BACKGROUND

Platelet-rich fibrin matrices release a natural mixture of growth factors that play central roles in the complex processes of tendon healing.

OBJECTIVE

Application of autologous platelet-rich matrices during Achilles tendon surgery may promote healing and functional recovery.

METHODS

Case-control study and descriptive laboratory study; Level of evidence, 3.

METHODS

Twelve athletes underwent open suture repair after complete Achilles tendon tear. Open suture repair in conjunction with a preparation rich in growth factors (PRGF) was performed in 6 athletes and retrospectively compared with a matched group that followed conventional surgical procedure. The outcomes were evaluated on the basis of range of motion, functional recovery, and complications. Achilles tendons were examined by ultrasound at 50 +/- 11 months in retrospective controls and 32 +/- 10 months in the PRGF group. In the laboratory portion of the study, PRGF treatment was characterized by the number of platelets and concentration of insulin (IGF-I), transformed (TGF-beta1), platelet-derived (PDGF-AB), vascular endothelial (VEGF), hepatocyte (HGF), and epidermal (EGF) growth factors in patients affected by musculoskeletal traumatic injuries.

RESULTS

Athletes receiving PRGF recovered their range of motion earlier (7 +/- 2 weeks vs 11 +/- 3 weeks, P = .025), showed no wound complication, and took less time to take up gentle running (11 +/- 1 weeks vs 18 +/- 3 weeks, P = .042) and to resume training activities (14 +/- 0.8 weeks vs 21 +/- 3 weeks, P = .004). The cross-sectional area of the PRGF-treated tendons increased less (t = 3.44, P = .009). TGF-beta1 (74.99 +/- 32.84 ng/mL), PDGF-AB (35.62 +/- 14.57 ng/mL), VEGF (383.9 +/- 374.9 pg/mL), EGF (481.5 +/- 187.5 pg/mL), and HGF (593.87 +/- 155.76 pg/mL) significantly correlated with the number of platelets (677 +/- 217 platelets/microL, P < .05).

CONCLUSIONS

The operative management of tendons combined with the application of autologous PRGF may present new possibilities for enhanced healing and functional recovery. This needs to be evaluated in a randomized clinical trial.

NRPs (neuropilins) are co-receptors for class 3 semaphorins, polypeptides with key roles in axonal guidance, and for members of the VEGF (vascular endothelial growth factor) family of angiogenic cytokines. They lack a defined signalling role, but are thought to mediate functional responses as a result of complex formation with other receptors, such as plexins in the case of semaphorins and VEGF receptors (e.g. VEGFR2). Mutant mouse studies show that NRP1 is essential for neuronal and cardiovascular development, whereas NRP2 has a more restricted role in neuronal patterning and lymphangiogenesis, but recent findings indicate that NRPs may have additional biological roles in other physiological and disease-related settings. In particular, NRPs are highly expressed in diverse tumour cell lines and human neoplasms and have been implicated in tumour growth and vascularization in vivo. However, despite the wealth of information regarding the probable biological roles of these molecules, many aspects of the regulation of cellular function via NRPs remain uncertain, and little is known concerning the molecular mechanisms through which NRPs mediate the functions of their various ligands in different cell types.

OBJECTIVE

Visfatin is a novel adipokine whose plasma concentrations are altered in obesity and obesity-related disorders; these states are associated with an increased incidence of cardiovascular disease. We therefore investigated the effect of visfatin on vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP-2, MMP-9) production and the potential signalling cascades.

RESULTS

In human umbilical vein endothelial cells (HUVECs), visfatin significantly and dose-dependently up-regulated gene expression and protein production of VEGF and MMPs and down-regulated expression of tissue inhibitors of MMPs (TIMP-1 and TIMP-2). The gelatinolytic activity of MMPs (analysed by zymography) correlated with mRNA and western blot findings. Interestingly, visfatin significantly up-regulated VEGF receptor 2 expression. Inhibition of VEGFR2 and VEGF [by soluble FMS-like tyrosine kinase-1 (sFlt1)] down-regulated visfatin-induced MMP induction. Visfatin induced dose- and time-dependent proliferation and capillary-like tube formation. Importantly, visfatin was noted to have anti-apoptotic effects. In HUVECs, visfatin dose-dependently activated PI3K/Akt (phosphatidylinositol 3-kinase/Akt) and ERK(1/2) (extracellular signal-regulated kinase) pathways. The functional effects and MMP/VEGF induction were shown to be dependent on the MAPK/PI3K-Akt/VEGF signalling pathways. Inhibition of PI3K/Akt and ERK(1/2) pathways led to significant decrease of visfatin-induced MMP and VEGF production and activation, along with significant reduction in endothelial proliferation and capillary tube formation.

