treated with either silymarin or EGb exhibited an improvement in the liver architecture compared to NDEA group.

Conclusions

The obtained findings suggested that silymarin and EGb may have beneficial chemopreventive roles against hepatocarcinogenesis through their antioxidant, antiangiogenic and antigenotoxic activities.     

Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity

Background

Eosinophils are involved in various inflammatory processes including allergic inflammation during which angiogenesis has been documented. Angiogenesis is most likely connected to the hypoxia which characterizes inflamed tissues. Eosinophils produce VEGF and are pro-angiogenic. However, to the best of our knowledge no study has been performed to verify the existence of a direct link between eosinophils, hypoxia and angiogenesis in allergic inflammation.

Objective

To characterize eosinophil function and angiogenic potential under hypoxic conditions.

Methods

Human peripheral blood eosinophils were cultured in normoxic or hypoxic conditions with or without cytokines. Viability and apoptosis were assessed by Annexin V/PI staining. Anti- or pro-apoptotic protein levels, HIF-1α levels and MAPK phosphorylation were analyzed by immunoblot analysis. Angiogenic mediator release was evaluated by ELISA.

Results

Hypoxic eosinophils were more viable than normoxic ones after up to three days. In addition in hypoxia, anti-apoptotic Bcl-XL protein levels increased more than pro-apoptotic Bax levels. Hypoxia increased VEGF and IL-8 release. In hypoxic eosinophils high levels of HIF-1α were observed, particularly in the presence of GM-CSF. MAPK, particularly ERK1/2 inhibitors, decreased hypoxia-mediated VEGF release and HIF-1α expression.

Conclusion

Eosinophils respond to hypoxia by up-regulation of survival and of some of their pro-angiogenic functions indicating a correlation between eosinophilic inflammation and angiogenesis.     

Up-regulation of intestinal vascular endothelial growth factor by Afa/Dr diffusely adhering Escherichia coli

Background

Angiogenesis has been recently described as a novel component of inflammatory bowel disease pathogenesis. The level of vascular endothelial growth factor (VEGF) has been found increased in Crohn''s disease and ulcerative colitis mucosa. To question whether a pro-inflammatory Escherichia coli could regulate the expression of VEGF in human intestinal epithelial cells, we examine the response of cultured human colonic T84 cells to infection by E. coli strain C1845 that belongs to the typical Afa/Dr diffusely adhering E. coli family (Afa/Dr DAEC).

Methodology

VEGF mRNA expression was examined by Northern blotting and q-PCR. VEGF protein levels were assayed by ELISA and its bioactivity was analysed in endothelial cells. The bacterial factor involved in VEGF induction was identified using recombinant E. coli expressing Dr adhesin, purified Dr adhesin and lipopolysaccharide. The signaling pathway activated for the up-regulation of VEGF was identified using a blocking monoclonal anti-DAF antibody, Western blot analysis and specific pharmacological inhibitors.

Principal Findings

C1845 bacteria induce the production of VEGF protein which is bioactive. VEGF is induced by adhering C1845 in both a time- and bacteria concentration-dependent manner. This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells. Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways.

Conclusions

Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells. Thus, these results suggest a link between an entero-adherent, pro-inflammatory E. coli strain and angiogenesis which appeared recently as a novel component of IBD pathogenesis.     

Protective role of vascular endothelial growth factor in endotoxin-induced acute lung injury in mice

Background

Vascular endothelial growth factor (VEGF), a substance that stimulates new blood vessel formation, is an important survival factor for endothelial cells. Although overexpressed VEGF in the lung induces pulmonary edema with increased lung vascular permeability, the role of VEGF in the development of acute lung injury remains to be determined.

Methods

To evaluate the role of VEGF in the pathogenesis of acute lung injury, we first evaluated the effects of exogenous VEGF and VEGF blockade using monoclonal antibody on LPS-induced lung injury in mice. Using the lung specimens, we performed TUNEL staining to detect apoptotic cells and immunostaining to evaluate the expression of apoptosis-associated molecules, including caspase-3, Bax, apoptosis inducing factor (AIF), and cytochrome C. As a parameter of endothelial permeability, we measured the albumin transferred across human pulmonary artery endothelial cell (HPAEC) monolayers cultured on porous filters with various concentrations of VEGF. The effect of VEGF on apoptosis HPAECs was also examined by TUNEL staining and active caspase-3 immunoassay.

