Recipient-Site Preconditioning with Deferoxamine Increases Fat Graft Survival by Inducing VEGF and Neovascularization in a Rat Model.

BACKGROUND: The authors hypothesize that ischemic preconditioning of the recipient site with deferoxamine will increase fat graft survival by enhancing angiogenesis in a rat model. METHODS: Cell viability, tube formation, and mRNA expression were measured in human umbilical vein endothelial cells treated with deferoxamine. A total of 36 rats were then used for an in vivo study. A dose of 100 mg/kg of deferoxamine was injected subcutaneously into the rat scalp every other day for five treatments. On the day after the final injection, the scalp skin was harvested from half the animals to evaluate the effects of deferoxamine on the recipient site. In the remaining animals, inguinal fat tissue was transplanted to the scalp. Eight weeks after transplantation, the grafts were harvested to evaluate the effects of deferoxamine preconditioning on fat graft survival. RESULTS: In human umbilical vein endothelial cells, treatment with a deferoxamine concentration higher than 400 µM decreased cell viability compared with the control (p = 0.002). Treatment with 100 and 200 µM deferoxamine increased endothelial tube formation (p = 0.001) and mRNA levels of angiogenesis-related factors (p = 0.02). Rat scalps treated with deferoxamine exhibited increased capillary neoformation (p = 0.001) and vascular endothelial growth factor protein expression (p = 0.024) compared with controls. Fat graft volume retention, capillary density (p < 0.001), and adipocyte viability (p < 0.001) in the grafted fat increased when the recipient site was preconditioned with deferoxamine. CONCLUSION: This study demonstrated that recipient site preconditioning with deferoxamine increases fat graft survival by inducing vascular endothelial growth factor and neovascularization.

Elevated blood/lymphatic vessel ratio in pterygium and its relationship with vascular endothelial growth factor (VEGF) distribution.

INTRODUCTION: Pterygium is a conjunctival fibrovascular tissue growth on the cornea. The pathogenesis of pterygium involves several factors such as the presence of active angiogenic factors. Expansion of the lymphatic microvasculature has also been hypothesized. This study examines the activity of the angiogenic/lymphangiogenic factor VEGF and the expression of vascular and lymphatic endothelial proteins in pterygia and normal conjunctival tissues. MATERIAL AND METHODS: Primary grade 2 pterygium (n=20) and normal conjunctiva (n=20) biopsies were obtained during surgery after written informed consent. mRNA expression for CD31, podoplanin, and VEGF (isoforms VEGF-A and VEGF-165) were determined by qRT-PCR. Tissue samples were also processed for immunohistochemical techniques to examine the lymphatic and vascular endothelium (anti-D2-40, anti-CD31 respectively) and VEGF-A and VEGF-C levels and distribution. RESULTS: VEGF-A gene expression levels failed to differ between the healthy and pterygium tissues. However, expression of its more angiogenic isoform, VEGF-165, was significantly higher in the pterygia. Immunohistochemistry revealed the greater presence of VEGF-A, compared to VEGF-C, in pterygium than conjunctiva, both in blood vessels and extracellular matrix. In addition, pterygia showed higher expression levels of the endothelial junction protein CD31. Lymphatic marker D2-40 expression was slightly augmented in this pathological tissue. The ratio between blood and lymphatic vessel counts was 1.05 in the normal conjunctiva and 3-fold this value in pterygium. CONCLUSION: In pterygium, while both lymphangiogenesis and angiogenesis take place, the formation of new blood vessels is the most relevant event, correlating with the increased expression of vascular endothelial CD31 and an elevated blood/lymphatic vessel ratio. The presence of high levels of VEGF-A in both vessel networks and extracellular matrix in human pterygium tissue may have a major impact on angiogenesis in this pathological tissue.

Quantification of STAT3 and VEGF expression for molecular diagnosis of lymph node metastasis in breast cancer.