CONCLUSIONS

Our data provide the first evidence of visfatin-induced endothelial VEGF and MMP production and activity. Further, we show for the first time the involvement of the MAPK and PI3K/Akt signalling pathways in mediating these actions, as well as endothelial cell proliferation. Collectively, our findings provide novel insights into visfatin-induced endothelial angiogenesis.

Angiogenesis is an essential component of skeletal development and VEGF signaling plays an important if not pivotal role in this process. Previous attempts to examine the roles of VEGF in vivo have been largely unsuccessful because deletion of even one VEGF allele leads to embryonic lethality before skeletal development is initiated. The availability of mice expressing only the VEGFVEGF during embryonic skeletal development. Our study of these mice provides new in vivo evidence for multiple important roles of VEGF in both endochondral and intramembranous bone formation, as well as some insights into isoform-specific functions. There are two key differences in vascularization of developing bones between wild-type and VEGF(120/120) mice. VEGF(120/120) mice have not only a delayed recruitment of blood vessels into the perichondrium but also show delayed invasion of vessels into the primary ossification center, demonstrating a significant role of VEGF at both an early and late stage of cartilage vascularization. These findings are the basis for a two-step model of VEGF-controlled vascularization of the developing skeleton, a hypothesis that is supported by the new finding that VEGF is expressed robustly in the perichondrium and surrounding tissue of cartilage templates of future bones well before blood vessels appear in these regions. We also describe new in vivo evidence for a possible role of VEGF in chondrocyte maturation, and document that VEGF has a direct role in regulating osteoblastic activity based on in vivo evidence and organ culture experiments.

Vascular endothelial growth factors (VEGFs) are key regulators of permeability. The principal evidence behind how they increase vascular permeability in vivo and in vitro and the consequences of that increase are addressed here. Detailed analysis of the published literature has shown that in vivo and in vitro VEGF-mediated permeability differs in its time course, but has common involvement of many specific signalling pathways, in particular VEGF receptor-2 activation, calcium influx through transient receptor potential channels, activation of phospholipase C gamma and downstream activation of nitric oxide synthase. Pathways downstream of endothelial nitric oxide synthase appear to involve the guanylyl cyclase-mediated activation of the Rho-Rac pathway and subsequent involvement of junctional signalling proteins such as vascular endothelial cadherin and the tight junctional proteins zona occludens and occludin linked to the actin cytoskeleton. The signalling appears to be co-ordinated through spatial organization of the cascade into a signalplex, and arguments for why this may be important are considered. Many proteins have been identified to be involved in the regulation of vascular permeability by VEGF, but still the mechanisms through which these are thought to interact to control permeability are dependent on the experimental system, and a synthesis of existing data reveals that in intact vessels the co-ordination of the pathways is still not understood.

The oncogene c-maf is translocated in approximately 5%-10% of multiple myelomas. Unexpectedly, we observed c-maf expression in myeloma cell lines lacking c-maf translocations and in 50% of multiple myeloma bone marrow samples. By gene expression profiling, we identified three c-maf target genes: cyclin D2, integrin beta7, and CCR1. c-maf transactivated the cyclin D2 promoter and enhanced myeloma proliferation, whereas dominant inhibition of c-maf blocked tumor formation in immunodeficient mice. c-maf-driven expression of integrin beta7 enhanced myeloma adhesion to bone marrow stroma and increased production of VEGF. We propose that c-maf transforms plasma cells by stimulating cell cycle progression and by altering bone marrow stromal interactions. The frequent overexpression of c-maf in myeloma makes it an attractive target for therapeutic intervention.

To provide 2-year results comparing anti-vascular endothelial growth factor (VEGF) agents for center-involved diabetic macular edema (DME) using a standardized follow-up and retreatment regimen.

Randomized clinical trial.

Six hundred sixty participants with visual acuity (VA) impairment from DME.

Randomization to 2.0-mg aflibercept, 1.25-mg repackaged (compounded) bevacizumab, or 0.3-mg ranibizumab intravitreous injections performed up to monthly using a protocol-specific follow-up and retreatment regimen. Focal/grid laser photocoagulation was added after 6 months if DME persisted. Visits occurred every 4 weeks during year 1 and were extended up to every 4 months thereafter when VA and macular thickness were stable.

Change in VA, adverse events, and retreatment frequency.