Results

Exogenous VEGF significantly decreased LPS-induced extravascular albumin leakage and edema formation. Treatment with anti-VEGF antibody significantly enhanced lung edema formation and neutrophil emigration after intratracheal LPS administration, whereas extravascular albumin leakage was not significantly changed by VEGF blockade. In lung pathology, pretreatment with VEGF significantly decreased the numbers of TUNEL positive cells and those with positive immunostaining of the pro-apoptotic molecules examined. VEGF attenuated the increases in the permeability of the HPAEC monolayer and the apoptosis of HPAECs induced by TNF-α and LPS. In addition, VEGF significantly reduced the levels of TNF-α- and LPS-induced active caspase-3 in HPAEC lysates.

Conclusion

These results suggest that VEGF suppresses the apoptosis induced by inflammatory stimuli and functions as a protective factor against acute lung injury.     

Dysregulated angiogenesis in B-chronic lymphocytic leukemia: Morphologic, immunohistochemical, and flow cytometric evidence

Background

The extent of enhanced bone marrow angiogenesis in chronic lymphocytic leukemia (CLL) and relationship to proangiogenic factors and prognostic indicators is largely unexplored.

Methods

To further investigate the role of angiogenesis in CLL by evaluating the topography and extent of angiogenesis in a group of CLL bone marrow biopsies, to study the expression of pro and antiangiogenic vascular factors in CLL cells to more precisely document the cell types producing these factors, and to evaluate the role, if any, of localized hypoxia in upregulation of angiogenesis in CLL We used immunohistochemistry (IHC) (n = 21 pts) with antibodies to CD3 and CD20, proangiogenic (VEGF, HIF-1a) and antiangiogenic (TSP-1) factors, and VEGF receptors -1 and -2 to examine pattern/extent of CLL marrow involvement, microvessel density (MVD), and angiogenic characteristics; flow cytometry (FC) was performed on 21 additional cases for VEGF and TSP-1.

Results

CLL patients had higher MVD (23.8 vs 14.6, p~0.0002) compared to controls (n = 10). MVD was highest at the periphery of focal infiltrates, was not enhanced in proliferation centers, and was increased irrespective of the presence or absence of cytogenetic/immunophenotypic markers of aggressivity. By IHC, CLL cells were VEGF(+), HIF-1a (+), TSP-1(-), VEGFR-1(+), and VEGFR-2(+). By FC, CLL cells were 1.4–2.0-fold brighter for VEGF than T cells and were TSP-1(-).

Conclusion

CLL demonstrates enhanced angiogenesis, with increased MVD, upregulated VEGF and downregulated TSP-1. Upregulation of HIF-1a in all CLL cases suggests localized tissue hypoxia as an important stimulant of microvessel proliferation. The presence of VEGF receptors on CLL cells implies an autocrine effect for VEGF. Differences in MVD did not correlate with traditional genetic/immunophenotypic markers of aggressiveness.     

Lipocalin 2 is protective against E. coli pneumonia

Background

Little is known about airway remodelling in bronchial biopsies (BB) in smokers and chronic obstructive pulmonary disease (COPD). We conducted an initial pilot study comparing BB from COPD patients with nonsmoking controls. This pilot study suggested the presence of reticular basement membrane (Rbm) fragmentation and altered vessel distribution in COPD.

Methods

To determine whether Rbm fragmentation and altered vessel distribution in BB were specific for COPD we designed a cross-sectional study and stained BB from 19 current smokers and 14 ex-smokers with mild to moderate COPD and compared these to 15 current smokers with normal lung function and 17 healthy and nonsmoking subjects.

Results

Thickness of the Rbm was not significantly different between groups; although in COPD this parameter was quite variable. The Rbm showed fragmentation and splitting in both current smoking groups and ex-smoker COPD compared with healthy nonsmokers (p < 0.02); smoking and COPD seemed to have additive effects. Rbm fragmentation correlated with smoking history in COPD but not with age. There were more vessels in the Rbm and fewer vessels in the lamina propria in current smokers compared to healthy nonsmokers (p < 0.05). The number of vessels staining for vascular endothelial growth factor (VEGF) in the Rbm was higher in both current smoker groups and ex-smoker COPD compared to healthy nonsmokers (p < 0.004). In current smoker COPD VEGF vessel staining correlated with FEV1% predicted (r = 0.61, p < 0.02).