BACKGROUND: Axillary lymph node metastasis is associated with increased risk of regional recurrence, distant metastasis, and poor survival in breast malignant neoplasm. Expression of signal transducer and activator of transcription 3 (STAT3) is significantly associated with tumor formation, migration, and invasion in various cancers. In addition, vascular endothelial growth factor (VEGF) expression could promote angiogenesis and increase the risk of tumorigenesis. To determine correlations among STAT3 expression, VEGF, and clinicopathological data on lymph node involvement in breast cancer patients after surgery. METHODS: The mRNA expression levels of STAT3 and VEGFs were measured in 45 breast invasive ductal carcinoma tissues, 45 peritumoral tissues, and 45 adjacent nontumor tissues by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Postoperative pathological examination revealed explicit axillary lymph node involvement in all patients. RESULTS: Average mRNA levels of STAT3 and VEGFs were the highest in breast invasive ductal carcinoma tissues, followed by peritumoral tissues. High expression of STAT3 showed significant positive correlation with high axillary lymph node involvement and progesterone receptor (PR), VEGF-C, VEGF-D, and vascular endothelial growth factor receptor (VEGFR)-3 expression. The expression levels of STAT3, VEGF-C, and VEGFR-3 were significantly higher in the tumor tissues of patients with axillary lymph node metastasis than in those of patients without the metastasis. Expression levels of VEGF-C and VEGFR-3 were also significantly higher in peritumoral tissues of patients with axillary lymph node metastasis. Positive correlations were found between STAT3 and VEGF-C/-D mRNA levels. CONCLUSION: These data suggest that STAT3/VEGF-C/VEGFR-3 signaling pathway plays an important role in carcinogenesis and lymph-angiogenesis. Our findings suggest that STAT3 may be a potential molecular biomarker for predicting the involvement of axillary lymph nodes in breast cancer, and therapies targeting STAT3 may be important for preventing breast cancer metastasis.

A Mini-Review on Thalidomide: Chemistry, Mechanisms of Action, Therapeutic Potential and Anti-Angiogenic Properties in Multiple Myeloma.

Thalidomide is a drug with interesting therapeutic properties but also with severe side effects which require a careful and monitored use. Potential immunomodulatory, antiinflammatory, anti-angiogenic and sedative properties make thalidomide a good candidate for the treatment of several diseases such as multiple myeloma. Through an increase in the degradation of TNFα-mRNA, thalidomide reduces the production of TNFα by monocytes and macrophages stimulated by lipopolysaccharide or by T lymphocytes induced by mitogenic stimuli. The decreased level of TNFα alters the mechanisms of intracellular transduction by preventing the activation of NF-kB and by decreasing the synthesis of proteins, in particular IL-6, involved in cell proliferation, inflammation, angiogenesis and protection from apoptosis. Furthermore, thalidomide affects VEGF levels by down-regulating its expression. Nowadays, new safer and less toxic drugs, analogs of thalidomide, are emerging as beneficial for a more targeted treatment of multiple myeloma and several other diseases such as Crohn';s disease, rheumatoid arthritis, sarcoidosis, erythema nodosum leprosum, graft-versus-host disease.

Formononetin, an active compound of Astragalus membranaceus (Fisch) Bunge, inhibits hypoxia-induced retinal neovascularization via the HIF-1α/VEGF signaling pathway.

BACKGROUND: It has been reported that formononetin (FMN), one of the main ingredients from famous traditional Chinese medicine "Huang-qi" (Astragalus membranaceus [Fisch] Bunge) for Qi-tonifying, exhibits the effects of immunomodulation and tumor growth inhibition via antiangiogenesis. Furthermore, A. membranaceus may alleviate the retinal neovascularization (NV) of diabetic retinopathy. However, the information of FMN on retinal NV is limited so far. In the present study, we investigated the effects of FMN on the hypoxia-induced retinal NV and the possible related mechanisms. MATERIALS AND METHODS: The VEGF secretion model of acute retinal pigment epithelial-19 (ARPE-19) cells under chemical hypoxia was established by the exposure of cells to 150 µM CoCl2 and then cells were treated with 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1, a potent HIF-1α inhibitor, 1.0 µg/mL) or different concentrations of FMN (0.2 µg/mL, 1.0 µg/mL, and 5.0 µg/mL). The supernatants of cells were collected 48 hours later to measure the VEGF concentrations, following the manufacturer's instruction. The mRNA expressions of VEGF, HIF-1α, PHD-2, and ß-actin were analyzed by quantitative reverse transcription polymerase chain reaction, and the protein expressions of HIF-1α and PHD-2 were determined by Western blot analysis. Furthermore, the rats with retinopathy were treated by intraperitoneal administration of conbercept injection (1.0 mg/kg) or FMN (5.0 mg/kg and 10.0 mg/kg) in an 80% oxygen atmosphere. The retinal avascular areas were assessed through visualization of the retinal vasculature by adenosine diphosphatase staining and hematoxylin and eosin staining. RESULTS: FMN can indeed inhibit the VEGF secretion of ARPE-19 cells under hypoxia, downregulate the mRNA expression of VEGFA and PHD-2, and decrease the protein expression of VEGF, HIF-1α, and PHD-2 in vitro. Furthermore, FMN can prevent hypoxia-induced retinal NV in vivo. CONCLUSION: FMN can ameliorate retinal NV via the HIF-1α/VEGF signaling pathway, and it may become a potential drug for the prevention and treatment of diabetic retinopathy.

Association between VEGF-A, C and D expression and lymph node involvement in breast cancer: a meta-analysis.

BACKGROUND: Metastasis is the primary cause of death in patients with breast cancer. Although VEGF-A, C and D are considered to be prime factors in lymph node metastasis in breast cancer, the published studies have conflicting conclusions. METHODS: To resolve this conflict, we conducted a meta-analysis of 37 studies (n = 5,001 patients) evaluating the correlation between VEGF-A, C and D immunohistochemical expression and lymph node metastasis (LNM). The meta-analysis included 22 studies of VEGF-A, 17 of VEGF-C, and 6 of VEGF-D. The relationships between VEGF-A, C and D and clinicopathological parameters were also examined. RESULTS: The results showed a significant association between VEGF-A or VEGF-C overexpression and LNM (risk ratio [RR] = 1.28 [95% CI 1.04-1.58], p = 0.02; and RR = 1.36 [95% CI 1.07-1.72], p = 0.01, respectively). Subgroup evaluation showed a significant association between VEGF-A, C and D overexpression and LNM when analyses were limited to Asian patients (RR = 1.78 [95% CI 1.28-2.46], p = 0.0005; RR = 1.38 [95% CI 1.04-1.84], p = 0.03, and RR = 2.62 [95% CI 1.35-5.09], p = 0.004, respectively). VEGF-A overexpression was significantly associated with lymph vessel invasion (RR = 1.86 [95% CI 1.33-2.60], p = 0.0003). Overexpression of VEGF-C or VEGF-D was significantly associated with HER-2 positivity (RR = 1.30 [95% CI 1.06-1.59], p = 0.01; and RR = 1.75 [95% CI 1.01-3.03], p = 0.05, respectively). CONCLUSIONS: With some limitations, our meta-analysis indicated that VEGF-A and C could predict LNM in patients with breast cancer, particularly Asian patients.

Prognostic impact of elevation of vascular endothelial growth factor family expression in patients with non-small cell lung cancer: an updated meta-analysis.

BACKGROUND: The vascular endothelial growth factor family has been implicated in tumorigenesis and metastasis. The prognostic value of each vascular endothelial growth factor family member, particular VEGF/ VEGFR co-expression, in patients with non-small lung cancer remains controversial. MATERIALS AND METHODS: Relevant literature was identified by searching PubMed, EMBASE and Web of Science. Studies evaluating expression of VEGFs and/or VEGFRs by immunohistochemistry or ELISA in lung cancer tissue were eligible for inclusion. Hazard ratios (HRs) and 95% confidence intervals (CIs) from individual study were pooled by using a fixed- or random-effect model, heterogeneity and publication bias analyses were also performed. RESULTS: 74 studies covering 7,631 patients were included in the meta-analysis. Regarding pro-angiogenesis factors, the expression of VEGFA (HR=1.633, 95%CI: 1.490-1.791) and VEGFR1 (HR=1.924, 95%CI: 1.220-3.034) was associated separately with poor survival. Especially, VEGFA over-expression was an independent prognostic factor in adenocarcinoma (ADC) (HR=1.775, 95%CI: 1.384-2.275) and SCC (HR=2.919, 95%CI: 2.060-4.137). Co-expression of VEGFA/VEGFR2 (HR=2.011, 95%CI: 1.405-2.876) was also significantly associated with worse survival. For lymphangiogenesis factors, the expression of VEGFC (HR=1.611, 95%CI: 1.407-1.844) predicted a poor prognosis. Co-expression of VEGFC/VEGFR3 (HR=2.436, 95%CI: 1.468-4.043) emerged as a preferable prognostic marker. CONCLUSIONS: The expression of VEGFA (particularly in SCC and early stage NSCLC), VEGFC, VEGFR1 indicates separately an unfavorable prognosis in patients with NSCLC. Co-expression VEGFA/ VEGFR2 is comparable with VEGFC/VEGFR3, both featuring sufficient discrimination value as preferable as prognostic biologic markers.

Lower values of VEGF in endometrial secretion are a possible cause of subfertility in non-atopic asthmatic patients.

OBJECTIVE: Using endometrial secretion analysis, we assessed whether altered inflammatory cytokine levels can be detected in the uterine environment in asthma patients, thereby providing a possible cause of reduced fertility in asthmatics. METHODS: Forty-four unexplained infertile women (aged 28-44) underwent asthma and allergy testing, questionnaires, endometrial secretion and blood samples in the mid-secretory phase of the menstrual cycle (day 19-23) during assisted reproduction. Differences in cytokines and growth factors were analyzed. RESULTS: Mean log-VEGF in uteri was lower in asthma patients compared with controls (2.29 versus 2.70, p = 0.028). This was mainly due to lower values of VEGF among women with non-atopic asthma compared with women with atopic asthma (1.86 versus 2.72, p = 0.009) and with healthy controls (1.86 versus 2.70, p = 0.01). Asthma treatment status had no effect on VEGF levels in uteri. Serum high sensitivity CRP was negatively correlated with VEGF in endometrial secretions. No other significant correlations were observed between peripheral blood values and markers found in utero. CONCLUSION: Asthma is associated with lower values of VEGF in uterine endometrial secretions, which might affect the receptiveness of the endometrium and thereby increase time to pregnancy. The effect appears to be associated with non-atopic asthma with general increased systemic inflammation.

Expression of tissue factor signaling pathway elements correlates with the production of vascular endothelial growth factor and interleukin-8 in human astrocytoma patients.

The expression levels of tissue factor (TF), the clotting initiator protein, have been correlated with angiogenesis and the histological grade of malignancy in glioma patients. The pro-tumor function of TF is linked to a family of G protein-coupled receptors known as protease-activated receptors (PARs), which may be activated by blood coagulation proteases. Activation of PARs elicits a number of responses, including the expression of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). In the present study, we analyzed the expression of TF signaling pathway elements (TF, PAR1 and PAR2) and evaluated their correlation with the expression of downstream products (VEGF and IL-8) in human astrocytoma patients. Quantitative PCR (qPCR) showed a significant increase in TF expression in grade IV (glioblastoma) tumors, which was inversely correlated with the expression of the tumor-suppressor PTEN. Immunohistochemistry and qPCR analyses demonstrated a highly significant elevation in the expression of PAR1, but not PAR2, in tumor samples from high-grade astrocytoma patients. The elevated VEGF expression levels detected in the high-grade astrocytoma samples were positively correlated with TF, PAR1 and PAR2 expression. In addition, IL-8 was significantly increased in glioblastoma patients and positively correlated with TF and PAR2 expression. Further in vitro assays employing the human glioma cell lines U87-MG and HOG demonstrated that a synthetic peptide PAR2 agonist stimulated VEGF and IL-8 production. Our findings suggest a role for TF signaling pathway elements in astrocytoma progression, particularly in glioblastoma. Therefore, TF/PAR signaling elements may be suitable targets for the development of new therapies for the treatment of aggressive glioma.

DcR3 regulates the growth and metastatic potential of SW480 colon cancer cells.

Decoy receptor 3 (DcR3) is considered to have anti­apoptotic and pro-metastatic functions, suggesting it might be a therapeutic target. We examined the role and mechanisms of DcR3 on growth and the metastatic ability of SW480 colon cancer cells to provide therapeutic information for targeting DcR3 by RNA interference (RNAi) technology. Growth and the metastatic ability were inhibited, apoptosis was induced and cell cycle profile was changed after decreasing DcR3 expression, with lower levels of vascular endothelial growth factors (VEGFs) and matrix metalloproteinases (MMPs) expression. Our results implied the therapeutic potential of silencing DcR3 expression by RNAi in colon cancer.

A structure-activity relationship study of 1,2,4-triazolo[1,5-a][1,3,5]triazin-5,7-dione and its 5-thioxo analogues on anti-thymidine phosphorylase and associated anti-angiogenic activities.

Thirty-three 1,2,4-triazolo[1,5-a][1,3,5]triazin-5,7-dione and its 5-thioxo analogues were designed and synthesized which contained different substituents at meta- and/or para-positions of 2-phenyl or 2-benzyl ring attached to the fused ring structure. The preliminary pharmacological evaluation demonstrated that the 5-thioxo analogues of 1,2,4-triazolo[1,5-a][1,3,5]triazine exhibited a varying degree of inhibitory activity towards thymidine phosphorylase, comparable or better than reference compound, 7-Deazaxanthine (7-DX, 2) (IC50 value = 42.63 µM). Moreover, compounds 5q and 6i displayed a mixed-type of inhibitory mechanism in the presence of variable concentrations of thymidine (dThd). In addition, selected compounds were found to have a noticeable inhibitory effect on the expression of angiogenesis markers, including VEGF and MMP-9 in MDA-MB-231 breast cancer cells.

Association of single nucleotide polymorphisms of ß2-adrenergic receptor gene with clinicopathological features of pancreatic carcinoma.

ß2-Adrenoceptor agonists induce pancreatic cancer occurrence and progression through ß2-AR. Polymorphisms in ß2-AR gene lead to modified sensitivity to agonists and variable tumorigenic potential. In this study, pancreatic carcinoma and non-neoplastic pancreatic tissues were genotyped at codons 16 and 27 by PCR-restriction fragment length polymorphism and DNA sequencing. Expressions of ß2-AR, EGFR, VEGF and MMP-2 were detected by immunohistochemistry. The frequencies of genotypes and alleles at codon 16 between pancreatic carcinoma and non-neoplastic pancreatic tissues showed no difference. The genotype frequencies were associated with TNM grade, lymph node metastasis, and one-year survival rate. The allele G at codon 16 frequently appeared in tumors with high TNM grade, lymph node metastasis, poor prognosis, high expression levels of ß2-AR, EGFR, VEGF and MMP-2. The genotype and allele frequencies of codon 27 were not associated with clinicopathological features and down-stream protein expressions. Collectively, SNPs of ß2-AR gene at codon 16 were associated with the biological behavior of pancreatic carcinoma. The allele G at codon 16 could facilitate the progression and metastasis of pancreatic carcinoma through elevating vascularization and activating the EGFR pathway. SNPs at codon 16 of ß2-AR are new useful biomarkers for predicting biological behavior and survival of pancreatic carcinoma and might be used as a new gene therapeutic target.

mTORC1 and mTORC2 play different roles in the functional survival of transplanted adipose-derived stromal cells in hind limb ischemic mice via regulating inflammation in vivo.

Poor cell survival severely limits the beneficial effects of stem cell therapy for peripheral arterial disease (PAD). This study was designed to investigate the role of mammalian target of rapamycin (mTOR) in the survival and therapeutic function of transplanted murine adipose-derived stromal cells (mADSCs) in a murine PAD model. mADSCs (1.0 × 10(7)) were isolated from dual-reporter firefly luciferase and enhanced green fluorescent protein-positive transgenic mice, intramuscularly implanted into the hind limb of C57BL/6 mice after femoral artery ligation/excision, and monitored using noninvasive bioluminescence imaging (BLI). Although engrafted mADSCs produced antiapoptotic/proangiogenic effects in vivo by modulating the inflammatory and angiogenic cytokine response involving the mTOR pathway, longitudinal BLI revealed progressive death of post-transplant mADSCs within ~4 weeks in the ischemic hind limb. Selectively targeting mTOR complex-1 (mTORC1) using low-dose rapamycin treatment with mADSCs attenuated proinflammatory cytokines (interleukin [IL]-1ß and tumor necrosis factor-alpha [TNF-α]) expression and neutrophil/macrophage infiltration, which overtly promoted mADSCs viability and antiapoptotic/proangiogenic efficacy in vivo. However, targeting dual mTORC1/mTORC2 using PP242 or high-dose rapamycin caused IL-1ß/TNF-α upregulation and anti-inflammatory IL-10, IL-6, and vascular endothelial growth factor/vascular endothelial growth factor receptor 2 downregulation, undermining the survival and antiapoptotic/proangiogenic action of mADSCs in vivo. Furthermore, low-dose rapamycin abrogated TNF-α secretion by mADSCs and rescued the cells from hypoxia/reoxygenation-induced death in vitro, while PP242 or high-dose rapamycin exerted proinflammatory effects and promoted cell death. In conclusion, mTORC1 and mTORC2 may differentially regulate inflammation and affect transplanted mADSCs' functional survival in ischemic hind limb. These findings uncover that mTOR may evolve into a promising candidate for mechanism-driven approaches to facilitate the translation of cell-based PAD therapy.

Altered gene expression profile in cumulus cells of mature MII oocytes from patients with polycystic ovary syndrome.

STUDY QUESTION: Oocyte developmental competence is altered in patients with polycystic ovary syndrome (PCOS); is gene expression in cumulus cells (CCs) from mature metaphase II oocytes of patients with PCOS altered as well? SUMMARY ANSWER: Compared with CCs from non-PCOS patients, the gene expression profile of CCs isolated from mature oocytes of patients with PCOS present alterations that could explain the abnormal folliculogenesis and reduced oocyte competence in such patients. WHAT IS KNOWN ALREADY: Abnormal mRNA expression of several members of the insulin-like growth factor (IGF) family in CCs from PCOS patients was previously reported. Moreover, the whole transcriptome has been investigated in cultured CCs from PCOS patients. STUDY DESIGN, SIZE AND DURATION: This retrospective study included six PCOS patients diagnosed following the Rotterdam Criteria and six non-PCOS patients who all underwent ICSI for male infertility in the assisted reproduction technique (ART) Department of Montpellier University Hospital, between 2009 and 2011. PARTICIPANTS/MATERIALS, SETTING AND METHODS: CCs from PCOS and non-PCOS patients who underwent controlled ovarian stimulation (COS) were isolated mechanically before ICSI. Gene expression profiles were analysed using the microarray technology and the Significance Analysis of Microarray was applied to compare the expression profiles of CCs from PCOS and non-PCOS patients. MAIN RESULTS: The gene expression profile of CCs from patients with PCOS was significantly different from that of CCs from non-PCOS patients. Specifically, CCs from women with PCOS were characterized by abnormal expression of many growth factors, including members of the epidermal growth factor-like (EGFR, EREG and AREG) and IGF-like families (IGF1R, IGF2R, IGF2BP2 and IGFBP2), that are known to play a role in oocyte competence. In addition, mRNA transcripts of factors involved in steroid metabolism, such as CYP11A1, CYP1B1, CYP19A1 and CYP2B7P1, were deregulated in PCOS CCs, and this could explain the abnormal steroidogenesis observed in these women. Functional annotation of the differentially expressed genes suggests that defects in the transforming growth factor ß and estrogen receptors signalling cascades may contribute to the reduced oocyte developmental competence in patients with PCOS. LIMITATIONS AND REASONS FOR CAUTION: Owing to the strict selection criteria (similar age, weight and reasons for ART), this study included a small sample size (six cases and six controls), and thus, further investigations using a large cohort of patients are needed to confirm these results. WIDER IMPLICATIONS OF THE FINDINGS: This study opens a new perspective for understanding the pathogenesis of PCOS. STUDY FUNDING/COMPETING INTERESTS: This work was partially supported by a grant from the Ferring Pharmaceutical. The authors of the study have no competing interests to report. TRIAL REGISTRATION NUMBER: Not applicable.

In vitro expression of vascular endothelial growth factor and its receptors by placental macrophages.

The expression of VEGF and membrane-bound and soluble forms of the VEGF-R1 receptor in cultured placental macrophages (trimesters I and III of pregnancy) was studied by flow cytometry, cytometric bead array, and ELISA. Nearly all population of placental macrophages (98%) was capable of producing VEGF during the early and late gestational periods. However, the expression of cellular VEGF-R1 varied from 3.4 to 92%. VEGF secretion was relatively low in the first and third trimesters (0.5 and 1.1 pg/10(5) cells, respectively). Cultured placental macrophages produced soluble receptor sVEGF-R1 in the first and third trimesters (86.4 and 36.4 pg/10(5) cells, respectively). Stimulation with LPS was followed by a 4-fold increase in sVEGF-R1 secretion. Our results indicate that placental macrophages are involved in the autocrine and paracrine regulation in chorionic villi. The data suggest that these cells have a physiological and pathogenetic role in gestation.

Protection against titanium particle-induced inflammatory osteolysis by the proteasome inhibitor bortezomib in vivo.

Wear particle-induced vascularized granulomatous inflammation and subsequent inflammatory osteolysis is the most common cause of aseptic loosening after total joint replacement (TJR); however, the precise mechanism by which this occurs is unclear. This study investigates the effects of the proteasome inhibitor bortezomib (Bzb) on the expression of key biochemical markers of bone metabolism and vascularised granulomatous tissues, such as receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), vascular endothelial growth factor (VEGF) and tumor necrosis factor receptor-associated factor 6 (TRAF6). In addition, the effect of Bzb on apoptosis of CD68+ cells was examined. A total of 32 female BALB/C mice were randomly divided into four groups. After implantation of calvaria bone from syngeneic littermates, titanium (Ti) particles were injected into established air pouches for all mice (excluding negative controls) to provoke inflammatory osteolysis. Subsequently, Bzb was administered at a ratio of 0, 0.1, or 0.5 mg/kg on day 1, 4, 8, and 11 post-surgery to alleviate this response. All of the air pouches were harvested 14 days after the surgical procedure and were processed for molecular and histological analysis. The results demonstrated that Ti injection elevated the expression of RANKL, OPG, VEGF, and TRAF6 at both the gene and protein levels, increased counts of infiltrated cells and thickness of air pouch membranes, and elevated the apoptosis index (AI) of CD68+ cells. Bzb treatment significantly improved Ti particle-induced implanted bone osteolysis, attenuated vascularised granulomatous tissues and elevated AI of CD68+ cells. Therefore, the proteasome pathway may represent an effective therapeutic target for the prevention and treatment of aseptic loosening.

Breaking the 'harmony' of TNF-α signaling for cancer treatment.

Tumor necrosis factor-alpha (TNF-α) binds to two distinct receptors, TNFR1/p55 and TNFR2/p75. TNF-α is implicated in the processes of tumor growth, survival, differentiation, invasion, metastases, secretion of cytokines and pro-angiogenic factors. We have shown that TNFR2/p75 signaling promotes ischemia-induced angiogenesis via modulation of several angiogenic growth factors. We hypothesized that TNFR2/p75 may promote tumor growth and angiogenesis. Growth of mouse Lewis lung carcinoma (LLC1) and/or mouse melanoma B16 cell was evaluated in wild type (WT), p75 knockout (KO) and double p55KO/p75KO mouse tumor xenograft models. Compared with WT and p55KO/p75KO mice, growth of tumors in p75KO mice was significantly decreased (twofold) in both LLC and B16 tumors. Tumor growth inhibition was correlated with decreases in vascular endothelial growth factor (VEGF) expression and capillary density, as well as bone marrow-derived endothelial progenitor cells incorporation into the functional capillary network, and an increase in apoptotic cells in LLC xenografts. Gene array analysis of tumor tissues showed a decrease in gene expression in pathways that promote tumor angiogenesis and cell survival. Blocking p75 by short-hairpin RNA in cultured LLCs led to increases in TNF-mediated apoptosis, as well as decreases in the constitutive and TNF-mediated expression of angiogenic growth factors (VEGF, HGF, PLGF), and SDF-1α receptor CXCR4. In summary, p75 is essential for tumor angiogenesis and survival in highly vascularized murine lung tumor xenografts. Blocking p75 expression may lead to tumor regression. This may represent new and effective therapy against lung neoplasms and potentially tumors of other origin.