Median numbers of injections were 5, 6, and 6 in year 2 and 15, 16, and 15 over 2 years in the aflibercept, bevacizumab, and ranibizumab groups, respectively (global P = 0.08). Focal/grid laser photocoagulation was administered in 41%, 64%, and 52%, respectively (aflibercept vs. bevacizumab, P < 0.001; aflibercept vs. ranibizumab, P = 0.04; bevacizumab vs. ranibizumab, P = 0.01). At 2 years, mean VA improved by 12.8, 10.0, and 12.3 letters, respectively. Treatment group differences varied by baseline VA (P = 0.02 for interaction). With worse baseline VA (20/50 to 20/320), mean improvement was 18.1, 13.3, and 16.1 letters, respectively (aflibercept vs. bevacizumab, P = 0.02; aflibercept vs. ranibizumab, P = 0.18; ranibizumab vs. bevacizumab, P = 0.18). With better baseline VA (20/32 to 20/40), mean improvement was 7.8, 6.8, and 8.6 letters, respectively (P>> 0.10, for pairwise comparisons). Anti-Platelet Trialists' Collaboration (APTC) events occurred in 5% with aflibercept, 8% with bevacizumab, and 12% with ranibizumab (global P = 0.047; aflibercept vs. bevacizumab, P = 0.34; aflibercept vs. ranibizumab, P = 0.047; ranibizumab vs. bevacizumab, P = 0.20; global P = 0.09 adjusted for potential confounders).

All 3 anti-VEGF groups showed VA improvement from baseline to 2 years with a decreased number of injections in year 2. Visual acuity outcomes were similar for eyes with better baseline VA. Among eyes with worse baseline VA, aflibercept had superior 2-year VA outcomes compared with bevacizumab, but superiority of aflibercept over ranibizumab, noted at 1 year, was no longer identified. Higher APTC event rates with ranibizumab over 2 years warrants continued evaluation in future trials.

Inadequate function of dendritic cells (DCs) in tumor-bearing hosts is one mechanism of tumor escape from immune system control and may compromise the efficacy of cancer immunotherapy. Vascular endothelial growth factor (VEGF), produced by most tumors, not only plays an important role in tumor angiogenesis but also can inhibit the maturation of DCs from hematopoietic progenitors. Here, we investigate a novel combination of antiangiogenic and immunotherapy based on this dual role of VEGF. Two s.c. mouse tumor models were used: D459 cells, expressing mutant human p53; and MethA sarcoma with point mutations in the endogenous murine p53 gene. Therapy with anti-mouse VEGF antibody (10 microg i.p. twice a week over 4 weeks) was initiated when tumors became palpable. Treatment of established tumors with anti-VEGF antibody alone did not affect the rate of tumor growth. However, anti-VEGF antibody significantly improved the number and function of lymph node and spleen DCs in these tumor-bearing animals. To investigate the possible effects of this antibody on the immunotherapy of established tumors, tumor-bearing mice were immunized with DCs pulsed with the corresponding mutation-specific p53 peptides, together with injections of anti-VEGF antibody. Therapy with peptide-pulsed DCs alone resulted in considerable slowing of tumor growth but only during the period of treatment, and tumor growth resumed after the end of the therapy. Combined treatment with peptide-pulsed DCs and anti-VEGF antibody resulted in a prolonged and much more pronounced antitumor effect. This effect was associated with the induction of significant anti-p53 CTL responses only in this group of mice. These data suggest that inhibition of VEGF may be a valuable adjuvant in the immunotherapy of cancer.

Neuropilins (NP1 and NP2) are vascular endothelial growth factor (VEGF) receptors that mediate developmental and tumor angiogenesis. Transgenic mice, in which both NP1 and NP2 were targeted (NP1(-/-)NP2(-/-)) died in utero at E8.5. Their yolk sacs were totally avascular. Mice deficient for NP2 but heterozygous for NP1 (NP1(+/-)NP2(-/-)) or deficient for NP1 but heterozygous for NP2 (NP1(-/-)NP2(+/-)) were also embryonic lethal and survived to E10-E10.5. The E10 yolk sacs and embryos were easier to analyze for vascular phenotype than the fragile poorly formed 8.5 embryos. The vascular phenotypes of these E10 mice were very abnormal. The yolk sacs, although of normal size, lacked the larger collecting vessels and had less dense capillary networks. PECAM staining of yolk sac endothelial cells showed the absence of branching arteries and veins, the absence of a capillary bed, and the presence of large avascular spaces between the blood vessels. The embryos displayed blood vessels heterogeneous in size, large avascular regions in the head and trunk, and blood vessel sprouts that were unconnected. The embryos were about 50% the length of wild-type mice and had multiple hemorrhages. These double NP1/NP2 knockout mice had a more severe abnormal vascular phenotype than either NP1 or NP2 single knockouts. Their abnormal vascular phenotype resembled those of VEGF and VEGFR-2 knockouts. These results suggest that NRPs are early genes in embryonic vessel development and that both NP1 and NP2 are required.

The central role of VEGF (vascular endothelial growth factor A) in angiogenesis is dependent upon its ability to co-ordinately regulate multiple endothelial functions. The multifunctionality of VEGF at the cellular level results from its ability to initiate a diverse, complex and integrated network of signalling pathways via its major receptor, kinase-insert-domain-containing receptor (KDR). Activation of phospholipase C-gamma, protein kinase C, Ca(2+), ERK (extracellular-signal-regulated protein kinase), Akt, Src, focal adhesion kinase and calcineurin pathways has been implicated in mediating multiple VEGF functions, including survival, proliferation, migration, vascular permeability, tubulogenesis, NO and prostanoid synthesis, and gene expression. NO and prostanoids in turn play paracrine and autocrine roles in linking post-receptor signalling to biological functions. Integration between biologically important signalling cascades occurs at several points. Akt and ERK, for example, are key junction points linking together signal transduction involved in survival and NO generation, and proliferation and prostanoid biosynthesis. Together, the multiplicity, functional versatility and integration of VEGF signalling provide a useful framework for understanding the mechanisms underlying the endothelial biological response to this key factor.

Tumors of the central nervous system (CNS) often have sustained expression of labile genes, including angiogenic growth factors and immunosuppressive cytokines, which promote tumor progression. Stabilization of the RNA transcripts for these genes, such as vascular endothelial growth factor (VEGF), is an important molecular pathway for this up-regulation. HuR, a member of the Elav family of RNA-binding proteins, has been implicated in this pathway through its binding to adenine and uridine (AU)-rich stability elements (ARE) located in the 3' untranslated regions (3'-UTRs) of the mRNA. Whereas three of the Elav family members (Hel-N1, HuC, and HuD) are restricted to young and mature neurons, HuR is more broadly expressed, including proliferating cells of the developing CNS. Because RNA stabilization of labile genes may promote tumor growth, we analyzed and compared the expression pattern of HuR in 35 freshly resected and cultured CNS tumors to determine whether there was any correlation with tumor grade or histological type. We found that HuR mRNA was consistently expressed in all of the tumors, regardless of cell origin or degree of malignancy. Using a novel HuR-specific polyclonal antibody, we found that strong HuR protein expression was limited to high-grade malignancies (glioblastoma multiforme and medulloblastoma). Within the glioblastoma multiforme, prominent HuR expression was also detected in perinecrotic areas in which angiogenic growth factors are up-regulated. To further define its role as a potential RNA stabilizer, we analyzed whether HuR could bind to the stability motifs within the 3'-UTRs of cytokines and growth factors linked to brain tumor progression. We used a novel ELISA-based RNA binding assay and focused on the 3'-UTRs of angiogenic factors VEGF, COX-2, and (interleukin) IL-8 as well as the immunomodulating factors IL-6, transforming growth factor (TGF)-beta and tumor necrosis factor (TNF)-alpha as potential RNA ligands. Our results indicated overall a very high binding affinity to these RNA targets. A comparison of these ligands revealed a hierarchy of binding affinities with the angiogenic factors, and TGF-beta showing the highest (Kd of 1.8-3.4 nM), and TNF-alpha the lowest (Kd of 18.3 nM). The expression pattern of HuR, coupled with the RNA binding data, strongly suggests a role for this protein in the posttranscriptional regulation of these genes in CNS tumors.

Several growth factors such as vascular endothelial growth factor (VEGF)-A and placental growth factor (PlGF) are involved in the placental vascular development. We investigated whether dysregulation in the VEGF family may explain the defective uteroplacental vascularization characterizing preeclampsia. We compared pregnancies complicated by early onset severe preeclampsia or intrauterine growth retardation to normal pregnancies. Maternal plasma, placentas, and placental bed biopsies were collected. The mRNA levels of VEGF-A, PlGF, and their receptors were quantified in placentas and placental beds. Levels of VEGF-A, PlGF, and soluble VEGF receptor (VEGFR) were assessed in maternal plasma. In compromised pregnancies, elevated levels of VEGF-A and VEGFR-1 mRNAs may reflect the hypoxic status of the placenta. On contrast, the membrane-bound VEGFR-1 was decreased in the placental bed of preeclamptic patients. Preeclampsia was associated with low levels of circulating PlGF and increased levels of total VEGF-A and soluble VEGFR-1. Free VEGF-A was undetectable in maternal blood. Immunohistochemical studies revealed that VEGF-A and PlGF were localized in trophoblastic cells. Altogether, our results suggest two different pathophysiological mechanisms associated with preeclampsia. The first one is related to an overproduction of competitive soluble VEGFR-1 that may lead to suppression of VEGF-A and PlGF effects. The second one is the down-regulation of its membrane bound form (VEGFR-1) in the placental bed, which may result in the defective uteroplacental development.

