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Patients suffering from both diabetes and PAD (peripheral arterial disease) are at risk of developing critical limb ischaemia and ulceration, and potentially requiring limb amputation. In addition, diabetes complicates surgical treatment of PAD and impairs arteriogenesis. Arteriogenesis is defined as the remodelling of pre-existing arterioles into conductance vessels to restore the perfusion distal to the occluded artery. Several strategies to promote arteriogenesis in the peripheral circulation have been devised, but the mechanisms through which diabetes impairs arteriogenesis are poorly understood. The present review provides an overview of the current literature on the deteriorating effects of diabetes on the key players in the arteriogenesis process. Diabetes affects arteriogenesis at a number of levels. First, it elevates vasomotor tone and attenuates sensing of shear stress and the response to vasodilatory stimuli, reducing the recruitment and dilatation of collateral arteries. Secondly, diabetes impairs the downstream signalling of monocytes, without decreasing monocyte attraction. In addition, EPC (endothelial progenitor cell) function is attenuated in diabetes. There is ample evidence that growth factor signalling is impaired in diabetic arteriogenesis. Although these defects could be restored in animal experiments, clinical results have been disappointing. Furthermore, the diabetes-induced impairment of eNOS (endothelial NO synthase) strongly affects outward remodelling, as NO signalling plays a key role in several remodelling processes. Finally, in the structural phase of arteriogenesis, diabetes impairs matrix turnover, smooth muscle cell proliferation and fibroblast migration. The review concludes with suggestions for new and more sophisticated therapeutic approaches for the diabetic population.

BACKGROUND AND METHODS: Novel approaches to treat chemo-refractory metastatic breast cancer (MBC) are currently under investigation. This retrospective series reviews the outcome of 70 MBC patients who have participated in 30 phase I trials at the Royal Marsden Hospital from 2002 to 2009.RESULTS: The median treatment lines before phase I trial entry for MBC was 5 (range: 1-12 lines). The overall response rate was 11.4% (95% CI: 4.0-18.9%) and the clinical benefit rate at 4 months was 20% (95% CI: 10.6-29.3). The median time to progression was 7.0 weeks (95% CI: 6.4-7.5) and median overall survival was 8.7 months (95% CI: 7.6-9.8) from start of first phase I treatment. No patients discontinued trial because of treatment-related toxicities. Abnormal lactate dehydrogenase, serum albumin or=5 previous treatment lines, liver metastases and Eastern Cooperative Group performance status >or=2 at study entry were significantly associated with poor overall survival in multivariate analysis.CONCLUSION: This retrospective analysis provides evidence that patients with MBC tolerate phase I clinical trials and a significant proportion of patients with chemo-refractory disease, particularly those with triple-negative or Her2-positive breast cancer, may benefit from treatment.

Summary Background In animal models, inhaled nitric oxide improved gas exchange and lung structural development, but its use in premature infants at risk of developing bronchopulmonary dysplasia remains controversial. We therefore tested the hypothesis that inhaled nitric oxide at a low concentration, started early and maintained for an extended period in babies with mild respiratory failure, might reduce the incidence of bronchopulmonary dysplasia. Methods 800 preterm infants with a gestational age at birth of between 24 weeks and 28 weeks plus 6 days (inclusive), weighing at least 500 g, requiring surfactant or continuous positive airway pressure for respiratory distress syndrome within 24 h of birth were randomly assigned in a one-to-one ratio to inhaled nitric oxide (5 parts per million) or placebo gas (nitrogen gas) for a minimum of 7 days and a maximum of 21 days in a double-blind study done at 36 centres in nine countries in the European Union. Care providers and investigators were masked to the computer-generated treatment assignment. The primary outcome was survival without development of bronchopulmonary dysplasia at postmenstrual age 36 weeks. Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00551642. Findings 399 infants were assigned to inhaled nitric oxide, and 401 to placebo. 395 and 400, respectively, were analysed. Treatment with inhaled nitric oxide and placebo did not result in significant differences in survival of infants without development of bronchopulmonary dysplasia (258 [65%] of 395 vs 262 [66%] of 400, respectively; relative risk 1·05, 95% CI 0·78–1·43); in survival at 36 weeks' postmenstrual age (343 [86%) of 399 vs 359 [90%] of 401, respectively; 0·74, 0·48–1·15); and in development of bronchopulmonary dysplasia (81 [24%] of 339 vs 96 [27%] of 358, respectively; 0·83, 0·58–1·17). Interpretation Early use of low-dose inhaled nitric oxide in very premature babies did not improve survival without bronchopulmonary dysplasia or brain injury, suggesting that such a preventive treatment strategy is unsuccessful. Funding INO Therapeutics.

The hypoxia-inducible factor 1 (HIF-1) pathway is induced in many tumors and associated with poorer outcome. The hypoxia-responsive transcription factor HIF-1alpha dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT), which is also an important binding partner for the aryl hydrocarbon receptor (AhR). AhR is an important mediator in the metabolic activation and detoxification of carcinogens, such as the environmental pollutant benzo[a]pyrene (BaP). We hypothesized that HIF-1alpha activation attenuates BaP-induced AhR-mediated gene expression, which may lead to increased genetic instability and malignant progression. Human lung carcinoma cells (A549) were simultaneously stimulated with CoCl(2), which leads to HIF-1alpha stabilization and varying concentrations of BaP. Both quantitative PCR and immunoblot analysis indicated that induction of the hypoxia response pathway significantly reduced the levels of AhR downstream targets CYP1A1 and CYP1B1 and AhR protein binding to ARNT. We further demonstrate that the BaP-induced hypoxanthine-guanine phosphoribosyltransferase mutation frequency and gamma-H2AX foci were markedly amplified when the HIF-1 pathway was induced. BaP-DNA adducts were only marginally increased, and transient strand breaks were diminished by HIF-1 induction, indicating changes in DNA repair. These data indicate that concurrent exposure of tumor cells to hypoxia and exogenous genotoxins can enhance genetic instability.

Angiogenesis is a critical step in tumor development, in which vascular endothelial growth factor (VEGF) is a key growth aspect. Heat shock protein 90 (HSP90), a molecular chaperone, is essential for the activity of key proteins involved in VEGF transcription. Currently, no biomarkers to predict the effect of, or monitor, HSP90 inhibition therapy in individual patients exist. (89)Zr-bevacizumab PET provides a noninvasive tool to monitor tumor VEGF levels. The aim of this study was to investigate (89)Zr-bevacizumab PET for early antiangiogenic tumor response evaluation of treatment with the new HSP90 inhibitor NVP-AUY922. In xenografts of A2780 and its cisplatin-resistant CP70 human ovarian cancer subline, (89)Zr-bevacizumab small-animal PET was performed before and after NVP-AUY922 treatment and verified with histologic response and ex vivo tumor VEGF levels. Compared with pretreatment values, 2 wk of NVP-AUY922 treatment decreased (89)Zr-bevacizumab uptake by 44.4% (P = 0.0003) in A2780 xenografts, whereas tumor uptake was not affected in CP70 xenografts. The same pattern was observed in A2780 and CP70 tumor VEGF levels, measured with enzyme-linked immunosorbent assay, and mean vessel density after NVP-AUY922 treatment. These findings coincided with reduction in the proliferation rate, assessed by Ki67 staining, in A2780 tumor tissue only. CONCLUSION: (89)Zr-bevacizumab PET was in line with the antiangiogenic response and direct antitumor effects after NVP-AUY922 treatment, supporting the specificity of (89)Zr-bevacizumab PET as a sensitive technique to monitor the antiangiogenic response of HSP90 inhibition in vivo.

Background:The importance of angiogenesis in solid tumour growth is well recognised. Tumour angiogenesis is considered the result of an imbalance between pro- and anti-angiogenic factors produced by both the malignancy and normal cells. Endoglin (CD105) is a proliferation-associated, hypoxia-inducible glycoprotein that seems to be clinically superior to other pan-endothelial markers in the selective evaluation of tumour angiogenesis. Several studies have revealed CD105 up-regulation in a wide range of tumour endothelia. Since 2002, endothelial CD105 expression has also been retrospectively investigated in patients with head and neck squamous cell carcinoma (HNSCC).Methods:An exhaustive literature review was performed to investigate available evidence on CD105 expression and its biological role and therapeutic potential in HNSCC.Results:The available evidence supports the hypothesis that CD105 expression in HNSCC may be a valuable parameter for pinpointing patients at greater risk of recurrent malignancy and with a worse prognosis. A high CD105 expression in HNSCC was associated with metastatic lymph nodes in most of the studies.Conclusions:Prospective studies are mandatory to confirm that CD105 expression is a significant prognostic hallmark in HNSCC. The results of prospective studies could be relevant for the adoption of stricter follow-up protocols and/or alternative therapeutic regimens for patients with a high CD105 expression in HNSCC. Great interest is currently being focused on vascular targeting for therapeutic purposes. Preclinical studies on appropriate animal models resembling HNSCC to investigate the effects of inhibiting CD105 may show the efficacy of combined treatment strategies associating angiogenic-targeted with conventional therapies for HNSCC.