Effect of heparin-binding EGF-like growth factor and amphiregulin on the MAP kinase-induced production of vascular endothelial growth factor by human granulosa cells.

The function of granulosa cells is regulated by various hormones and growth factors. Our aim is to clarify the regulation of vascular endothelial growth factor (VEGF) production induced by heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR) in a human granulosa cell line, KGN. KGN cells were cultured and incubated for 24 h with HB-EGF and AR. The levels of VEGF in the culture media were measured by an enzyme-linked immunosorbent assay. The activation of MAP kinase in KGN cells was detected by Western blot analysis. VEGF production was significantly increased by HB-EGF or AR alone in a dose-dependent manner, whereas it was decreased by AG1478 or U0126. The MAP kinase activity was increased by treatment with HB-EGF or AR. The results suggested that VEGF is induced by HB-EGF and AR through mechanisms involving MAP kinase. The increase in VEGF may contribute to neovascularization, which in turn would promote various ovulation phenomena as well as follicular growth.

Effect of methylprednisolone on perivascular pulmonary edema, inflammatory infiltrate, VEGF and TGF-beta immunoexpression in the remaining lungs of rats after left pneumonectomy

Pneumonectomy is associated with high rates of morbimortality, with postpneumonectomy pulmonary edema being one of the leading causes. An intrinsic inflammatory process following the operation has been considered in its physiopathology. The use of corticosteroids is related to prevention of this edema, but no experimental data are available to support this hypothesis. We evaluated the effect of methylprednisolone on the remaining lungs of rats submitted to left pneumonectomy concerning edema and inflammatory markers. Forty male Wistar rats weighing 300 g underwent left pneumonectomy and were randomized to receive corticosteroids or not. Methylprednisolone at a dose of 10 mg/kg was given before the surgery. After recovery, the animals were sacrificed at 48 and 72 h, when the pO2/FiO2 ratio was determined. Right lung perivascular edema was measured by the index between perivascular and vascular area and neutrophil density by manual count. Tissue expression of vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-β) were evaluated by immunohistochemistry light microscopy. There was perivascular edema formation after 72 h in both groups (P = 0.0031). No difference was observed between operated animals that received corticosteroids and those that did not concerning the pO2/FiO2 ratio, neutrophil density or TGF-β expression. The tissue expression of VEGF was elevated in the animals that received methylprednisolone both 48 and 72 h after surgery (P = 0.0243). Methylprednisolone was unable to enhance gas exchange and avoid an inflammatory infiltrate and TGF-β expression also showed that the inflammatory process was not correlated with pulmonary edema formation. However, the overexpression of VEGF in this group showed that methylprednisolone is related to this elevation.

Differential dependency of human cancer cells on vascular endothelial growth factor-mediated autocrine growth and survival.

Analysis using the public microarray database Gene Expression Omnibus indicates significantly higher mRNA expression of VEGF and VEGFRs in colorectal cancer and high grade astrocytoma but not in hepatocellular carcinoma compared to normal tissue. Human malignant astrocytoma cell lines (U251-MG and U373-MG) and HT-1080 fibrosarcoma cells expressed relatively higher levels of VEGF and VEGFRs compared to hepatocellular and colorectal cancer cell lines. Administration of exogenous VEGF-A induced cell growth in a dose-dependent fashion in astrocytoma and fibrosarcoma cells but not in colorectal and hepatocellular cancer cells. The blockade of VEGF inhibited cell survival only in U251-MG, U373-MG and HT-1080 cells. These results collectively suggest the role of autocrine VEGF signaling in various cancer cells and provide a basis for the variable clinical responses to antiangiogenic therapy observed in different types of malignancies.