Conclusions

Airway remodelling in smokers and mild to moderate COPD is associated with fragmentation of the Rbm and altered distribution of vessels in the airway wall. Rbm fragmentation was also present to as great an extent in ex-smokers with COPD. These characteristics may have potential physiological consequences.     

Treatment with the arginase inhibitor Nw-hydroxy-nor-L-arginine restores endothelial function in rat adjuvant-induced arthritis

Introduction

Endothelial dysfunction (ED) participates to atherogenesis associated to rheumatoid arthritis. We recently reported increased arginase activity/expression in vessels from adjuvant-induced arthritis (AIA) rats. In the present study, we investigated the effects of a curative treatment with the arginase inhibitor N_w-hydroxy-nor-L-arginine (nor-NOHA) on vascular dysfunction in AIA rats.

Methods

AIA rats were treated with nor-NOHA (40 mg/kg/d, ip) for 21 days after the onset of arthritis. A group of untreated AIA rats and a group of healthy rats served as controls. ED was assessed by the vasodilatory effect of acetylcholine (Ach) on aortic rings. The role of superoxide anions, prostanoids, endothelium-derived hyperpolarizing factor (EDHF) and nitric oxide synthase (NOS) pathway was studied. Plasma levels of IL-6 and vascular endothelial growth factor (VEGF) were determined by ELISA kits. Arthritis severity was estimated by a clinical, radiological and histological analysis.

Results

Nor-NOHA treatment fully restored the aortic response to Ach to that of healthy controls. The results showed that this beneficial effect is mediated by an increase in NOS activity and EDHF and reduced superoxide anion production as well as a decrease in the activity of cyclooxygenase (COX)-2, thromboxane and prostacyclins synthases. In addition, nor-NOHA decreased IL-6 and VEGF plasma levels in AIA rats. By contrast, the treatment did not modify arthritis severity in AIA rats.

Conclusions

The treatment with an arginase inhibitor has a potent effect on ED in AIA independently of the severity of the disease. Our results suggest that this new pharmacological approach has the potential as a novel add-on therapy in the treatment of RA.     

Mechanistic insight into activation of MAPK signaling by pro-angiogenic factors

Background

Angiogenesis is important in physiological and pathological conditions, as blood vessels provide nutrients and oxygen needed for tissue growth and survival. Therefore, targeting angiogenesis is a prominent strategy in both tissue engineering and cancer treatment. However, not all of the approaches to promote or inhibit angiogenesis lead to successful outcomes. Angiogenesis-based therapies primarily target pro-angiogenic factors such as vascular endothelial growth factor-A (VEGF) or fibroblast growth factor (FGF) in isolation. However, pre-clinical and clinical evidence shows these therapies often have limited effects. To improve therapeutic strategies, including targeting FGF and VEGF in combination, we need a quantitative understanding of the how the promoters combine to stimulate angiogenesis.

Results

In this study, we trained and validated a detailed mathematical model to quantitatively characterize the crosstalk of FGF and VEGF intracellular signaling. This signaling is initiated by FGF binding to the FGF receptor 1 (FGFR1) and heparan sulfate glycosaminoglycans (HSGAGs) or VEGF binding to VEGF receptor 2 (VEGFR2) to promote downstream signaling. The model focuses on FGF- and VEGF-induced mitogen-activated protein kinase (MAPK) signaling and phosphorylation of extracellular regulated kinase (ERK), which promotes cell proliferation. We apply the model to predict the dynamics of phosphorylated ERK (pERK) in response to the stimulation by FGF and VEGF individually and in combination. The model predicts that FGF and VEGF have differential effects on pERK. Additionally, since VEGFR2 upregulation has been observed in pathological conditions, we apply the model to investigate the effects of VEGFR2 density and trafficking parameters. The model predictions show that these parameters significantly influence the response to VEGF stimulation.

Conclusions

The model agrees with experimental data and is a framework to synthesize and quantitatively explain experimental studies. Ultimately, the model provides mechanistic insight into FGF and VEGF interactions needed to identify potential targets for pro- or anti-angiogenic therapies.

     

Mural Cell Associated VEGF Is Required for Organotypic Vessel Formation

Background

Blood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics.

Methods and Findings

To better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF.

Conclusions

These results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation.