OBJECTIVE

To evaluate changes in expression levels of vascular endothelial growth factor (VEGF) mRNA in human endometrial explants in a chicken chorioallantoic membrane model of endometriosis.

METHODS

Experimental prospective study.

METHODS

University hospital.

METHODS

Endometrial biopsy samples were obtained from healthy, ovulating women undergoing elective surgery.

METHODS

Endometrial fragments were placed on the chicken chorioallantoic membrane and removed for analysis after 0, 24, 48, and 72 hours.

METHODS

Expression of different VEGF mRNA splice variants was tested. Expression of VEGF(165) mRNA was assessed by using competitive polymerase chain reaction and normalized to expression of the housekeeping gene human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA.

RESULTS

After 0, 24, 48, and 72 hours of incubation, all grafts expressed VEGF(121), VEGF(145), VEGF(165), and VEGF(189) mRNA. Expression of VEGF(165) mRNA increased up to 10-fold at 24 to 72 hours compared with precultivation values.

CONCLUSIONS

Levels of VEGF(165) mRNA in endometrial grafts increase after implantation on chicken chorioallantoic membrane. Hypoxic induction of VEGF mRNA expression in endometrial cell cultures has been reported previously. Induction of VEGF expression might indicate relative hypoxia of the specimen due to insufficient vascularization. Expression of VEGF may assist in vascularization of endometrial explants after retrograde menstruation.

<AbstractText>The current study was conducted with the central objective of investigating the expression of microRNA-<em>145</em> (miR-<em>145</em>) in renal <em>vascular</em> lesions (RVLs) in juvenile lupus nephritis (JLN) and its possible mechanism.</AbstractText><AbstractText>The clinical data of 49 JLN patients confirmed by renal biopsy were collected and followed by grouping according to the RVLs score after hematoxylin-eosin staining: mild, moderate, and severe groups. In situ hybridization was used to detect the expression of miR-<em>145</em> in renal vessels which was then being compared among different RVLs groups. Up-LV-miR-<em>145</em> and LV-miR-NC lentiviral vectors were constructed and transfected into human <em>vascular</em> smooth muscle cells (HVSMCs), respectively. After HVSMCs were treated with 10.0 µg/L platelet-derived <em>growth</em> <em>factor</em> (PDGF)-BB for 24 h, the proliferation, migration, and apoptosis of <em>endothelial</em> cells were detected by MTT, Transwell assay, and flow cytometry, respectively. Western blot was used to detect expression of alpha-smooth muscle actin (α-SM-actin) and osteopontin (OPN).</AbstractText><AbstractText>The expression of miR-<em>145</em> in renal <em>vascular</em> cells was statistically significant. The higher the inner membrane ratio, the lesser the miR-<em>145</em> expression. After treatment with PDGF-BB, expression of miR-<em>145</em> in HVSMCs decreased, proliferation and migration ability enhanced, apoptosis decreased, α-SM-actin decreased, and OPN increased. The proliferation and migration ability of HVSMCs in the LV-miR-<em>145</em> group suppressed, apoptosis enhanced, α-SM-actin increased, and OPN decreased.</AbstractText><AbstractText>Our study revealed that miR-<em>145</em> expression decreased with the increase of <em>vascular</em> damage. miR-<em>145</em> can inhibit proliferation, migration, and differentiation phenotypic transformation of HVSMCs induced by PDGF-BB. miR-<em>145</em> may be involved in the pathogenesis of RVLs and may be a new target for treatment of RVLs in lupus nephritis.</AbstractText>

Betulin derivatives exhibit an antiproliferative activity and have been tested for many cancer cell lines. This paper describes a new series of 3-phosphate derivatives of betulin bearing different substituents at C28 position. The synthesized compounds were tested in vitro for their antiproliferative effect against human leukemia (MV-4-11 and CCRF/CEM), lung carcinoma (A549), prostate cancer (DU <em>145</em>), melanoma (Hs 294T) cell lines, and murine leukemia P388. To explore the possible mechanism of anticancer activity for the most in vitro active compounds (4, 5, 7 and 8) and betulin, molecular docking was performed to the binding sites of potential anticancer targets, described for the various triterpene derivatives, including topoisomerase I and II, epidermal <em>growth</em> <em>factor</em> receptor (EGFR) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGFR), transcription <em>factor</em> NF-κB, anti-apoptotic protein Bcl-2 and peroxisome proliferator-activated receptor (PPARγ). According to the results of the docking, the best fit to the binding pocket of PPARγ was shown by compound 4.

The current study was designed to investigate the protective role of diosmin against cyclophosphamide-induced premature ovarian insufficiency (POI). Female Swiss albino rats received a single intraperitoneal dose of cyclophosphamide (200 mg/kg) followed by 8 mg/kg/day for the next 15 consecutive days either alone or in combination with oral diosmin at 50 or 100 mg/kg. Histopathological examination of ovarian tissues, hormonal assays for follicle stimulating hormone (FSH), estradiol (E2), and anti-Mullerian hormone (AMH), assessment of the oxidative stress status, as well as measurement of the relative expression of miRNA-<em>145</em> and its target genes [<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> B <i>(VEGF-B)</i> and regulator of cell cycle (<i>RGC32</i>)] were performed. Diosmin treatment ameliorated the levels of E2, AMH, and oxidative stress markers. Additionally, both low and high diosmin doses significantly reduced the histopathological alterations and nearly preserved the normal ovarian reserve. MiRNA-<em>145</em> expression was upregulated after treatment with diosmin high dose. miRNA-<em>145</em> target genes were over-expressed after both low and high diosmin administration. Based on our findings, diosmin has a dose-dependent protective effect against cyclophosphamide-induced ovarian toxicity in rats.

<strong class="sub-title"> Keywords: </strong> miRNA-<em>145</em>; oxidative stress; premature ovarian insufficiency.

OBJECTIVE

Macrophage migration inhibitory factor (MIF) and vascular endothelial growth factor (VEGF), as crucial parameters of angiogenesis and inflammation, were evaluated to identify the role of cyclic citrullinated peptide antibodies (anti-CCP) during angiogenesis in rheumatoid arthritis (RA) and psoriatic arthritis (PsA).

METHODS

A total of 145 patients with RA, 44 patients with PsA, and 73 healthy subjects were included in this study. The clinical features, total blood counts, and acute phase parameters of RA and PsA patients were recorded. Anti-CCP antibody, VEGF, and MIF levels were determined with enzyme-linked immunosorbent assay (ELISA).

RESULTS

Anti-CCP positivity was significantly higher in the RA group (69%) than in both PsA (20.6%) and controls (8.2%) (p values<0.001). There was no difference between anti-CCP-positive and -negative RA patients regarding the extra-articular manifestations (p>0.05). VEGF and MIF levels were similar in anti-CCP-positive and -negative RA patients (all p values>0.05). The specificity of anti-CCP antibodies for RA was found to be 87.2%. No relationship was found between anti-CCP antibody positivity and clinical features, disease activity, functional disability as assessed by health assessment questionnaire scores, and extra-articular manifestations. There was no relationship between parameters of angiogenesis and anti-CCP antibody positivity. Both RF and anti-CCP antibodies were observed to be positive in most patients with RA.

CONCLUSIONS

Either RF or anti-CCP antibody was positive in a considerable proportion of our RA patients. Therefore, anti-CCP antibodies are important in the diagnosis of RF-negative patients who present with clinical findings of RA.

Arteriogenic erectile dysfunction is associated with impairment of <em>vascular</em> perfusion to the erectile components of the penis. Animal studies have identified insulin-like <em>growth</em> <em>factor</em> (IGF-I) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) as penile angiogenic <em>growth</em> <em>factors</em>, but the role of these <em>factors</em> in humans is not well understood. We evaluated the ex vivo expression of IGF-I, VEGF, and their receptors (IGF-IR, Flt-1, and KDR) in human penile cavernosal smooth muscle cells (HCSMCs) to identify cellular and molecular pathways involved in the regulation of penile tissue <em>vascular</em>ity. Primary culture was initiated with explants of human corpora cavernosa, and early passage (3-5) cells were used for these evaluations. Cultures were examined to verify the presence of smooth muscle cells and the absence of <em>endothelial</em> cell contamination. Specific monoclonal antibodies were used to localize <em>growth</em> <em>factors</em> and their receptors. To evaluate gene expression of VEGF, Flt-1, and KDR, total RNA was extracted from cavernosal cells and subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using custom synthesized primers. To study the effect on cell proliferation, 10000 cells/well were exposed to varying concentrations of VEGF (0-50 ng/mL). At specified time periods the cells were trypsinized and counted. IGF-I and VEGF and their receptors were localized in the cultures, which were positive for the presence of smooth muscle cells and negative for <em>endothelial</em> cell contamination. RT-PCR evaluation revealed the expression of four splice variants of VEGF messenger RNA (VEGFs 121, <em>145</em>, 165, and 189) and two of its receptors (Flt-1 and KDR). VEGF165 and VEGF121 were the most abundant forms of messenger RNA and Flt-1 appeared to be the most prominent receptor type in these cells. Exposure to VEGF elicited a twofold to threefold increase in the proliferation of HCSMCs. HCSMCs express both IGF-I and VEGF and their receptors, which may be important in the control of <em>vascular</em>ity in human penile architecture.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF-A) is one of the most important proangiogenic <em>factors</em>. It has many isoforms encoded by one gene. The occurrence of these isoforms is associated with the process of alternative splicing of mRNA. Some of the splice forms are perceived as tissue specific. The aim of this study was to determine the alternative splicing of VEGF-A mRNA in dilated cardiomyopathy, especially at the level of particular myocardial layers. The assessment of post-transcriptional modifications of VEGF-A mRNA was made on specimens taken from the explanted hearts of patients undergoing cardiac transplantation. Molecular and histopathological studies were perfomed on particular layers of the myocardial muscle (endocardium, myocardium, epicardium). A molecular analysis of cardiac samples was performed by quantitative analysis of the mRNA of the studied VEGF-A isoforms (VEGF121,-<em>145</em>,-165,-183,-189, and-206) using QRTPCR with an ABI-PRISM 7700-TaqMan sequence detector. 72 cardiac specimens taken from the explanted hearts were analyzed. Each of the studied VEGF-A splice forms was present in the evaluated hearts, but the types of alternative splicing of mRNA were different in particular layers. Quantitative analysis revealed different amounts of the studied isoforms. Generally, significantly increased expression of the VEGF-A isoforms was observed in samples taken from hearts with post-inflammatory etiology of cardiomyopathy. Our conclusions are: 1. All the studied VEGF-A isoforms were found in the human hearts, including those thusfar considered characteristic for other tissues. 2. Significant differences were observed in the expression of the VEGF-A splice forms with respect to the myocardial layers and the location of the cardiac biopsy. 3. Repetitive and comparable results for samples with post-inflammatory etiology were obtained, and they revealed considerably higher amounts of VEGF-A isoforms compared to specimens with idiopathic etiology.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a multifunctional cytokine that plays a major role in angiogenesis. Alternative splicing causes the production of several different isoforms (VEGF121, <em>145</em>, 165, 183, 189, 206). VEGF is essential for tumor angiogenesis, and several studies have correlated elevated VEGF levels with tumor stage, metastases, and progression. We now report the isolation by phage display of human single-chain antibody fragment (scFv) anti-VEGF165. After four rounds of panning against VEGF165, 40 out of 90 phage clones displayed VEGF165-binding activity. One of the positive clones, designated B8, bound to VEGF165 with relatively high affinity and neutralized VEGF165 bioactivity in vitro. The B8 clone was expressed in the soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography. The purified scFv recognized VEGF165 with the K(D) of 1.80 x 10(-8) M without cross-reaction to VEGF121. In addition to binding, the purified scFv could does-dependently inhibit VEGF165-induced human umbilical vein-derived <em>endothelial</em> cells proliferation. Together with its fully human mature, B8 scFv may have therapeutic implications in therapy of angiogenesis-dependent diseases.

Ovarian cancer is the second most common gynaecological cancer worldwide, and its molecular mechanism has not been completely understood. Ets-1 is a member of the Ets transcription family and can play important roles in the regulation of extracellular matrix remodelling, invasion, angiogenesis and drug resistance in several malignancies, including ovarian cancer. In the current study, we downloaded two datasets from Gene Expression Omnibus database and sought to explore the regulation mechanism of Ets-1 in ovarian cancer by computational analysis of gene expression profiles. Microarray analysis identified a total of 548 genes that were regulated by Ets-1 in ovarian cancer. Functional annotation of these genes revealed that Ets-1 may be involved in several biological processes, both physiological and pathological, such as system development, response to stimulus, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) production, morphogenesis, cell proliferation, cell adhesion and signal transduction. Further, DNA methylation analysis of the DEGs found that 26.5% (<em>145</em>) of them were differentially methylated genes in ovarian cancer. Our results provide insight into the mechanism of Ets-1 regulating the transcription of its target genes in the complex and multistep process of ovarian cancer progression.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a multifunctional cytokine which plays a major role in angiogenesis. Alternative splicing causes the production of several different isoforms (VEGF-A 121, <em>145</em>, 165, 189, 206). VEGF is essential for tumor angiogenesis and several studies have correlated elevated VEGF levels with tumor stage, metastases and progression. Antibody phage display was employed to isolate two scFv antibody fragments, D8 and F10, with specificity for the VEGF165 isoform. It was shown by ELISA and competitive immunohistochemistry that each clone bound to VEGF165 but not VEGF121. Immunohistochemistry with D8 and F10 on colorectal carcinoma and adenoma sections revealed positive staining similar to that shown by a polyclonal VEGF antibody. The scFv antibody fragments, D8 and F10, will be useful in specifically detecting circulating VEGF165 in cancer patients as most studies to date have quantified the total level of circulating VEGF (121 and 165). These reagents will allow further elucidation of the role of VEGF in tumor angiogenesis.

Bevacizumab is a humanized monoclonal antibody that binds to <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and prevents tumor angiogenesis. Radionuclide imaging using radiolabeled bevacizumab might be useful for selection of patients for anti-VEGF therapy. This study describes preparation of a potential imaging agent, 111In-CHX-A"-DTPA-bevacizumab, and evaluation of specificity of its binding to three tumor cell lines, SKOV3, LS174T and DU <em>145</em>. Bevacizumab was conjugated with CHX-A"-DTPA and radiolabeled with 111In with high yield and excellent stability. Specificity of cellular binding was examined by a saturation assay using 100-fold excess of non-radiolabeled antibody. SKOV3 and LS174T tumor cell lines showed significantly specific binding, while DU <em>145</em> cells did not showed any specific binding. The specific binding is dependent to type of cell lines, which it is important for selection of tumor model for scintigraphic imaging of the VEGF expression.

<AbstractText>The purpose of the present study was to evaluate the long-term incidence and timing of reactivation in patients with type 3 neo<em>vascular</em>ization who were treated with three monthly anti-<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) injections.</AbstractText><AbstractText>A total of 179 patients (179 eyes) diagnosed with type 3 neo<em>vascular</em>ization with dry macula after three monthly anti-VEGF loading injections were included in this retrospective study. After the initial treatment, patients were followed up without further injection until the first reactivation. The incidence and timing of the first reactivation after the initial treatment were recorded, and <em>factors</em> predictive of early reactivation (≤ 6 months after the third anti-VEGF injection) were investigated.</AbstractText><AbstractText>During a mean follow-up of 37.5 ± 18.8 months, the first reactivation was noted in <em>145</em> patients (81.0%) at a mean of 6.6 ± 4.1 months after the third injection. In 94 eyes (64.8%), reactivation was noted 2-6 months after the third injection, while in 37 eyes (25.5%) it was noted 7-12 months after the third injection. In the remaining 14 eyes (9.7%), the reactivation was noted after this period. The incidence of early reactivation was higher in women (P = 0.014) and patients with thicker choroid (P = 0.026).</AbstractText><AbstractText>In patients with type 3 neo<em>vascular</em>ization, almost all reactivation was noted within 15 months of the third anti-VEGF injection, suggesting the need for close follow-up and detailed examination during this period. Female patients with thick choroid should be monitored more frequently during this early period.</AbstractText>

<AbstractText>We aimed to investigate the role of angiopoietin (Angpt) as a predictive biomarker for sepsis by evaluating associations between plasma Angpt and various inflammatory cytokines and mortality in critically ill patients with sepsis.</AbstractText><AbstractText>This study was a retrospective cohort study of the prospectively collected samples and clinical data of <em>145</em> patients with sepsis who were admitted to the medical intensive care unit (ICU) of a 2000-bed university tertiary referral hospital in South Korea. We collected plasma within 24 h of medical ICU admission, and several biomarkers (Angpt-1 and -2, Tie2, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>, interleukin (IL)-1β, IL-10, IL-18, IL-6, interferon gamma-induced protein-10, and tumor necrosis <em>factor</em>-α) were measured using a Human Magnetic Luminex Screening Assay kit.</AbstractText><p><div><b>RESULTS</b></div>Plasma Angpt-2 was correlated with IL-6 (r_s = 0.555) and tumor necrosis <em>factor</em>-α (r_s = 0.559). Plasma Angpt-2 (r_s = 0.530) and Angpt-2/1 (r_s = 0.562) were correlated with the Sequential Organ Failure Assessment (SOFA) score. The area under the curve (AUC) for the 28-day mortality prediction for the plasma Angpt-2/1 ratio was 0.736; AUCs for the Acute Physiology and Chronic Health Evaluation II (APACHE II) and SOFA scores were 0.659 and 0.745, respectively. Using multivariate Cox proportional hazard regression analysis for 28-day mortality, we found that acute respiratory distress syndrome (hazard ratio (HR) = 2.235, 95% CI = 1.163-4.296,p = 0.016), APACHE II score (HR = 1.127, 95% CI = 1.037-1.224,p = 0.005), and Angpt-2/1 > 3.2 (HR = 2.522, 95% CI = 1.205-5.278,p = 0.014) were risk <em>factors</em> for 28-day mortality.</p><AbstractText>Plasma Angpt-2 was related to cytokines, but Angpt-2/1 ratio was a good predictor of 28-day mortality in patients with sepsis.</AbstractText>

Nerve <em>Growth</em> <em>Factor</em> (NGF) and its high-affinity receptor tropomyosin receptor kinase A (TRKA) increase their expression during the progression of epithelial ovarian cancer (EOC), promoting cell proliferation and angiogenesis through several oncogenic proteins, such as c-MYC and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF). The expression of these proteins is controlled by microRNAs (miRs), such as miR-<em>145</em>, whose dysregulation has been related to cancer. The aims of this work were to evaluate in EOC cells whether NGF/TRKA decreases miR-<em>145</em> levels, and the effect of miR-<em>145</em> upregulation. The levels of miR-<em>145</em>-5p were assessed by qPCR in ovarian biopsies and ovarian cell lines (human ovarian surface epithelial cells (HOSE), A2780 and SKOV3) stimulated with NGF. Overexpression of miR-<em>145</em> in ovarian cells was used to evaluate cell proliferation, migration, invasion, c-MYC and VEGF protein levels, as well as tumor formation and metastasis in vivo. In EOC samples, miR-<em>145</em>-5p levels were lower than in epithelial ovarian tumors. Overexpression of miR-<em>145</em> decreased cell proliferation, migration and invasion of EOC cells, changes that were concomitant with the decrease in c-MYC and VEGF protein levels. We observed decreased tumor formation and suppressed metastasis behavior in mice injected with EOC cells that overexpressed miR-<em>145</em>. As expected, ovarian cell lines stimulated with NGF diminished miR-<em>145</em>-5p transcription and abundance. These results suggest that the tumoral effects of NGF/TRKA depend on the regulation of miR-<em>145</em>-5p levels in EOC cells, and that its upregulation could be used as a possible therapeutic strategy for EOC.

<strong class="sub-title"> Keywords: </strong> NGF; TRKA; VEGF; c-MYC; epithelial ovarian cancer; microRNA-<em>145</em>.

BACKGROUND

Vascular endothelial growth factor (VEGF) has been shown to induce neurogenesis in the brain and yield neuroprotective effects. It is hypothesized that chemotherapy reduces circulating VEGF levels and leads to cognitive decline among patients. This multicenter longitudinal study aimed to evaluate the impact of chemotherapy on VEGF levels and the association between VEGF levels and cognitive function.

METHODS

A total of 145 early-stage breast cancer patients were recruited and assessed before chemotherapy (T1), during chemotherapy (T2), and at the end of chemotherapy (T3). At each time point, plasma VEGF levels were assessed using a multiplex immunoassay. Cognitive function was assessed using both Functional Assessment of Cancer Therapy-Cognitive Function, Version 3 (FACT-Cog), and Headminder (a computerized, web-based neuropsychologic battery).

RESULTS

Generally, we observed higher-than-baseline plasma VEGF levels after the start of chemotherapy (P < .001). Among patients receiving anthracycline-based chemotherapy, the median plasma VEGF levels were significantly higher at T2 (T2: 37.3 pg/mL vs. T1: 21.3 pg/mL; P < .001) and T3 (T3: 35.5 pg/mL vs. T1: 21.3 pg/mL; P < .001) than at baseline. Plasma VEGF levels were not associated with chemotherapy-associated cognitive impairment.

CONCLUSIONS

Breast cancer patients experience an increasing trend in plasma VEGF levels during chemotherapy, and the regimen types may have a differential effect on circulating VEGF levels. Furthermore, changes in plasma VEGF levels during chemotherapy were not associated with cognitive impairment. VEGF may play a minor role in mediating the occurrence of chemotherapy-associated cognitive impairment.

For decades, lung cancer has been the leading cause of cancer-related death worldwide. Hypoxia-inducible <em>factors</em> (HIFs) play critical roles in mediating lung cancer development and metastasis. The present study aims to clarify how HIF's over-activation affects lung cancer angiogenesis not only in a normoxic condition, but also a hypoxic niche. Our study shows that human lung cancer exhibits elevated levels of ceruloplasmin (CP), which has a negative impact on the prognosis of patients. CP affects the cellular Fe²⁺ level, which inactivates prolyl hydroxylase (PHD) 1 and 2, resulting in HIF-2α enhancement. Increased HIF-2α leads to <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-A (VEGF-A) secretion and angiogenesis. The expression of CP is under the epigenetic control of miR-<em>145</em>-5p. Restoration of miR-<em>145</em>-5p by miRNA mimics transfection decreases CP expression, increases Fe²⁺ and PHD1/2 levels and HIF hydroxylation while reduced HIF-2α levels resulting in the inhibition of tumor angiogenesis. In contrast, inhibition of miR-<em>145</em>-5p by miRNA inhibitors increases the expression of CP and VEGF-A in lung cancer cells. Significantly, miR-<em>145</em>-5p expression is lost in the tumor samples of lung cancer patients, and low miR-<em>145</em>-5p expression is strongly correlated with a shorter overall survival time. In conclusion, the current study reveals the clinical importance and prognostic value of miR-<em>145</em>-5p and CP. It identifies a unique mechanism of HIF-2α over-activation, which is mediated by iron imbalance of the iron-PHD coupling that modulates tumor angiogenesis.

<strong class="sub-title"> Keywords: </strong> HIF-2α; angiogenesis; ceruloplasmin; lung cancer; miR-<em>145</em>-5p.

Bladder urothelial carcinoma (BC) is a fatal invasive malignancy and the most common malignancy of the urinary system. In the current study, we investigated the function and mechanisms of Neuropilin-1 (NRP1), the co-receptor for <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>, in BC pathogenesis and progression. The expression of NRP1 was evaluated using data extracted from GEO and HPA databases and examined in BC cell lines. The effect on proliferation, apoptosis, angiogenesis, migration, and invasion of BC cells were validated after <i>NRP1</i> knockdown. After identifying differentially expressed genes (DEGs) induced by <i>NRP1</i> silencing, GO/KEGG and IPA^® bioinformatics analyses were performed and specific predicted pathways and targets were confirmed <i>in vitro.</i> Additionally, the co-expressed genes and ceRNA network were predicted using data downloaded from CCLE and TCGA databases, respectively. High expression of NRP1 was observed in BC tissues and cells. <i>NRP1</i> knockdown promoted apoptosis and suppressed proliferation, angiogenesis, migration, and invasion of BC cells. Additionally, after <i>NRP1</i> silencing the activity of MAPK signaling and molecular mechanisms of cancer pathways were predicted by KEGG and IPA^® pathway analysis and validated using western blot in BC cells. <i>NRP1</i> knockdown also affected various biological functions, including antiviral response, immune response, cell cycle, proliferation and migration of cells, and neo<em>vascular</em>isation. Furthermore, the main upstream molecule of the DEGs induced by <i>NRP1</i> knockdown may be <i>NUPR1</i>, and <i>NRP1</i> was also the downstream target of <i>NUPR1</i> and essential for regulation of <i>FOXP3</i> expression to activate neo<em>vascular</em>isation. <i>DCBLD2</i> was positively regulated by <i>NRP1</i>, and PPAR signaling was significantly associated with low <i>NRP1</i> expression. We also found that NRP1 was a predicted target of miR-204, miR-143, miR-<em>145</em>, and miR-195 in BC development. Our data provide evidence for the biological function and molecular aetiology of NRP1 in BC and for the first time demonstrated an association between NRP1 and NUPR1, FOXP3, and DCBLD2. Specifically, downregulation of <i>NRP1</i> contributes to BC progression, which is associated with activation of MAPK signaling and molecular mechanisms involved in cancer pathways. Therefore, NRP1 may serve as a target for new therapeutic strategies to treat BC and other cancers.

Keywords: NRP1; apoptosis; bladder cancer; invasion; migration; neovascularisation; proliferation.

OBJECTIVE

To examine developmental changes in myocardial gene expression of previously identified regulators of vascular growth.

METHODS

Ovine left (LV) and right ventricle (RV) samples were obtained at four time points: 95 days' and 140 days' gestation (term = 145 days) and 7 days and 8 weeks postnatally. mRNA and protein levels of vascular endothelial growth factor (VEGF), its respective receptors (Flk-1 and Flt-1), basic fibroblast growth factor (bFGF), transforming growth factor-beta1 (TGF-beta1), and endothelial nitric oxide synthase (eNOS) were measured at these different time points.

RESULTS

RV but not LV VEGF mRNA levels decreased postnatally, although VEGF protein expression remained unchanged after birth. Flt-1 mRNA expression was divergent between ventricles, although the protein expression pattern was similar in RV and LV, decreasing with maturation. RV and LV Flk-1 mRNA decreased between 95 days and 140 days, remaining stable thereafter, while protein levels only decreased after birth. bFGF protein levels were highest in the LV at 140 days, and decreased after birth but remained unchanged in the RV throughout the period examined. TGF-beta1 and eNOS levels were highest early in gestation, decreasing with maturation in both ventricles.

CONCLUSIONS

Developmentally regulated ventricle-specific expression of VEGF, Flt-1, Flk-1, TGF-beta1, bFGF, and eNOS was demonstrated in the ovine myocardium. These findings suggest these proteins may participate in coronary vascular remodeling during the perinatal period and underscore the importance of studying the relationships among transcription factors, target genes, and anatomic/physiologic changes in the whole animal.

Purpose: To evaluate the anatomical correlation between fellow eyes for bilateral second-line anti-VEGF treatment in eyes with bilateral diabetic macular edema (DME) with incomplete response to first-line bevacizumab therapy.

Methods: Seventy-Four eyes (n=37 patients) with bilateral-DME having incomplete response to first-line bevacizumab therapy that were switched for bilateral treatment with ranibizumab were retrospectively evaluated. Data collected included demographics, visual acuity and macular thickness. We evaluate the correlation for the response of both eyes in terms of macular thickness and visual acuity.

<strong class="sub-title"> Results: </strong> The mean±SD age was 76±8 years. The mean±SD number of bevacizumab injections prior the switch was 11.03±5.1 in the first eye (FE) and 10.9±5.2 in the second eye (SE). The central subfield thickness (CST) reduced from 472±171 microns at baseline to 418±161 after the last bevacizumab injection and 365±74 after 3 ranibizumab injections in the FE (p=0.016, p=0.004, respectively), and from 463±<em>145</em> microns to 446±123, and 421±103 in the SE (p=0.112, p=0.001, respectively). There was strong positive correlation between the eyes for the CST reduction under bevacizumab and ranibizumab treatments in each visit. BCVA± SD at baseline was 0.41±0.30 LogMAR in the FE, and 0.42±0.29 in the SE (p=0.44). After 3 injections of bevacizumab, the BCVA was 0.37±0.26 and 0.42±0.23 in FE and SE respectively (p=0.013, p=0.132, respectively).

Conclusions: This study demonstrated a strong anatomical correlation responses between the eyes in patients with bilateral DME for both first-line bevacizumab therapy and second-line ranibizumab therapy. Response to second-line therapy was favorable and correlated among eyes regardless they were from the same or different individuals.

Keywords: Anti Vascular Endothelial Growth Factor; Diabetes Macular Edema (DME); Second line treatment; bilateral treatment.

Purpose: The current study was conducted to explore the clinical features and risk factors of patients with asthma complicated by obstructive sleep apnea-hypopnea syndrome (OSAHS).

Methods: Patients with asthma who underwent polysomnography in our hospital from August 2017 to December 2019 were enrolled in the study. Data on demographics, pulmonary function testing, polysomnography, blood gases, mean pulmonary artery pressure, and vascular endothelial growth factor (VEGF) were compared between the two groups.

Results: Of 238 patients with asthma, 93 who also had OSAHS formed the observation group and were subclassified into mild (n = 33), moderate (n = 41), and severe (n = 19) categories, while 145 patients with asthma alone were assigned to the control group. No significant differences were found in sex, age, course of disease, or pulmonary function between the two groups (P > 0.05), while the observation group showed more frequent allergic rhinitis and had greater BMI, neck circumference, mean pulmonary artery pressure (mPAP), and VEGF than those in the control group (P < 0.001). The peak expiratory flow (PEF), forced expiratory volume in 1 s (FEV₁), forced vital capacity (FVC), and FEV₁/FVC in the mild group and the moderate group were higher than those in the severe group (P < 0.001). The durations of AHI and SaO₂ < 90% in the mild group and the moderate group were shorter than that in the severe group, and the lowest level of SaO₂ in the mild group and the moderate group was higher than that in the severe group (P < 0.05). The mPAP and VEGF of the mild and moderate groups were lower than those of severe group (P < 0.001), with mild group lower than moderate group (P < 0.001).

Conclusion: Significant differences in allergic rhinitis, BMI, neck circumference, AHI, SaO2, mPAP, and VEGF were observed in patients with asthma complicated by OSAHS. These parameters are risk factors associated with asthma complicated by OSAHS.

Keywords: Analysis; Asthma; Clinical features; Inducement; Obstructive sleep apnea-hypopnea syndrome; Risk factors.

Background: In human subcutaneous adipose tissue, the superficial fascia distinguishes superficial and deep microenvironments showing extensions called retinacula cutis. The superficial subcutaneous adipose tissue has been described as hyperplastic and the deep subcutaneous adipose tissue as inflammatory. However, few studies have described stromal-vascular fraction (SVF) content and adipose-derived stromal/stem cells (ASCs) behavior derived from superficial and deep subcutaneous adipose tissue. In this study, we analyzed a third conjunctive microenvironment: the retinacula cutis superficialis derived from superficial subcutaneous adipose tissue.

Methods: The samples of abdominal human subcutaneous adipose tissue were obtained during plastic aesthetic surgery in France (Declaration DC-2008-162) and Brazil (Protocol 145/09).

Results: The SVF content was characterized in situ by immunofluorescence and ex vivo by flow cytometry revealing a high content of pre-adipocytes rather in superficial subcutaneous adipose tissue microenvironment. Adipogenic assays revealed higher percentage of lipid accumulation area in ASCs from superficial subcutaneous adipose tissue compared with retinacula cutis superficialis (p < 0.0001) and deep subcutaneous adipose tissue (p < 0.0001). The high adipogenic potential of superficial subcutaneous adipose tissue was corroborated by an up-regulation of adipocyte fatty acid-binding protein (FABP4) compared with retinacula cutis superficialis (p < 0.0001) and deep subcutaneous adipose tissue (p < 0.0001) and of C/EBPα (CCAAT/enhancer-binding protein alpha) compared with retinacula cutis superficialis (p < 0.0001) and deep subcutaneous adipose tissue (p < 0.0001) microenvironments. Curiously, ASCs from retinacula cutis superficialis showed a higher level of adiponectin receptor gene compared with superficial subcutaneous adipose tissue (p = 0.0409), widely known as an anti-inflammatory hormone. Non-induced ASCs from retinacula cutis superficialis showed higher secretion of human vascular endothelial growth factor (VEGF), compared with superficial (p = 0.0485) and deep (p = 0.0112) subcutaneous adipose tissue and with adipogenic-induced ASCs from superficial (p = 0.0175) and deep (p = 0.0328) subcutaneous adipose tissue. Furthermore, ASCs from retinacula cutis superficialis showed higher secretion of Chemokine (C-C motif) ligand 5 (CCL5) compared with non-induced (p = 0.0029) and induced (p = 0.0089) superficial subcutaneous adipose tissue.

Conclusions: This study highlights the contribution to ASCs from retinacula cutis superficialis in their angiogenic property previously described for the whole superficial subcutaneous adipose tissue besides supporting its adipogenic potential for superficial subcutaneous adipose tissue.

Keywords: Adipose stromal/stem cells; Deeper microenvironment; Human subcutaneous adipose tissue; Retinacula cutis microenvironment; Stromal vascular fraction; Superficial microenvironment.

OBJECTIVE: To study the expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) mRNA isoforms in ovarian carcinoma and to explore their role in tumorigenesis and development of ovarian carcinoma. METHODS: The types and levels of VEGF mRNA isoforms of surgical samples from 30 patients with ovarian carcinoma were determined by relatively quantative RT-PCR, nest PCR and sequence analysis. RESULTS: VEGF(121), VEGF(<em>145</em>), VEGF(165) and VEGF(189)mRNA were detected in normal ovaries and ovarian carcinoma tissues. The expression level of VEGF(121) was significantly higher than that of VEGF(<em>145</em>), VEGF(165) and VEGF(189) (P<0.001, respectively). The expression of all 4 isoforms in carcinoma tissues was increased significantly compared with that in normal ovaries (P<0.05). CONCLUSION: Overexpression of VEGF(121), VEGF(<em>145</em>), VEGF (165) and VEGF(189) mRNA, especially VEGF(121), was found in varian carcinoma tissues. This findings suggest that all 4 VEGF isoforms may be involved in the tumorigenesis and development of ovarian carcinoma and VEGF(121) may play a key role.

Hypoxia-inducible <em>factor</em> 1α (HIF1α) is a key regulator of oxygen homeostasis, and its target genes mediate adaptive, protective, and pathological processes. The role of HIF1α in neuronal survival is controversial and the brain maturation stage is important in determining its function in brain ischemia or hypoxia-ischemia (HI). In this study, we used neuron-specific HIF1α knockout mice at postnatal day 9 (P9), and immature cortical neurons (days 7-8 in vitro) treated with the HIF1α inhibitor 2-methoxyestradiol (2ME2) or stabilizer dimethyloxalylglycine (DMOG), to examine the function of neuronal HIF1α in neonatal HI in vivo (Vannucci model) and in vitro (oxygen glucose deprivation, OGD). Inhibition of HIF1α with 2ME2 in primary neurons or deletion of neuronal HIF1α in P9 mice increased both necrotic and apoptotic cell death following HI, as evaluated by the protein levels of <em>145</em>/150-kDa and 120-kDa spectrin breakdown products 24 h after HI. DMOG attenuated neuronal death right after OGD. Acute pharmacological manipulation of HIF1α synchronously regulated the expression of its targets, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and erythropoietin (Epo), in the same manner. The in vivo findings agree with our previous data using the same HIF1α-deficient mice at an earlier age. This study confirms the role of neuronal HIF1α signaling in the endogenous protective responses following HI in the developing brain.

<strong class="sub-title"> Background: </strong> Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. It has been demonstrated that microRNA-<em>145</em> (miR-<em>145</em>) is correlated with the progression of various cancers by regulating the expression of multiple target genes, especially a number of genes that regulate angiogenesis and proliferation. However, the underlying mechanisms of miR-<em>145</em> in tumor angiogenesis of UM are still not well illustrated. Thus, we aimed to explore the potential target genes or pathways regulated by miR-<em>145</em> in UM and the effect of miR-<em>145</em> on invasion and angiogenesis.

<strong class="sub-title"> Methods: </strong> Totally, 24 choroid samples were collected in our study, including 12 UM samples and 12 normal uveal tissues. The expression of neuroblastoma RAS viral oncogene homolog (N-RAS), phosphorylated protein kinase B (p-AKT), and vascular endothelial growth factor (VEGF) in UM tissues and normal uveal tissues was analyzed using Western blotting analysis. Lentivirus expression system was used to construct MUM-2B and OCM-1 cell lines with stable overexpression of miR-<em>145</em>. Transwell and endothelial cell tube formation assay were used to measure the effects of miR-<em>145</em> on the invasion and angiogenesis of UM in vitro. The downstream target genes of miR-<em>145</em> were predicted by bioinformatics and confirmed using a luciferase assay. BALB/c nude mice models were established to investigate the mechanisms of miR-<em>145</em> on tumor growth and angiogenesis in vivo. Group data comparisons were performed using analysis of Student's t test. A two-tailed P < 0.05 was considered as statistically significant.

<strong class="sub-title"> Results: </strong> The results of Western blotting analysis indicated that the expressions of N-RAS (1.10 ± 0.35 vs. 0.41 ± 0.36, t = 3.997, P = 0.012), p-AKT (1.16 ± 0.22 vs. 0.57 ± 0.03, t = 7.05, P = 0.001), and VEGF (0.97 ± 0.32 vs. 0.45 ± 0.21, t = 3.314, P = 0.008) in UM tumor tissues were significantly higher than those in normal uveal tissue. Luciferase assay demonstrated N-RAS and VEGF as downstream targets of miR-<em>145</em>. Moreover, tube formation assay revealed that miR-<em>145</em>-transfected human microvascular endothelial cell line formed shorter tube length (36.10 ± 1.51 mm vs. 42.91 ± 0.94 mm, t = 6.603, P = 0.003) and less branch points (350.00 ± 19.97 vs. 406.67 ± 17.62, t = 3.685, P = 0.021) as compared with controls. In addition, the numbers of invaded MUM-2B and OCM-1 cells with miR-<em>145</em> overexpression were significantly lower than the controls (35.7 ± 3.3 vs. 279.1 ± 4.9, t = 273.75, P < 0.001 and 69.5 ± 4.4 vs. 95.6 ± 4.7, t = 21.27, P < 0.001, respectively). In vivo, xenografts expressing miR-<em>145</em> had smaller sizes (miR-<em>145</em> vs. miR-scr, 717.41 ± 502.62 mmvs. 1694.80 ± 904.33 mm, t = 2.314, P = 0.045) and lower weights (miR-<em>145</em> vs. miR-scr, 0.74 ± 0.46 g vs. 1.65 ± 0.85 g, t = 2.295, P = 0.045).

<strong class="sub-title"> Conclusion: </strong> Our results indicated that miR-<em>145</em> is an important tumor suppressor and the inhibitory strategies against N-RAS/VEGF signaling pathway might be potential therapeutic applications for UM in the future.