Endometrial Mesenchymal Stromal Cells (endMSCs) are multipotent cells with immunomodulatory and pro-regenerative activity which is mainly mediated by a paracrine effect. The exosomes released by MSCs have become a promising therapeutic tool for the treatment of immune-mediated diseases. More specifically, extracellular vesicles derived from endMSCs (EV-endMSCs) have demonstrated a cardioprotective effect through the release of anti-apoptotic and pro-angiogenic factors. Here we hypothesize that EV-endMSCs may be used as a co-adjuvant to improve in vitro fertilization outcomes and embryo quality. Firstly, endMSCs and EV-endMSCs were isolated and phenotypically characterized for in vitro assays. Then, in vitro studies were performed on murine embryos co-cultured with EV-endMSCs at different concentrations. Our results firstly demonstrated a significant increase on the total blastomere count of expanded murine blastocysts. Moreover, EV-endMSCs triggered the release of pro-angiogenic molecules from embryos demonstrating an EV-endMSCs concentration-dependent increase of VEGF and PDGF-AA. The release of VEGF and PDGF-AA by the embryos may indicate that the beneficial effect of EV-endMSCs could be mediating not only an increase in the blastocyst's total cell number, but also may promote endometrial angiogenesis, vascularization, differentiation and tissue remodeling. In summary, these results could be relevant for assisted reproduction being the first report describing the beneficial effect of human EV-endMSCs on embryo development. This work was supported in part by CIBER-CV (CB16/11/00494). One grant from Junta de Extremadura (Ayuda a grupos catalogados de la Junta de Extremadura, GR15175). Two grants from Junta de Extremadura to JGC (TA13042 and 1616168 co-financed by FEDER/FSE). One grant to B-MC (IB16159 co-financed by FEDER/FSE). One grant ``Miguel Servet I´´ from Instituto de Salud Carlos III to JGC (CP17/00021 co-financed by FEDER/FSE). One grant ``Juan de la Cierva´´ to B-MC from Spanish Ministry of Economy, Industry and Competitiveness (IJCI-2014-19428). The funders had no role in study designs, data collection and analysis, decision to publish or preparation of the manuscript. Sí

Objective: Transplant vasculopathy consists of neointima formation in graft vasculature resulting from vascular smooth muscle cell recruitment and proliferation. Variation in the severity of vasculopathy has been demonstrated. Genetic predisposition is suggested as a putative cause of this variation, although cellular mechanisms are still unknown. Using a rat aorta transplant model we tested the hypothesis that kinetics of development of transplant vasculopathy are related to neointimal smooth muscle cell proliferative capacity and fibrocyte frequency, the latter being putative neointimal smooth muscle ancestral cells.Methods: Aortic allografts were transplanted in Lewis and Brown Norway, as well as MHC-congenic Lewis. 1N and Brown Norway. 1L recipients. Severity of transplant vasculopathy was quantified 4, 8, 12 and 24 weeks after transplantation. Host-endothelial chimerism, as a reflection of vascular injury, was determined by specific immunofluorescence. Neointimal smooth muscle cell proliferative capacity was determined in vitro and in situ. Fibrocyte frequency and phenotype were determined after in vitro culture by cell counting, immunofluorescence and in situ zymography.Results: Compared to Lewis, Brown Norway recipients developed accelerated transplant vasculopathy which is dependent on the presence of Brown Norway non-MHC-encoded determinants. Accelerated transplant vasculopathy was associated with increased levels of host-endothelial chimerism and increased neointimal smooth muscle cell proliferation, the latter being accompanied by increased endothelial and smooth muscle cell-derived neuropilin-like protein mRNA expression. Moreover, accelerated transplant vasculopathy was associated with increased frequency of circulating gelatinase-expressing CD45(+) vimentin(+) fibrocytes.Conclusion: Susceptibility for transplant vasculopathy appears to be genetically controlled and correlates with neointimal smooth muscle cell proliferative capacity and circulating fibrocyte frequency. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Angiogenesis plays an important role in the pathogenesis of inflammatory diseases, including rheumatoid arthritis ( RA). The site and extent of inflammation and subsequent joint destruction in the rheumatoid synovium is dependent on the development of new vasculature. Inhibition of angiogenesis, extensively studied in cancer, might therefore be of interest as treatment option for RA. Hypoxia-inducible factor-1 (HIF-1) has been reported to play a critical role in the regulation of hypoxia driven angiogenesis. HIF-1 is a transcription factor that is constitutively expressed in many cells. It gains transcriptional activity in hypoxic cells leading to the expression of genes involved in angiogenesis. The synovium is hypoxic, but also in an inflammatory environment such as seen in RA, inflammatory cytokines may be important inducers of HIF-1 expression and/or activation. Many drugs currently used in the treatment of RA have anti-angiogenic effects, which are exerted at different levels. Blocking of TNF-alpha, for instance, reduces TNF-alpha induced VEGF production. Studies aiming at direct inhibition of pro-angiogenic factors, such as inhibiting VEGF- or FGF-receptor signalling or blocking VEGF by monoclonal anti-VEGF antibody therapeutics, are in progress. Inhibition of HIF-1 expression or activation, either by blocking signal transduction pathways leading to HIF-1 induction or by inhibiting accumulation of HIF-1 protein, represents a new strategy, which is of interest for the treatment of RA. This review will concisely summarize the general knowledge on the molecular control of gene expression by HIF-1, its involvement in RA, and potential for therapeutic intervention at the level of HIF-1 activity.

Endocardial cushion defects (ECDs) of the cardiac outflow tract are among the most common congenital heart disease phenotypes. VEGF is essential for endocardial cushion formation and derangements in VEGF synthesis lead to ECD. Three functional single nucleotide polymorphisms (SNPs) in the VEGF gene -2578 C>A, -1154 G>A, and -634 G>C play a role in cardiogenesis. In a Dutch case-control family study of triads, 190 case and 317 control children with both parents, we investigated linkage and association between these VEGF SNPs and ECD. Allele frequencies for the three VEGF SNPs were comparable between ECD children and controls. However, VEGF alleles -2578 C and 1154 G were transmitted more frequently to children with ECD (p = 0.003 and p = 0.002), in particular perimembranous ventricular septal defects (p = 0.012 and p = 0.006). The -2578A/-1154A/-634G haplotype was associated with a reduced risk of ECD (OR 0.7: 95% CI, 0.6-1.0) and was significantly less transmitted to children with ECD U) = 0.002). In a Dutch population, we show that the VEGF 2578 C, -1154 G alleles, and the AAG haplotype are associated with ECD. Possible VEGF gene-enviromnent interactions exposures are discussed. (Pediatr Res 67: 23-28, 2010)

Relaxin is a naturally occurring peptide hormone that plays a central role in the hemodynamic and renovascular adaptive changes that occur during pregnancy. Triggering similar changes could potentially be beneficial in the treatment of patients with heart failure. The effects of relaxin include the production of nitric oxide, inhibition of endothelin, inhibition of angiotensin II, production of VEGF, and production of matrix metalloproteinases. These effects lead to systemic and renal vasodilation, increased arterial compliance, and other vascular changes. The recognition of this has led to the study of relaxin for the treatment of heart failure. An initial pilot study has shown favorable hemodynamic effects in patients with heart failure, including reduction in ventricular filling pressures and increased cardiac output. The ongoing RELAX-AHF clinical program is designed to evaluate the effects of relaxin on the symptoms and outcomes in a large group of patients admitted to hospital for acute heart failure. This review will summarize both the biology of relaxin and the data supporting its potential efficacy in human heart failure.

Background. Daily peritoneal exposure to peritoneal dialysis fluid (PDF) induces severe morphological alterations including fibrosis and angiogenesis that lead to a loss of peritoneal ultrafiltration (UF) capacity. Since cyclooxygenase (COX)-2 is involved in fibrosis and angiogenesis, we investigated the in vivo effects of a selective COX-2 inhibitor (celecoxib) in a rat-PD model.Methods. Sixteen rats daily received 10 ml of conventional PDF for 4-5 weeks intraperitoneally. Half of them (n = 8) daily received celecoxib (20 mg/kg BW) via oral gavage, and the other half (n = 8) received vehicle via oral gavage. The study also included two control groups (no PDF instillations), each consisting of n = 8 animals that daily received celecoxib or vehicle, respectively, via oral gavage. Functional, morphological and cellular parameters were analysed.Results. PDF exposure induced an inflammatory condition evidenced by the increased leucocyte number and synthesis of MCP-1, VEGF and hyaluronic acid. After PDF exposure, the omentum showed intense angiogenesis and milky spots formation. Parietal peritoneum showed increased angiogenesis, lymphangiogenesis, submesothelial matrix thickness and enhanced expression of mesothelial aquaporin1 (Aqp1). Concomitant PDF and celecoxib exposure drastically reduced PGE2 levels, angiogenesis, lymphangiogenesis, fibrosis and milky spot formation in studied tissues, but did not modify mesothelial Aqp1 expression nor the tissue expression of VEGF and inflammatory markers. PDF exposure induced severe UF failure that celecoxib treatment completely prevented.Conclusions. Altogether, celecoxib treatment improves UF capacity and reduces morphological alterations in our rat PD model.

Combining photodynamic therapy (PDT) using verteporfin (Visudyne(R)) with ranibizumab (Lucentis(R)) can optimize the overall treatment outcome by providing more efficacy in vessel closure, and thus reduce the need for retreatment in patients with wet age-related macular degeneration. In this preclinical study in the chorioallantoic membrane (CAM) of the chicken embryo, we compare the vascular occlusion effects of verteporfin and ranibizumab as monotherapies with those observed in the combined therapy. In order to optimize the combination therapy, we varied the timing and sequence of the PDT and antivascular endothelial growth factor modalities. We observed that 1 day after PDT, the smaller blood vessels (null set < 70 mu m) of the CAM were selectively occluded, but as early as 2 days after PDT, both significant reperfusion and regrowth of new vessels were observed. Both these phenomena could be significantly delayed by application of ranibizumab. Ranibizumab itself did not induce any vascular occlusion. Under the applied conditions of combination therapy, the occlusion of the targeted blood vessels could be significantly extended to 3 days in this model compared with 1 day in the case of verteporfin monotherapy. Thus, in the present preclinical study, we demonstrate that for the applied conditions, the optimal time to administer ranibizumab is 24 h after PDT.

Purpose To identify genetic markers in the pharmacokinetic and pharmacodynamic pathways of sunitinib that predispose for development of toxicities: thrombocytopenia, leukopenia, mucosal inflammation, hand-foot syndrome, and any toxicity according to National Cancer Institute Common Toxicity Criteria higher than grade 2. Patients and Methods A multicenter pharmacogenetic association study was performed in 219 patients treated with single-agent sunitinib. A total of 31 single nucleotide polymorphisms in 12 candidate genes, together with several nongenetic variants, were analyzed for a possible association with toxicity. In addition, genetic haplotypes were developed and related to toxicity. Results The risk for leukopenia was increased when the G allele in CYP1A1 2455A/G (odds ratio [OR], 6.24; P = .029) or the T allele in FLT3 738T/C (OR, 2.8; P = .008) were present or CAG in the NR1I3 (5719C/T, 7738A/C, 7837T/G) haplotype (OR, 1.74; P = .041) was absent. Any toxicity higher than grade 2 prevalence was increased when the T allele of vascular endothelial growth factor receptor 2 1191C/T (OR, 2.39; P = .046) or a copy of TT in the ABCG2 (−15622C/T, 1143C/T) haplotype (OR, 2.63; P = .016) were present. The risk for mucosal inflammation was increased in the presence of the G allele in CYP1A1 2455A/G (OR, 4.03; P = .021) and the prevalence of hand-foot syndrome was increased when a copy of TTT in the ABCB1 (3435C/T, 1236C/T, 2677G/T) haplotype (OR, 2.56; P = .035) was present. Conclusion This exploratory study suggests that polymorphisms in specific genes encoding for metabolizing enzymes, efflux transporters, and drug targets are associated with sunitinib-related toxicities. A better understanding of genetic and nongenetic determinants of sunitinib toxicity should help to optimize drug treatment in individual patients.

Background & Aims: Vascular endothelial growth factor (VEGF) is expressed robustly in human colon neoplasia and is a major new "rational" target of therapy for cancers of the colon and other organs. Nonetheless, the mechanism(s) of action of VEGF-targeted therapies and the biologic roles of VEGF in tumorigenesis have not been well defined. We used a transgenic approach to directly test the hypothesis that augmented VEGF expression can drive progression of intestinal neoplasia. Methods: Transgenic mouse lines were generated with moderate (vilVEGF1) and high (vilVEGF2) VEGF expression from the intestinal epithelium. vilVEGF1 mice were bred to Min mice (adenomatous polyposis coli [APC] +/-). Colon epithelial cells from an APC patient were cocultured with endothelial cells and fibroblasts. Results: viIVEGF mice were generally healthy but displayed red small intestines. Vessels were larger and more numerous in the submucosa but not the mucosa. The mucosa showed striking stromal and epithelial hypercellularity, with increased epithelial proliferation. Many crypts formed cysts composed of relatively undifferentiated epithelial cells surrounded by cells with endothelial and myofibroblast markers. Compared with Min controls, vilVEGF1-Min mice developed 6-fold more intestinal adenomas of all sizes, with more advanced histologic features. Polycystic masses were also observed. Coculture of human colonocytes with endothelial cells and fibroblasts directly stimulated colonocyte proliferation. Conclusions: Augmented VEGF expression from intestinal epithelium potently stimulated cross talk with mesenchymal cells and proliferation of normal and neoplastic epithelium. These effects of VEGF, largely occurring prior to the canonical angiogenic switch in tumors, may be in part independent of angiogenesis.

Our group has shown that 1-year smoking cessation persisted or increased airway inflammation in chronic obstructive pulmonary disease (COPD). We compared adenosine and adenosine receptor (AR) expression in COPD and asymptomatic smokers (AS) before and after 1-year smoking cessation. Sputum cytospins and bronchial biopsies of (ex)smoking COPD patients and AS were studied for A(1)R, A(2A)R, A(2B)R, and A(3)R expression. Adenosine and inflammatory mediators were measured in sputum supernatants. At baseline, COPD patients had lower levels of adenosine and higher levels of vascular endothelial growth factor in sputum than AS. Smoking cessation induced significantly different effects in COPD than in AS, i.e. an increase in percentages of A(3)R expressing neutrophils and A(1)R expressing macrophages in COPD as increase in adenosine and monocyte chemoattractant protein-1 levels in sputum. Adenosine-related effector mechanisms may contribute to the persistence and progression of airway inflammation in COPD following 1-year smoking cessation.

Background. Brain death and cold preservation are major alloantigen-independent risk factors for transplantation Outcome. The present study was conducted to assess the influence of these factors on transplantation-associated injury independently or in combination.Methods. Brain death was induced in F344 rats. Renal grafts were harvested after 6 hr and either directly transplanted in unilateral nephrectomized Lewis recipient or Subjected to 24 hr of cold preservation in University of Wisconsin solution before implantation. Allografts obtained from living donor rats were also subjected to cold preservation or not. DNA damage was assessed before implantation by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining. Ten days after transplantation, renal histology was performed according to Banff '97 classification. The expressions of cytokines and adhesion molecules were analyzed by quantitative polymerase chain reaction.Results. Cold preservation significantly increased the number of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling positive cells in renal allografts. Ten days after transplantation, histology revealed a higher degree of tubulitis and vasculitis scores when the grafts were Subjected to cold storage. Vasculitis was aggravated when the graft was obtained from brain death (BD) donors. BD, but not cold preservation alone, was associated with papillary necrosis. This was more frequently observed after cold preservation. Immunohistology showed an increase in MHC class II+ cells after cold preservation. The combination of BD and cold preservation revealed a higher degree of VEGF and IL-10 expression.Conclusions. Our Study emphasizes that cold ischemia time should be limited when renal allografts from brain-dead donors are transplanted.

Object. In the present study, the authors analyzed the ANGPT1/ANGPT2 balance in the context of therapeutic outcome in 62 patients with primary glioblastomas multiforme (GBMs).Methods. The tumor tissue used was obtained in adult patients who underwent neurosurgical debulking. Microvessel density was assessed by morphometric analysis, Double immunostaining for Ki 67/CD34 and cleaved caspase-3/CD34 was used to investigate the proliferation and apoptotic fraction of both endothelial and tumor cells. The expression of VEGFs (A-D) was evaluated on immunohistochemistry. To measure tumor vascular stabilization, the ANGPT1/ANGPT2 mRNA balance was determined using real-time reverse transcriptase polymerase chain reaction.Results. Within the hypoxic perinecrotic tumor area, the apoptotic fraction of endothelial cells was positively correlated with VEGFA expression (p Conclusions. The results of the present study suggest that the ANGPT1 /ANGPT2 balance has prognostic value in patients with primary GBMs. The authors' findings support the need for further studies of the feasibility of antiangiogenic therapy in primary GBMs, with a special focus on the normalization of tumor vasculature. (DOI: 10.3171/2008.6.17612)

Vascular endothelial growth factor (VEGF), a potent growth factor for endothelial cells and inducer of angiogenesis, is important for endothelial integrity and thus for vascular function. On the other hand, VEGF may enhance the pathophysiologic mechanism of plaque formation and plaque destabilization. In this review we discuss the data available so far for VEGF as angiogenic and/or inflammatory cytokine in the vulnerable atherosclerotic plaque.

Vascular endothelial growth factor (VEGF), a potent growth factor for endothelial cells and inducer of angiogenesis, is important for endothelial integrity and thus for vascular function. On the other hand, VEGF may enhance the pathophysiologic mechanism of plaque formation and plaque destabilization. In this review we discuss the data available so far for VEGF as angiogenic and/or inflammatory cytokine in the vulnerable atherosclerotic plaque.

Erythropoietin is a haematopoietic hormone with extensive non-haematopoietic effects. The discovery of an erythropoietin receptor outside the haematopoietic system has fuelled the research into the beneficial effects of erythropoietin for various conditions, predominantly in cardiovascular disease. Experimental evidence has revealed the cytoprotective and angiogenic properties of erythropoietin and it seems that the erythropoietin-erythropoietin receptor system provides a powerful backbone against acute and chronic myocardial ischemia, each gaining from the different properties of erythropoietin. Clinical trials in which erythropoietin was titrated to achieve certain haematocrit levels have generated equivocal results. It has been suggested that a (too) high haematocrit is undesirable in cardiovascular disease. We have shown that intermittent (low-dose) erythropoietin administration, that does not increase haematocrit substantially, suffices to activate the beneficial downstream pathways of erythropoietin. We postulate that intermittent administration or a lower than conventional dose of erythropoietin, not only aimed at increasing haemoglobin at high levels, will provide powerful cellular protection and will improve cardiac outcome, without the side effects of erythropoietin associated with increased haematocrit. (C) 2008 Elsevier B.V. All rights reserved.

Acute myeloid leukemia (AML) is a disease with a poor prognosis. It has been demonstrated that AML cells express the vascular endothelial growth factors, VEGFA and VEGFC, as well as kinase insert domain-containing receptor (VEGFR2), the main receptor for downstream effects, resulting in an autocrine pathway for cell survival. This study investigates the role of the VEGFR inhibitor PTK787/ZK 222584 in leukemic cell death, and the possibility of an additional effect on cell death by a chemotherapeutic drug, amsacrine. In three AML cell lines and 33 pediatric AML patient samples, we performed total cell-kill assays to determine the percentages of cell death achieved by PTK787/ZK 222584 and/or amsacrine. Both drugs induced AML cell death. Using a response surface analysis, we could show that, in cell lines as well as in primary AML blasts, an equal magnitude of leukemic cell death could be obtained when lower doses of the more toxic amsacrine were combined with low dosages of the less toxic VEGFR inhibitor. This study shows that PTK787/ ZK 222584 might have more clinical potential in AML when combined with a chemotherapeutic drug such as amsacrine. In future, it will be interesting to study whether the complications and the long-term effects of chemotherapy can be reduced by lowering the dosages of amsacrine, and by replacing it with other drugs with lower toxicity profiles, such as PTK787/ZK 222584.

The advanced glycation end product (AGE)-receptor for AGE (RAGE) pathway is involved in the pathogenesis of diabetic microvascular damage. The special distribution of RAGE and its engagement has an impact on the development of diabetic retinopathy. In the present study, we used immunofluorescence and confocal laser microscopy to study RAGE expression with special emphasis on Muller glia in Sprague Dawley rats. RAGE expression was low in nondiabetic retinae and was found in ganglion cells and Muller cell end feet. In diabetic retinae, upregulated RAGE was predominantly expressed in retinal glia. Since Muller cells are important in the regulation of important features of early retinal vascular damage, such as vascular permeability, homeostasis, and response to stress, RAGE appears to be a central modulator in diabetic retinopathy.

Erythropoietin (EPO) mobilises endothelial progenitor cells and promotes neovascularisation in heart failure. The present authors studied the effects of EPO on pulmonary vascular and cardiac remodelling in a model for flow-associated pulmonary arterial hypertension (PAH). PAH was induced in adult male Wistar rats by the injection of monocrotaline combined with an abdominal aortocaval shunt 1 week later (PAH or experimental group). Immediately afterwards, rats were randomised into those who received treatment with EPO (PAH+EPO group) and controls. Pulmonary and systemic haemodynamics, and right ventricular and pulmonary vascular remodelling were evaluated 3 weeks later. Vascular occlusion of the intra-acinar pulmonary vessels (13.4 +/- 0.7 versus 16.7 +/- 1.3% in PAH+EPO and PAH, respectively) and medial wall thickness of the pre-acinar arteries (wall-to-lumen ratio 0.13 +/- 0.01 versus 0.17 +/- 0.01 in PAH+EPO and PAH, respectively) decreased after treatment with EPO. Moreover, right ventricular capillary density was increased by therapy (2,322 +/- 61 versus 2,100 +/- 63 capillaries mm(-2) in PAH+EPO and PAH, respectively). Increased mean pulmonary arterial pressure and decreased right ventricular contractility in the model were not altered by EPO treatment. In this rat model of flow-associated pulmonary arterial hypertension, erythropoietin treatment beneficially affected pulmonary vascular and cardiac remodelling. These histopathological effects were not accompanied by significantly improved haemodynamics.

Semliki Forest virus (SFV) is an efficient vector for cardiac gene delivery. The relatively short transgene expression induced by SFV seems appropriate for angiogenic gene therapy. We tested the effects of SFV expressing vascular endothelial growth factor (VEGF) on cardiac angiogenesis and heart failure in the mRen2 transgenic rat.Six-week-old mRen2 rats received SFV-VEGF or control virus (n=7 each) administered intracoronarily. Twelve days after transfection, cardiac capillary density and function were assessed. Capillary density in cardiac regions where SFV expression was highest had decreased by 20% in the SFV-VEGF-treated group. The decrease in capillary density was accompanied by impaired systolic function as illustrated by increased endsystolic volumes and a 34% decrease in cardiac output.We conclude that the time frame of SFV expression is sufficient to induce structural alterations, but that VEGF in mRen2 transgenic rats did not elicit the expected angiogenic effect. Rather, capillary density was decreased and subsequently cardiac function was impaired. This paradoxical finding is possibly related to the pathophysiology associated with this model and warrants caution if one is to pursue VEGF-mediated, angiogenic therapy before proceeding to a clinical setting. (Neth Heart J 2007;15:335-41.).

CD34(+) progenitor cells hold promise for therapeutic neovascularization in various settings. In this study, the role of human peripheral blood CD34(+) cells in neovascularization and inflammatory cell recruitment was longitudinally studied in vivo. Human CD34(+) cells were incorporated in Matrigel, implanted subcutaneously in nude mice, and explanted after 2, 4, 7, or 14 days. Cell-free Matrigels served as controls. Histochemical analyses demonstrated that neovascularization occurred almost exclusively in CD34(+) implants. Cellular and capillary density were increased in cell-loaded Matrigels after 2 days and further increased at 14 days. Human CD34(+) cells did not incorporate in neovessels, but formed vWF(+)/ CD31(+)/NEGF(+) cell clusters that were present up to day 14. However, CD34(+) cells induced host neovascularization, as demonstrated by increased presence of murine CD31(+) and vWF(+) vasculature from day 7 to 14. Moreover, recruitment of murine monocytes/macrophages was significantly enhanced in CD34(+) implants at all time points. Gene expression of chemotactic cytokines MCPA and IL-8 was detected on CD34(+) cells in vitro and confirmed imummohistochemically in cell-loaded explants at all time points. Our data indicate that human CD34(+) cells, implanted in a hypoxic environment, generate an angiogenic niche by secreting chemotactic and angiogenic factors, enabling rapid neovascularization, possibly via recruitment of monocytes/macrophages. (C) 2007 Elsevier Inc. All rights reserved.

Background Angiogenesis can be enhanced by several growth factors, like vascular endothelial growth factor-165 (VEGF(165)) and basic fibroblast growth factor (bFGF). Delayed release of such growth factors could be provided by incorporation of growth factors in fibrin matrices. In this study, we present a slow release system for VEGF(165) and bFGF in fibrin sealant.Materials and methods In vitro: Pieces of Integra (TM) matrix of 15 mm in diameter were prepared. Integra (TM) matrices were divided into four groups (A=control; B=fibrin sealant; C=fibrin sealant+growth factors; D=growth factors). In vivo: The bioartificial dermal templates were transplanted into a full-skin defect of the back of nu-nu mice. Four different groups included each six matrices at 2 and 4 weeks.Results In vitro: In groups C and D, continuous release of VEGF(165) and bFGF was eminent. The incorporation of growth factors into fibrin sealant evoked a prolonged growth factor release (p Conclusions A model of slow protein release by combining VEGF(165) and bFGF with fibrin sealant was produced. This model resulted in a prolonged bioavailability of growth factors in vivo for functional purposes. Fibrin and collagen can release growth factors in vivo and induce significant and faster neovascularisation in bioartificial dermal templates.

Most solid tumors express the cyclooxygenase-2 (COX-2) protein, a target of NSAIDs. COX-2 overexpression in tumorsis considered a predictor of more advanced stage disease and of worse prognosis in a number of studies investigating solid malignancies. Therefore, NSAIDs are evaluated as anti-cancer drugs. NSAIDs inhibit proliferation, invasiveness of tumors, and angiogenesis and overcome apoptosis resistance in a COX-2 dependent and independent manner. This review will focus on the rationale behind NSAIDs, including selective COX-2 inhibitors, in combination with conventional chemotherapeutic drugs or novel molecular targeted drugs. Studies investigating anti-cancer effects of NSAIDs on cell lines and xenograft models have shown modulation of the Akt, NF-kappa B, tyrosine kinase and the death receptor-mediated apoptosis pathways. COX-2 expression in tumors is not yet used as biomarker in the clinic. Despite the increased risk on cardiovascular toxicity induced by selective COX-2 inhibitors, several ongoing clinical trials are still investigating the therapeutic benefits of NSAIDs in oncology. The anti-tumor effects in these trials balanced with the side effects data will define the precise role of selective COX-2 inhibitors in the treatment of cancer patients. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

The transcription factor hypoxia-inducible factor (HIF)-1 plays a central physiological role in oxygen and energy homeostasis, and is activated during hypoxia by stabilization of the subunit HIF-1 alpha. Activation can also occur by proinflammatory cytokines during inflammation. Hypoxia, as well as proinflammatory cytokines, plays an important role in the synovia in rheumatoid arthritis (RA) patients. Expression of HIF-1 alpha has been demonstrated in RA synovial lining layer. The aim of the study was to investigate the regulation of the intracellular signal transduction pathways, involved in the expression of HIF-1 alpha, and in the expression of genes regulated by HIF-1 alpha in rheumatoid synovial fibroblasts (RSF). RSF were cultured under proinflammatory conditions (IL-1 beta and TNF-alpha stimulation) and under chemical hypoxia (CoCl2 treatment). Expression of HIF-1 alpha was analyzed in nuclear extracts by Western blotting. The effect of inhibitors of the PI3K and the ERK pathway on HIF-1 alpha protein expression was measured. mRNA expression of HIF-1 alpha, COX-2, vascular endothelial growth factor (VEGF), and stromal cell-derived factor (SDF)-1 was determined by real-time RT-PCR, and protein production of VEGF and SDF-1 by ELISA. Treatment of the synovial fibroblasts with 150 MM CoCl2 as well as stimulation with 10 ng/mL IL-1 beta or TNF-alpha resulted in strong protein expression of HIF-1 alpha, measured with Western blotting. Pretreatment with the MEK1/2 inhibitor PD98059 as well as the PI3K inhibitor LY294002 resulted in inhibition of the cytokine-induced HIF-1 alpha expression. Furthermore, it was shown that cytokine-induced mRNA expression of HIF-1 alpha was inhibited by the PI3K inhibitor. We found that cytokine stimulation induced VEGF mRNA and protein production, but no significant effect of kinase inhibition was found on VEGF production in cytokine-stimulated RSF, Both the ERK pathway and the PI3K pathway are involved in the cytokine-induced HIF-1 alpha expression in RSF and in the expression of proangiogenic factors.

AIMS: Thioredoxin interacting protein (TXNIP) is an attractive candidate gene for diabetes or diabetic dyslipidaemia, since TXNIP is the strongest glucose-responsive gene in pancreatic B-cells, TXNIP deficiency in a mouse model is associated with hyperlipidaemia and TXNIP is located in the 1q21-1q23 chromosomal Type 2 diabetes mellitus (DM) locus. We set out to investigate whether metabolic effects of TXNIP that were previously reported in a murine model are also relevant in human Type 2 DM. METHODS: The frequency distribution of a 3' UTR single nucleotide polymorphism (SNP) in TXNIP was investigated in subjects with normal glucose tolerance (NGT; n = 379), impaired glucose tolerance (IGT; n = 228) and Type 2 DM (n = 230). Metabolic data were used to determine the effect of this SNP on parameters associated with lipid and glucose metabolism. RESULTS: The frequency of the TXNIP variation did not differ between groups, but within the group of diabetic subjects, carriers of the TXNIP-T variant had 1.6-fold higher triglyceride concentrations (P = 0.015; n = 136) and a 5.5-mmHg higher diastolic blood pressure (P = 0.02; n = 212) than homozygous carriers of the common C-allele, whereas in non-diabetic subjects fasting glucose was 0.26 mmol/l lower (P = 0.002; n = 478) in carriers of the T-allele. Moreover, a significant interaction between plasma glucose concentrations and TXNIP polymorphism on plasma triglycerides was observed (P = 0.012; n = 544). CONCLUSION: This is the first report to implicate TXNIP in a human disorder of energy metabolism, Type 2 diabetes. The effect of TXNIP on triglycerides is influenced by plasma glucose concentrations, suggesting that the biological relevance of TXNIP variations may be particularly relevant in recurrent episodes of hyperglycaemia.

Most solid tumors express the cyclooxygenase-2 (COX-2) protein, a target of NSAIDs. COX-2 overexpression in tumorsis considered a predictor of more advanced stage disease and of worse prognosis in a number of studies investigating solid malignancies. Therefore, NSAIDs are evaluated as anti-cancer drugs. NSAIDs inhibit proliferation, invasiveness of tumors, and angiogenesis and overcome apoptosis resistance in a COX-2 dependent and independent manner. This review will focus on the rationale behind NSAIDs, including selective COX-2 inhibitors, in combination with conventional chemotherapeutic drugs or novel molecular targeted drugs. Studies investigating anti-cancer effects of NSAIDs on cell lines and xenograft models have shown modulation of the Akt, NF-kappa B, tyrosine kinase and the death receptor-mediated apoptosis pathways. COX-2 expression in tumors is not yet used as biomarker in the clinic. Despite the increased risk on cardiovascular toxicity induced by selective COX-2 inhibitors, several ongoing clinical trials are still investigating the therapeutic benefits of NSAIDs in oncology. The anti-tumor effects in these trials balanced with the side effects data will define the precise role of selective COX-2 inhibitors in the treatment of cancer patients. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

OBJECTIVE Behcet's disease (BD) is a systemic inflammatory disease with an incompletely understood etiology. Despite the identification of multiple common genetic variants associated with BD, rare genetic variants have been less explored. We undertook this study to investigate the role of rare variants in BD by performing whole exome sequencing in BD patients of European descent. METHODS Whole exome sequencing was performed in a discovery set comprising 14 German BD patients of European descent. For replication and validation, Sanger sequencing and Sequenom genotyping were performed in the discovery set and in 2 additional independent sets of 49 German BD patients and 129 Italian BD patients of European descent. Genetic association analysis was then performed in BD patients and 503 controls of European descent. Functional effects of associated genetic variants were assessed using bioinformatic approaches. RESULTS Using whole exome sequencing, we identified 77 rare variants (in 74 genes) with predicted protein-damaging effects in BD. These variants were genotyped in 2 additional patient sets and then analyzed to reveal significant associations with BD at 2 genetic variants detected in all 3 patient sets that remained significant after Bonferroni correction. We detected genetic association between BD and LIMK2 (rs149034313), involved in regulating cytoskeletal reorganization, and between BD and NEIL1 (rs5745908), involved in base excision DNA repair (P = 3.22 × 10(-4) and P = 5.16 × 10(-4) , respectively). The LIMK2 association is a missense variant with predicted protein damage that may influence functional interactions with proteins involved in cytoskeletal regulation by Rho GTPase, inflammation mediated by chemokine and cytokine signaling pathways, T cell activation, and angiogenesis (Bonferroni-corrected P = 5.63 × 10(-14) , P = 7.29 × 10(-6) , P = 1.15 × 10(-5) , and P = 6.40 × 10(-3) , respectively). The genetic association in NEIL1 is a predicted splice donor variant that may introduce a deleterious intron retention and result in a noncoding transcript variant. CONCLUSION We used whole exome sequencing in BD for the first time and identified 2 rare putative protein-damaging genetic variants associated with this disease. These genetic variants might influence cytoskeletal regulation and DNA repair mechanisms in BD and might provide further insight into increased leukocyte tissue infiltration and the role of oxidative stress in BD.

Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely. Eight healthy smokers aged 40–65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis. A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL. The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression. NHS Health Research Authority ( 13/LO/0256 ).

Impaired angiogenesis has been implicated in adipose tissue dysfunction and the development of obesity and associated metabolic disorders. Here, we report the unexpected finding that vascular endothelial growth factor B (VEGFB) gene transduction into mice inhibits obesity-associated inflammation and improves metabolic health without changes in body weight or ectopic lipid deposition. Mechanistically, the binding of VEGFB to VEGF receptor 1 (VEGFR1, also known as Flt1) activated the VEGF/VEGFR2 pathway and increased capillary density, tissue perfusion, and insulin supply, signaling, and function in adipose tissue. Furthermore, endothelial Flt1 gene deletion enhanced the effect of VEGFB, activating the thermogenic program in subcutaneous adipose tissue, which increased the basal metabolic rate, thus preventing diet-induced obesity and related metabolic complications. In obese and insulin-resistant mice, Vegfb gene transfer, together with endothelial Flt1 gene deletion, induced weight loss and mitigated the metabolic complications, demonstrating the therapeutic potential of the VEGFB/VEGFR1 pathway.

Corrigendum: Experimental Eye Research 169 (2018) 60 doi: 10.1016/j.exer.2018.01.006 WOS: 000430158400008 Intravitreal administration is the method of choice in drug delivery to the retina and/or choroid. Rabbit is the most commonly used animal species in intravitreal pharmacokinetics, but it has been criticized as being a poor model of human eye. The critique is based on some anatomical differences, properties of the vitreous humor, and observed differences in drug concentrations in the anterior chamber after intravitreal injections. We have systematically analyzed all published information on intravitreal pharmacokinetics in the rabbit and human eye. The analysis revealed major problems in the design of the pharmacokinetic studies. In this review we provide advice for study design. Overall, the pharmacokinetic parameters (clearance, volume of distribution, half-life) in the human and rabbit eye have good correlation and comparable absolute values. Therefore, reliable rabbit-to-man translation of intravitreal pharmacokinetics should be feasible. The relevant anatomical and physiological parameters in rabbit and man show only small differences. Furthermore, the claimed discrepancy between drug concentrations in the human and rabbit aqueous humor is not supported by the data analysis. Based on the available and properly conducted pharmacokinetic studies, the differences in the vitreous structure in rabbits and human patients do not lead to significant pharmacokinetic differences. This review is the first step towards inter-species translation of intravitreal pharmacokinetics. More information is still needed to dissect the roles of drug delivery systems, disease states, age and ocular manipulation on the intravitreal pharmacokinetics in rabbit and man. Anyway, the published data and the derived pharmacokinetic parameters indicate that the rabbit is a useful animal model in intravitreal pharmacokinetics. (C) 2015 The Authors. Published by Elsevier Ltd.

A low-grade inflammatory response is commonly seen in the peripheral blood of major depressive disorder (MDD) patients, especially those with refractory and chronic disease courses. However, electroconvulsive therapy (ECT), the most drastic intervention reserved for these patients, is closely associated with an enhanced haematogenous as well as neuroinflammatory immune response, as evidenced by both human and animal studies. A related line of experimental evidence further shows that inflammatory stimulation reinforces neurotrophin expression and may even mediate dramatic neurogenic and antidepressant- like effects following exposure to chronic stress. The current review therefore attempts a synthesis of our knowledge on the neurotrophic and immunological aspects of ECT and other electrically based treatments in psychiatry. Perhaps contrary to contemporary views, we conclude that targeted potentiation, rather than suppression, of inflammatory responses may be of therapeutic relevance to chronically depressed patients or a subgroup thereof.

Purpose To report the incidence and characteristics of endophthalmitis after intravitreal injections of anti–vascular endothelial growth factor agents or corticosteroids and to describe the clinical and bacteriologic characteristics, management, and outcome of these eyes with acute endophthalmitis in France. Design Retrospective, nationwide multicenter case series. Methods From January 2, 2008 to June 30, 2013, a total of 316 576 intravitreal injections from 25 French ophthalmic centers were included. For each center, the number of intravitreal injections was determined using billing codes and the injection protocol was recorded. A registry and hospital records were reviewed to identify patients treated for endophthalmitis after injection during the same time period. The main outcome measures were the incidence of clinical endophthalmitis and visual acuity of endophthalmitis cases. Results During the study period, 65 cases of presumed endophthalmitis were found, giving an overall incidence of 0.021% (2.1 in 10 000 injections) (95% confidence interval [CI], 0.016%–0.026%). The median number of days from injection to presentation was 4 [1–26] days. The most common symptom was vision loss. Bacterial identification was achieved in 43.4%. The most frequent pathogens were gram-positive bacteria (91.3%), including coagulase-negative Staphylococcus in 78.3%. Neither the interval between injection and presentation for endophthalmitis nor the clinical signs differentiated culture-positive from culture-negative cases. In multivariate analysis, the use of a disposable conjunctival mould assist device and the use of prophylaxis with an antibiotic or antiseptic were significantly associated with an increased incidence of endophthalmitis ( P = .001). The majority of patients had worse visual acuity after 3 months of follow-up when compared with acuity before endophthalmitis. Conclusions The incidence of presumed endophthalmitis after intravitreal injections of anti–vascular endothelial growth factors or corticosteroids was low and the prognosis poor. Prevention and management remain challenging. It remains to be determined whether the findings of this study are relevant for other countries using different techniques for intravitreal injections.

Renal cell carcinoma (RCC) is an immunogenic tumor, but uses several immune-suppressive mechanisms to shift the balance from tumor immune response toward tumor growth. Although RCC has traditionally been considered to be radiation resistant, recent evidence suggests that hypofractionated radiotherapy contributes to systemic antitumor immunity. Because the efficacy of antitumor immune responses depends on the complex balance between diverse immune cells and progressing tumor cells, radiotherapy alone is unlikely to induce persistent antitumor immunity. Therefore, the combination of radiotherapy with drugs having synergistic immunomodulatory properties holds great promise with the optimal timing and sequence of modalities depending on the agent used. We highlight the immunomodulatory properties of targeted therapies, such as tyrosine kinase inhibitors, mammalian target of rapamycin (mTOR) inhibitors and vascular endothelial growth factor (VEGF) neutralizing antibodies, and will suggest a combination schedule with radiotherapy based on the available literature. We also address the combination of radiotherapy with innovative treatments in the field of immunotherapy.

The human gammaherpesvirus family includes Epstein-Barr virus (EBV) and human herpesvirus (HHV)-8, also known as Kaposi sarcoma-associated herpesvirus (KSHV). In human immunodeficiency virus (HIV)-infected patients, both EBV and KSHV have been implicated in the development of a wide range of tumors. KSHV-associated diseases include Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). EBV has been associated with the development of several malignancies, including Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). The introduction of highly active antiretroviral therapy (HAART) has dramatically modified the natural history of HIV infection, causing a decline in the incidence of acquired immunodeficiency syndrome (AIDS)-defining malignancies, including KS. However, it has had a less favorable impact on EBV-related malignancies and NHLs remain the most common tumors in the HAART era. In this review, we briefly summarize the pathogenesis, epidemiology, clinical features, and therapeutic approach to EBV- and KSHV-associated tumors in the setting of HIV infection.

The greatest challenge in treating acute myeloid leukemia (AML) is refractory disease. With approximately 60-80% of AML patients dying of relapsed disease, there is an urgent need to define and target mechanisms of drug resistance. Unfortunately, targeting cell-intrinsic resistance has failed to improve clinical outcomes in AML Emerging data show that cell-extrinsic factors in the bone marrow microenvironment protect and support AML cells. The vascular niche, in particular, regulates AML cell survival and cell cycling by both paracrine secretion and adhesive contact with endothelial cells. Moreover, AML cells can functionally integrate within vascular endothelia, undergo quiescence, and resist cytotoxic chemotherapy. Together, these findings support the notion of blood vessels as sanctuary sites for AML Therefore, vascular targeting agents may serve to remit AML. Several early phase clinical trials have tested anti-angiogenic agents, leukemia mobilizing agents, and vascular disrupting agents in AML patients. In general, these agents can be safely administered to AML patients and cardiovascular side effects were reported. Response rates to vascular targeting agents in AML have been modest; however, a majority of vascular targeting trials in AML are monotherapy in design and indiscriminate in patient recruitment. When considering the chemosensitizing effects of targeting the microenvironment, there is a strong rationale to build upon these early phase clinical trials and initiate phase IB/II trials of combination therapy where vascular targeting agents are positioned as priming agents for cytotoxic chemotherapy. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

Combining a modified (m)FOLFOX-6 (leucovorin, 5-fluorouracil [5-FU], and 85 mg/kg(2) oxaliplatin) regimen with antiangiogenic therapy is a standard treatment option in advanced colorectal cancer. In this phase Ib study, safety, pharmacokinetics, and antitumor activity of tivozanib with mFOLFOX-6 were assessed. Tivozanib could be combined at its recommended dose Of 1.5 mg with mFOLFOX-6, demonstrating antitumor activity, A randomized study in advanced colorectal Cancer comparing bevacizumab and tivozanib with mFOLFOX-6 has been performed.Background: Tivozanib hydrochloride (tivozanib) is a potent and selective tyrosine kinase inhibitor of all 3 vascular endothelial growth factor receptors with antitumor activity additive to 5-fluorouracil in preclinical models. This study was conducted to determine maximum tolerated dose (MTD), dose-limiting toxicities (DLTs), pharmacokinetics (PKs), and antitumor activity of escalating doses of tivozanib with a modified (m)FOLFOX-6 (leucovorin, 5-fluorouracil [5-FU], and 85 mg/kg(2) oxaliplatin) regimen in patients with advanced gastrointestinal tumors. Patients and Methods: Tivozanib was administered orally once daily for 21 days in 28-day cycles, with mFOLFOX-6 administered every 14 days. Patients were allowed to continue tivozanib after discontinuation of mFOLFOX-6. Results: Thirty patients were assigned to tivozanib 0.5 mg (n = 9); 1.0 mg (n = 3); or 1.5 mg (n = 18) with mFOLFOX-6. Patients received a median of 5.2 (range, 0.03-26.9) months of tivozanib. DLTs were observed in 2 patients: Grade 3/4 transaminase level increases with tivozanib 0.5 mg, and Grade 3 dizziness with tivozanib 1.5 mg. Other Grade 3/4 adverse events included hypertension (n = 8); fatigue (n = 8), and neutropenia (n = 6): MTD for tivozanib with mFOLFOX-6 was confirmed as 1.5 mg. No PK interactions between tivozanib and mFOLFOX-6 were observed. One patient had an ongoing clinical complete response, 10 had a partial response, and 11 obtained prolonged stable disease. Conclusion: Tivozanib and mFOLFOX-6 is feasible and appears to be safe. The recommended dose for tivozanib with mFOLFOX-6 is 1.5 mg/d. Observed clinical activity merits further exploration in gastrointestinal tumors.

A dual-action ligand targeting both integrin alpha(V)beta(3) and vascular endothelial growth factor receptors (VEGFRs), was synthesized via conjugation of a cyclic peptidomimetic alpha(V)beta(3) Arg-Gly-Asp (RGD) ligand with a decapentapeptide. The latter was obtained from a known VEGFR antagonist by acetylation at the Lys13 side chain. Functionalization of the precursor ligands was carried out in solution and in the solid phase, affording two fragments: an alkyne VEGFR ligand and the azide integrin alpha(V)beta(3) ligand, which were conjugated by click chemistry. Circular dichroism studies confirmed that both the RGD and VEGFR ligand portions of the dual-action compound substantially adopt the biologically active conformation. In vitro binding assays on isolated integrin alpha(V)beta(3) and VEGFR-1 showed that the dual-action conjugate retains a good level of affinity for both its target receptors, although with one order of magnitude (10/20 times) decrease in potency. The dual-action ligand strongly inhibited the VEGF-induced morphogenesis in Human Umbilical Vein Endothelial Cells (HUVECs). Remarkably, its efficiency in preventing the formation of new blood vessels was similar to that of the original individual ligands, despite the worse affinity towards integrin alpha(V)beta(3) and VEGFR-1.

Thalidomides were initially thought to be broad-range drugs specifically for curing insomnia and relieving morning sickness in pregnant women. However, its use was discontinued because of a major drawback of causing teratogenicity. In this study, we found that a thalidomide derivative, 5-hydroxy-2-(2,6-diisopropylphenyl)-1H-isoindole-1,3-dione (5HPP-33), inhibited the proliferation of MCF-7 with a half-maximal inhibitory concentration of 4.5 +/- 0.4 mu M. 5HPP-33 depolymerized microtubules and inhibited the reassembly of cold-depolymerized microtubules in MCF-7 cells. Using time-lapse imaging, the effect of 5HPP-33 on the dynamics of individual microtubules in live MCF-7 cells was analyzed. 5HPP-33 (5 mu M) decreased the rates of growth and shortening excursions by 34 and 33%, respectively, and increased the time microtubules spent in the pause state by 92% as compared to that of the vehicle-treated MCF-7 cells. 5HPP-33 (5 mu M) reduced the dynamicity of microtubules by 62% compared to the control. 5HPP-33 treatment reduced the distance between the two poles of a bipolar spindle, induced multipolarity in some of the treated cells, and blocked cells at mitosis. In vitro, 5HPP-33 bound to tubulin with a weak affinity. Vinblastine inhibited the binding of 5HPP-33 to tubulin, and 5HPP-33 inhibited the binding of BODIPY FL-vinblastine to tubulin. Further, a molecular docking analysis suggested that 5HPP-33 shares its binding site on tubulin with vinblastine. The results provided significant insight into the antimitotic mechanism of action of 5HPP-33 and also suggest a possible mechanism for the teratogenicity of thalidomides.

Item does not contain fulltext Major depressive disorder is a heterogeneous disorder, mostly diagnosed on the basis of symptomatic criteria alone. It would be of great help when specific biomarkers for various subtypes and symptom clusters of depression become available to assist in diagnosis and subtyping of depression, and to enable monitoring and prognosis of treatment response. However, currently known biomarkers do not reach sufficient sensitivity and specificity, and often the relation to underlying pathophysiology is unclear. In this review, we evaluate various biomarker approaches in terms of scientific merit and clinical applicability. Finally, we discuss how combined biomarker approaches in both preclinical and clinical studies can help to make the connection between the clinical manifestations of depression and the underlying pathophysiology.

Purpose Pharmacologic control of neovascularization is a promising approach for the treatment of retinal angiogenesis. UPARANT, an inhibitor of the urokinase-type plasminogen activator receptor (uPAR), inhibits VEGF-driven angiogenesis in vitro and in vivo. This study investigates for the first time the effectiveness of UPARANT in counteracting pathologic neovascularization in the retina. Methods Murine retinal fragments and a mouse model of oxygen-induced retinopathy (OIR) were used. In mice with OIR, UPARANT-treated retinas were analyzed for avascular area and neovascular tuft formation. Levels of transcription and proangiogenic factors were determined. UPARANT effects on the blood-retinal barrier (BRB), visual function, retinal cytoarchitecture, and inflammatory markers were also assessed. Human umbilical vein endothelial cells (HUVECs) and chick embryo chorioallantoic membrane (CAM) in which angiogenesis was induced by the vitreous fluid from patients with proliferative diabetic retinopathy (PDR) were also used. Results UPARANT reduced VEGF-induced angiogenesis in retinal fragments. In mice with OIR, UPARANT decreased neovascular response, VEGF, and VEGF receptor-2 activity. Transcription factors regulating VEGF expression were also reduced. UPARANT restored BRB integrity, recovered visual loss, and reduced levels of inflammatory markers. Restored electroretinogram does not involve any rescue in the retinal cytoarchitecture. Finally, UPARANT blocked PDR vitreous fluid-induced angiogenesis in HUVEC and CAM assays. Conclusions The finding that UPARANT is effective against neovascularization may help to establish uPAR as a target in the treatment of proliferative retinopathies. The potential application of UPARANT in retinal diseases is further supported by UPARANT capacity to counteract the angiogenic activity of PDR vitreous fluid.