BACKGROUND

Olaparib is a poly(ADP-ribose) polymerase inhibitor and cediranib is an anti-angiogenic agent with activity against VEGF receptor (VEGFR) 1, VEGFR2, and VEGFR3. Both oral agents have antitumour activity in women with recurrent ovarian cancer, and their combination was active and had manageable toxicities in a phase 1 trial. We investigated whether this combination could improve progression-free survival (PFS) compared with olaparib monotherapy in women with recurrent platinum-sensitive ovarian cancer.

METHODS

In our randomised, open-label, phase 2 study, we recruited women (aged ≥18 years) who had measurable platinum-sensitive, relapsed, high-grade serous or endometrioid ovarian, fallopian tube, or primary peritoneal cancer, or those with deleterious germline BRCA1/2 mutations from nine participating US academic medical centres. We randomly allocated participants (1:1) according to permuted blocks, stratified by germline BRCA status and previous anti-angiogenic therapy, to receive olaparib capsules 400 mg twice daily or the combination at the recommended phase 2 dose of cediranib 30 mg daily and olaparib capsules 200 mg twice daily. The primary endpoint was progression-free survival analysed in the intention-to-treat population. The phase 2 trial is no longer accruing patients. An interim analysis was conducted in November, 2013, after 50% of expected events had occurred and efficacy results were unmasked. The primary analysis was performed on March 31, 2014, after 47 events (66% of those expected). The trial is registered with ClinicalTrials.gov, number NCT01116648.

RESULTS

Between Oct 26, 2011, and June 3, 2013, we randomly allocated 46 women to receive olaparib alone and 44 to receive the combination of olaparib and cediranib. Median PFS was 17·7 months (95% CI 14·7-not reached) for the women treated with cediranib plus olaparib compared with 9·0 months (95% CI 5·7-16·5) for those treated with olaparib monotherapy (hazard ratio 0·42, 95% CI 0·23-0·76; p=0·005). Grade 3 and 4 adverse events were more common with combination therapy than with monotherapy, including fatigue (12 patients in the cediranib plus olaparib group vs five patients in the olaparib monotherapy group), diarrhoea (ten vs none), and hypertension (18 vs none).

CONCLUSIONS

Cediranib plus olaparib seems to improve PFS in women with recurrent platinum-sensitive high-grade serous or endometrioid ovarian cancer, and warrants study in a phase 3 trial. The side-effect profile suggests such investigations should include assessments of quality of life and patient-reported outcomes to understand the effects of a continuing oral regimen with that of intermittent chemotherapy.

BACKGROUND

American Recovery and Reinvestment Act grant from the National Institutes of Health (NIH) (3 U01 CA062490-16S2); Intramural Program of the Center for Cancer Research; and the Division of Cancer Treatment and Diagnosis, National Cancer Institute, NIH.

Despite substantial evidence that nitric oxide (NO) and/or endogenous S-nitrosothiols (SNOs) exert protective effects in a variety of cardiovascular diseases, the molecular details are largely unknown. Here we show that following left coronary artery ligation, mice with a targeted deletion of the S-nitrosoglutathione reductase gene (GSNOR(-/-)) have reduced myocardial infarct size, preserved ventricular systolic and diastolic function, and maintained tissue oxygenation. These profound physiological effects are associated with increases in myocardial capillary density and S-nitrosylation of the transcription factor hypoxia inducible factor-1alpha (HIF-1alpha) under normoxic conditions. We further show that S-nitrosylated HIF-1alpha binds to the vascular endothelial growth factor (VEGF) gene, thus identifying a role for GSNO in angiogenesis and myocardial protection. These results suggest innovative approaches to modulate angiogenesis and preserve cardiac function.

Multiple cellular pathways influence the growth and metastatic potential of tumors. This creates heterogeneity, redundancy, and the potential for tumors to bypass signaling pathway blockade, resulting in primary or acquired resistance. Combining therapies that inhibit different signaling pathways has the potential to be more effective than inhibition of a single pathway and to overcome tumor resistance. Vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) inhibitors have become key therapies in several tumor types. Close relationships between these factors exist: VEGF signaling is up-regulated by EGFR expression and, conversely, VEGF up-regulation independent of EGFR signaling seems to contribute to resistance to EGFR inhibition. Therefore, inhibition of both pathways could improve antitumor efficacy and overcome resistance to EGFR inhibition. Preclinical studies have shown that VEGF and EGFR inhibitors can have additive effects and that combined inhibition is effective in EGFR inhibitor-resistant cell lines. Clinical trials have also produced promising data: combining the anti-VEGF monoclonal antibody bevacizumab with the anti-EGFR antibody cetuximab or the EGFR tyrosine kinase inhibitor erlotinib increases benefit compared with either of these anti-EGFR agents alone or combined with chemotherapy. The potential of this novel approach to anticancer therapy will be elucidated by large, ongoing clinical trials.

Vascular endothelial growth factor (VEGF), produced by almost all tumor cells, affects the ability of hemopoietic progenitor cells (HPC) to differentiate into functional dendritic cells (DC) during the early stages of their maturation. In this study we demonstrate specific binding of VEGF to HPC. This binding was efficiently competed by placenta growth factor (PIGF), a ligand reportedly specific for the Flt-1 receptor. The number of binding sites for VEGF decreased during DC maturation in vitro associated with decreased levels of mRNA for Flt-1. VEGF significantly inhibited nuclear factor-kappa B (NF-kappa B)-dependent activation of reporter gene transcription during the first 24 h in culture. The presence of VEGF significantly decreased the specific DNA binding of NF-kappa B as early as 30 min after induction with TNF-alpha. This was followed on days 7 to 10 by decreases in the mRNA for RelB and c-Rel, two subunits of NF-kappa B. Blockade of NF-kappa B activity in HPC at early stages of differentiation with an adenovirus expressing a dominant I kappa B inhibitor of NF-kappa B reproduced the pattern of effects observed with VEGF. Thus, NF-kappa B plays an important role in maturation of HPCs to DC, and VEGF activation of the Flt-1 receptor is able to block the activation of NF-kappa B in this system. Blockade of NF-kappa B activation in HPCs by tumor-derived factors may therefore be a mechanism by which tumor cells can directly down-modulate the ability of the immune system to generate effective antitumor immune responses.

miR-210 is a key player of cell response to hypoxia, modulating cell survival, VEGF-driven endothelial cell migration, and the ability of endothelial cells to form capillary-like structures. A crucial step in understanding microRNA (miRNA) function is the identification of their targets. However, only few miR-210 targets have been identified to date. Here, we describe an integrated strategy for large-scale identification of new miR-210 targets by combining transcriptomics and proteomics with bioinformatic approaches. To experimentally validate candidate targets, the RNA-induced silencing complex (RISC) loaded with miR-210 was purified by immunoprecipitation along with its mRNA targets. The complex was significantly enriched in mRNAs of 31 candidate targets, such as BDNF, GPD1L, ISCU, NCAM, and the non-coding RNA Xist. A subset of the newly identified targets was further confirmed by 3'-untranslated region (UTR) reporter assays, and hypoxia induced down-modulation of their expression was rescued blocking miR-210, providing support for the approach validity. In the case of 9 targets, such as PTPN1 and P4HB, miR-210 seed-pairing sequences localized in the coding sequence or in the 5'-UTR, in line with recent data extending miRNA targeting beyond the "classic" 3'-UTR recognition. Finally, Gene Ontology analysis of the targets highlights known miR-210 impact on cell cycle regulation and differentiation, and predicts a new role of this miRNA in RNA processing, DNA binding, development, membrane trafficking, and amino acid catabolism. Given the complexity of miRNA actions, we view such a multiprong approach as useful to adequately describe the multiple pathways regulated by miR-210 during physiopathological processes.

The critical role of IL-17 has recently been reported in a variety of conditions. Since IL-17 deeply participates in the pathogenesis of psoriasis and keratinocyte production of certain cytokines, the involvement of T helper cell 17 (Th17) in atopic dermatitis (AD) is an issue to be elucidated. To evaluate the participation of Th17 cells in AD, we successfully detected circulating lymphocytes intracellularly positive for IL-17 by flow cytometry, and the IL-17+ cell population was found exclusively in CD3+CD4+ T cells. The percentage of Th17 cells was increased in peripheral blood of AD patients and associated with severity of AD. There was a significant correlation between the percentages of IL-17+ and IFN-gamma+ cells, although percentage of Th17 cells was not closely related to Th1/Th2 balance. Immunohistochemically, IL-17+ cells infiltrated in the papillary dermis of atopic eczema more markedly in the acute than chronic lesions. Finally, IL-17 stimulated keratinocytes to produce GM-CSF, TNF-alpha, IL-8, CXCL10, and VEGF. A marked synergistic effect between IL-17 and IL-22 was observed on IL-8 production. The number of Th17 cells is increased in the peripheral blood and acute lesional skin of AD. Th17 cells may exaggerate atopic eczema.

Pigment epithelium-derived factor (PEDF) is a potent angiogenic inhibitor. Reduced PEDF levels are associated with diabetic retinopathy. However, the mechanism for the protective effects of PEDF against diabetic retinopathy (DR) is presently unclear. As inflammation plays a role in DR, the present study determined the effect of PEDF on inflammation. Western blot analysis and ELISA demonstrated that retinal and plasma PEDF levels were drastically decreased in rats with endotoxin-induced uveitis (EIU), which suggests that PEDF is a negative acute-phase protein. Intravitreal injection of PEDF significantly reduced vascular hyper-permeability in rat models of diabetes and oxygen-induced retinopathy, correlating with the decreased levels of retinal inflammatory factors, including VEGF, VEGF receptor-2, MCP-1, TNF-alpha, and ICAM-1. In cultured retinal capillary endothelial cells, PEDF significantly decreased TNF-alpha and ICAM-1 expression under hypoxia. Moreover, down-regulation of PEDF expression by siRNA resulted in significantly increases of VEGF and TNF-alpha secretion in retinal Müller cells. These findings suggest that PEDF is a novel endogenous anti-inflammatory factor in the eye. The decrease of ocular PEDF levels may contribute to inflammation and vascular leakage in DR.

IL-17, which exerts strong pro-inflammatory effects, has emerged as an important mediator in inflammation-associated cancer. However, the characteristics of IL-17-producing cells, the relevance of IL-17 to clinical parameters and its function in the development and progression of colorectal carcinoma still remain to be explored. In the present study, we first found the levels of IL-17 producing cells were significantly increased in the tumor regions of samples from colorectal carcinoma patients compared with non-tumor regions. Confocal microscopic analysis showed co-staining of IL-17 with CD4 and CD68, indicating IL-17 in colorectal carcinoma was expressed by macrophage and Th17. High expression of IL-17 was associated with high microvessel density. Univariate and multivariate analysis revealed that IL-17 was an independent prognostic factor for overall survival. To explore the underlying mechanisms of IL-17 in angiogenesis, we used PCR-array to find pro-angiogenic factor in cancer cells specifically induced by IL-17, then validated VEGF as one of factors in IL-17-mediated angiogenesis with the use of quantitative RT-PCR, ELISA and VEGF immunohistochemistry. Our results propose IL-17 as a novel indicator of prognosis in the patients with colorectal carcinoma and could serve as a novel therapeutic target for colorectal carcinoma, furthermore our results indicate that IL-17 producing cells may facilitate development of colorectal carcinoma by fostering angiogenesis via promote VEGF production from cancer cells.

Vascular endothelial growth factor (VEGF) is a prime regulator of normal and pathological angiogenesis. Three related endothelial cell growth factors, VEGF-B, VEGF-C, and VEGF-D were recently cloned. We have here studied the regulation of VEGF-C, a lymphatic endothelial growth factor, by angiogenic proinflammatory cytokines. Interleukin (IL)-1beta induced a concentration- and a time-dependent increase in VEGF-C, but not in VEGF-B, mRNA steady-state levels in human lung fibroblasts. The increase in VEGF-C mRNA levels was mainly due to increased transcription rather than elevated mRNA stability as detected by the nuclear run-on method and by following mRNA decay in the presence of an inhibitor of transcription, respectively. In contrast, angiopoietin-1 mRNA, encoding the ligand for the endothelial-specific Tek/Tie-2 receptor, was down-regulated by IL-1beta. Tumor necrosis factor-alpha and IL-1alpha also elevated VEGF-C mRNA steady-state levels, whereas the IL-1 receptor antagonist and dexamethasone inhibited the effect of IL-1beta. Experiments with cycloheximide indicated that the effect of IL-1beta was independent of protein synthesis. Hypoxia, which is an important inducer of VEGF expression, had no effect on VEGF-B or VEGF-C mRNA levels. IL-1beta and tumor necrosis factor-alpha also stimulated the production of VEGF-C protein by the fibroblasts. Cytokines and growth factors have previously been shown to down-regulate VEGF receptors in vascular endothelial cells. We found that the mRNA for the VEGF- and VEGF-C-binding VEGFR-2 (KDR/Flk-1) was stimulated by IL-1beta in human umbilical vein endothelial cells, whereas the mRNA levels of VEGFR-1 (Flt-1) and VEGFR-3 (Flt-4) were not altered. Our data suggest that in addition to VEGF, VEGF-C may also serve as an endothelial stimulus at sites of cytokine activation. In particular, these results raise the possibility that certain proinflammatory cytokines regulate the lymphatic vessels indirectly via VEGF-C.

The maintenance of oxygen homeostasis is critical for survival, and the master regulator of this process in metazoan species is hypoxia-inducible factor 1 (HIF-1), which controls both O(2) delivery and utilization. Under conditions of reduced O(2) availability, HIF-1 activates the transcription of genes, whose protein products mediate a switch from oxidative to glycolytic metabolism. HIF-1 is activated in cancer cells as a result of intratumoral hypoxia and/or genetic alterations. In cancer cells, metabolism is reprogrammed to favor glycolysis even under aerobic conditions. Pyruvate kinase M2 (PKM2) has been implicated in cancer growth and metabolism, although the mechanism by which it exerts these effects is unclear. Recent studies indicate that PKM2 interacts with HIF-1α physically and functionally to stimulate the binding of HIF-1 at target genes, the recruitment of coactivators, histone acetylation, and gene transcription. Interaction with HIF-1α is facilitated by hydroxylation of PKM2 at proline-403 and -408 by PHD3. Knockdown of PHD3 decreases glucose transporter 1, lactate dehydrogenase A, and pyruvate dehydrogenase kinase 1 expression; decreases glucose uptake and lactate production; and increases O(2) consumption. The effect of PKM2/PHD3 is not limited to genes encoding metabolic enzymes because VEGF is similarly regulated. These results provide a mechanism by which PKM2 promotes metabolic reprogramming and suggest that it plays a broader role in cancer progression than has previously been appreciated.

BACKGROUND

Sunitinib malate (SUTENT) is an oral, multitargeted tyrosine kinase inhibitor, approved multinationally for the treatment of advanced RCC and of imatinib-resistant or - intolerant GIST. The purpose of this study was to explore potential biomarkers of sunitinib pharmacological activity via serial assessment of plasma levels of four soluble proteins from patients in a phase II study of advanced RCC: VEGF, soluble VEGFR-2 (sVEGFR-2), placenta growth factor (PlGF), and a novel soluble variant of VEGFR-3 (sVEGFR-3).

METHODS

Sunitinib was administered at 50 mg/day on a 4/2 schedule (4 weeks on treatment, 2 weeks off treatment) to 63 patients with metastatic RCC after failure of first-line cytokine therapy. Predose plasma samples were collected on days 1 and 28 of each cycle and analyzed via ELISA.

RESULTS

At the end of cycle 1, VEGF and PlGF levels increased >3-fold (relative to baseline) in 24/54 (44%) and 22/55 (40%) cases, respectively (P < 0.001). sVEGFR-2 levels decreased>>or= 30% in 50/55 (91%) cases and>>or= 20% in all cases (P < 0.001) during cycle 1, while sVEGFR-3 levels were decreased>>or= 30% in 48 of 55 cases (87%), and>>or= 20% in all but 2 cases. These levels tended to return to near-baseline after 2 weeks off treatment, indicating that these effects were dependent on drug exposure. Overall, significantly larger changes in VEGF, sVEGFR-2, and sVEGFR-3 levels were observed in patients exhibiting objective tumor response compared with those exhibiting stable disease or disease progression (P < 0.05 for each analyte; analysis not done for PlGF).

CONCLUSIONS

Sunitinib treatment in advanced RCC patients leads to modulation of plasma levels of circulating proteins involved in VEGF signaling, including soluble forms of two VEGF receptors. This panel of proteins may be of value as biomarkers of the pharmacological and clinical activity of sunitinib in RCC, and of angiogenic processes in cancer and other diseases.

Flk-1 (human counterpart, KDR) tyrosine kinase, which is one of the two VEGF receptors, is crucial for vascular development. Recently, we showed that, among tyrosine residues of KDR, tyrosine residues 1175 (Y1175, corresponding to Y1173 in murine Flk-1) and Y1214 (Y1212 in Flk-1) are autophosphorylated in response to VEGF, and that Y1175 is important for VEGF-dependent phospholipase Cgamma/PKC/mitogen-activated protein kinase activation leading to DNA synthesis in cultured endothelial cells. However, the importance of these tyrosine residues in Flk-1/KDR in vivo is not yet known. To examine the role of these Flk-1 tyrosine residues in vivo, we generated knock-in mice substituting Y1173 and Y1212 of the Flk-1 gene with phenylalanine, respectively. As a result, Flk-1(1173F) homozygous mice died between embryonic days 8.5 and 9.5 without any organized blood vessels or yolk sac blood islands, and hematopoietic progenitors were severely reduced, similar to the case of Flk-1 null mice. In contrast, Flk-1(1212F) homozygous mice were viable and fertile. These results suggest that the signaling via Y1173 of Flk-1 is essential for endothelial and hematopoietic development during embryogenesis.