Peritoneal dialysis (PD) is a form of renal replacement therapy whose repeated use can alter dialytic function through induction of epithelial-mesenchymal transition (EMT) and fibrosis, eventually leading to PD discontinuation. The peritoneum from Cav1(-/-) mice showed increased EMT, thickness, and fibrosis. Exposure of Cav1(-/-) mice to PD fluids further increased peritoneal membrane thickness, altered permeability, and increased the number of FSP-1/cytokeratin-positive cells invading the sub-mesothelial stroma. High-throughput quantitative proteomics revealed increased abundance of collagens, FN, and laminin, as well as proteins related to TGF-beta activity in matrices derived from Cav1(-/-) cells. Lack of Cav1 was associated with hyperactivation of a MEK-ERK1/2-Snail-1 pathway that regulated the Smad2-3/Smad1-5-8 balance. Pharmacological blockade of MEK rescued E-cadherin and ZO-1 inter-cellular junction localization, reduced fibrosis, and restored peritoneal function in Cav1(-/-) mice. Moreover, treatment of human PD-patient-derived MCs with drugs increasing Cav1 levels, as well as ectopic Cav1 expression, induced re-acquisition of epithelial features. This study demonstrates a pivotal role of Cav1 in the balance of epithelial versus mesenchymal state and suggests targets for the prevention of fibrosis during PD. We thank Dr. Kurzchalia, Dr. Pol, and Dr. Lisanti for providing us MEFs from WT and Cav1-/- mice. We thank Dr. Valeria Caiolfa, head of CNIC Microscopy unit, and Elvira Arza and Antonio Manuel Santos Beneit for technical assistance. We thank Cecilia Battistelli and Marta Loureiro Lopez por technical assistance in molecular biology and proteomics, respectively. This work was supported by grants from the MINECO (Spanish Ministry of Economy and Competitivity) to MADP (SAF2008-02100, SAF2011-25047 and CONSOLIDER CSD2009-00016) and to MLC (SAF2010-21249 and SAF2013-47611R), and from Fundacio la Marato TV3 (674/C/2013) to MADP. Raffaele Strippoli was supported by a Rio Hortega Contract (Instituto de Salud Carlos III). Ignacio Benedicto was recipient of a CIBERehd fellowship (Spanish Ministry of Health and Consumer Affairs). Susana Minguet was supported by the Ramon y Cajal program (Spanish Ministry of Science and Innovation). Simon Bartlett (CNIC) provided editorial assistance. The CNIC is supported by MINECO and the Pro-CNIC Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí

Objectives: As suggested by in-vitro data, we hypothesize that subtypes of ICRAS mutated non-small cell lung cancer (NSCLC) respond differently to chemotherapy regimens.Methods: Patients with advanced NSCLC and known KRAS mutation, treated with first-line platinumbased chemotherapy, were retrieved from hospital databases. Primary objective: to investigate overall response rate (ORR), progression free survival (PFS) and overall survival (OS) between different types of platinum-based chemotherapy per type of KRAS mutation.Results: 464 patients from 17 hospitals, treated between 2000 and 2013, were included. The majority of patients had stage IV disease (93%), had a history of smoking (98%) and known with an adenocarcinoma (91%). Most common types of ICRAS mutation were G12C (46%), G12V (20%) and G1 2D (10%). Platinum was combined with pemetrexed (n =334), taxanes (n = 68) or gemcitabine (n = 62). Patients treated with taxanes had a significant improved ORR (50%) compared to pemetrexed (21%) or gemcitabine (25%; p Conclusion: KRAS mutated NSCLC patients treated with taxane-based chemotherapy had best ORR Response to chemotherapy regimens was different in types of ICRAS mutation. Especially patients with G12V had better response to taxane treatment. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

Poorly differentiated thyroid cancer (PDTC) and undifferentiated thyroid cancer (UDTC) are still life-threatening pathologies, because of the lack of well-established diagnostic and therapeutic approaches. In the past, many attempts have been made to develop radiopharmaceutical to diagnose or treat radioactive iodine (RAI)-refractory metastases or recurrences, with limited results. Indeed, it was not possible to find a specific and overexpressed marker to be used as target of radiopharmaceuticals or targeted therapies. Nowadays, with novel advances in the field of tumor microenvironment, many new markers are available to be used as suitable targets for targeted therapies interfering with signalling pathways of cells involved in the mechanisms that favour tumor growth and metastatization. This opened new perspective in the use of radiopharmaceuticals targeting components of tumor microenvironment for early diagnosis, pre-operative staging or therapy planning and follow up with targeted drugs. In the present review we present the state of novel approaches to image thyroid cancer and its microenvironment, focusing on RAI-refractory thyroid cancer as a real clinical problem to be solved.

Purpose: The dose-response relationship for a relatively short length (4 mm) of rat spinal cord has been shown to be significantly modified by adjacent low-dose fields. In an additional series of experiments, we have now established the dose-volume dependence of this effect.Methods and Materials: Wistar rats were irradiated on the cervical spinal cord with single doses of unmodulated protons (150 MeV) to obtain sharp lateral penumbras, by use of the shoot-through technique, which employs the plateau of the depth-dose profile rather than the Bragg peak. Three types of inhomogeneous dose distributions were administered: Twenty millimeters of cervical spinal cord were irradiated with variable subthreshold (=bath) doses (4 and 18 Gy). At the center of the 20-mm segment, a short segment of 2 mm or 8 mm (=shower) was irradiated with variable single doses. These inhomogeneous dose distributions are referred to as symmetrical bath-and-shower experiments. An asymmetrical dose distribution was arranged by irradiation of 12 mm (=bath) of spinal cord with a dose of 4 Gy. The caudal 2 mm (=shower) of the 12-mm bath was additionally irradiated with variable single doses. This arrangement of inhomogeneous dose distribution is referred to as asymmetrical bath-and-shower experiment. The endpoint for estimation of the dose-response relationships was paralysis of the fore limbs or hind limbs and confirmation by histology.Results: The 2-mm bath-and-shower experiments with a 4-Gy bath dose showed a large shift of the dose-response curves compared with the 2-mm single field, which give lower ED50 values of 61.2 Gy and 68.6 Gy for the symmetrical and asymmetrical arrangement, respectively, compared with an ED50 of 87.8 Gy after irradiation of a 2-mm field only. If the bath dose is increased to 18 Gy, the ED50 value is decreased further to 30.9 Gy. For an 8-mm field, addition of a 4-Gy bath dose did not modify the ED50 obtained for an 8-mm field only (23.2 and 23.1 Gy).Conclusions: The spinal cord tolerance of relatively small volumes (shower) is strongly affected by low-dose irradiation (=bath) of adjacent tissue. The results of all bath-and-shower experiments show the effect of a low bath dose to be highest for a field of 2 mm, less for 4 mm, and absent for 8 mm. Adding a 4-Gy bath to only 1 side of a 2-mm field still showed a large effect. Because glial progenitor cells are known to migrate over at least 2 to 3 mm, this observation indicates that interference with stem cell migration is not the most likely mechanism of a bath effect. (C) 2006 Elsevier Inc.

Smokers develop metastatic prostate cancer more frequently than nonsmokers, suggesting that a tobacco-derived factor is driving metastatic progression. To identify smoking-induced alterations in human prostate cancer, we analyzed gene and protein expression patterns in tumors collected from current, past, and never smokers. By this route, we elucidated a distinct pattern of molecular alterations characterized by an immune and inflammation signature in tumors from current smokers that were either attenuated or absent in past and never smokers. Specifically, this signature included elevated immunoglobulin expression by tumor-infiltrating B cells, NF-κB activation, and increased chemokine expression. In an alternate approach to characterize smoking-induced oncogenic alterations, we also explored the effects of nicotine in human prostate cancer cells and prostate cancer–prone TRAMP mice. These investigations showed that nicotine increased glutamine consumption and invasiveness of cancer cells in vitro and accelerated metastatic progression in tumor-bearing TRAMP mice. Overall, our findings suggest that nicotine is sufficient to induce a phenotype resembling the epidemiology of smoking-associated prostate cancer progression, illuminating a novel candidate driver underlying metastatic prostate cancer in current smokers. Cancer Res; 76(5); 1055–65. ©2015 AACR.