Angiogenic Factor Vascular Endothelial Growth Factor Research Articles

Predictive biomarkers of response to the IRAK4/FLT3 inhibitor emavusertib in hematological malignancies.

6550 Background: Myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML) display a dynamic and diverse mutational landscape. Various mutations can induce the overexpression of a highly active long isoform of interleukin-1 receptor-associated kinase 4 (IRAK4), leading to downstream activation of transcription factors such as NFκB. This activation triggers inflammation, oncogenesis, and the survival of cancer cells. Emavusertib, a potent oral inhibitor of IRAK4 and FLT3, has demonstrated efficacy in pre-clinical leukemia models and in patients. In this abstract, we present data suggesting potential biomarkers of response to emavusertib based on clinical samples from the ongoing TakeAim Leukemia trial. Methods: In Phase I/IIa of the TakeAim Leukemia trial (NCT04278768), patients with relapsed/refractory (R/R) AML or high-risk MDS (hrMDS) received treatment with emavusertib monotherapy. Baseline and on treatment patient samples underwent RNA sequencing and proteomic analyses. RNA-seq was conducted on mononuclear cells from bone marrow or peripheral blood (n=42). Plasma proteins were quantified by the Luminex platform (n=51). Results: hrMDS patients responding to emavusertib monotherapy exhibited decreased gene expression levels of the NFκB target gene IL1β (P≤0.05) compared to non-responders. Protein analyses revealed that hrMDS responders had lower baseline levels of proliferation and migration-related factors, like Vascular Endothelial Growth Factor (VEGF-A) and CXCL12, in plasma compared to non-responders (P≤0.05). In AML samples, baseline VEGF-A levels were significantly higher in responders. The concentration of soluble PD1 (sPD1) increased in on-treatment samples in hrMDS patients (P≤0.05), but not in AML patient samples. Additionally, hrMDS samples presented lower IL2 and higher sCD47 levels compared to AML (P≤0.05). Conclusions: In this work, several baseline biomarkers indicative of a response to emavusertib were identified in patients with hematological malignancies. Responders in hrMDS showed lower baseline expression of the NFκB-associated pro-inflammatory gene, IL1β, and lower protein levels in plasma of the angiogenic factor VEGF-A and the chemokine CXCL12. In AML samples, responders have higher baseline levels of VEGF-A, indicating distinct predictive biomarkers for both pathologies. Other molecules of clinical interest, such as sPD1, showed increased concentrations in on-treatment plasma samples of hrMDS patients regardless of their response to emavusertib. Our findings highlight distinct baseline biomarkers for AML and hrMDS related to the pathophysiology of hematological malignancies. These results suggest that inflammatory and migration mediators, along with angiogenesis factors, hold potential as predictive biomarkers for assessing responses to emavusertib monotherapy treatment in hematological malignancies. Clinical trial information: NCT04278768 .

Inflammation mediated angiogenesis in chronic lymphocytic leukemia.

Chronic inflammation has been identified in leukemias as an essential regulator of angiogenesis. B-chronic lymphocytic leukemia (CLL) cells secrete high levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1 alpha (HIF1α). The aim was to assess the role of inflammation in activation of angiogenic factors: endothelial nitric oxide synthase (eNOS), HIF1α and VEGF via proliferation related signaling pathways and VEGF autocrine control. We isolated mononuclear cells (MNC) and CD19+ cells from peripheral blood of 60 patients with CLL. MNC were treated with pro-inflammatory interleukin-6 (IL-6) and VEGF, in combination with inhibitors of JAK1/2 (Ruxolitinib), mTOR (Rapamycin), NF-κB (JSH23), SMAD (LDN-193189) and PI3K/AKT (Ly294002) signaling pathways, to evaluate eNOS, VEGF and HIF1α expression by immunoblotting, immunocytochemistry and RT-qPCR. Also, we investigated IL-6 dependent neovascularization in human microvascular endothelial cells (HMEC-1) in co-culture with MNC of CLL. The angiogenic factors eNOS, VEGF and HIF1α had significantly higher frequencies in MNC of CLL in comparison to healthy controls (p < 0.001) and CD19+ cells of CLL. IL-6 increased the quantity of HIF1α (p < 0.05) and VEGF positive cells in the presence of JSH23 (p < 0.01). VEGF increased HIF1α (p < 0.05), and decreased eNOS gene expression (p < 0.01) in MNC of CLL. VEGF significantly (p < 0.001) increased the number of HIF1α positive MNC of CLL, prevented by inhibitors of JAK1/2, PI3K and mTOR signaling pathways. VEGF stimulation of SMAD (p < 0.05) and STAT5 (p < 0.01) signaling has been prevented by inhibitors of JAK1/2, mTOR, PI3K and SMAD signaling, individually (p < 0.01) or mutually (p < 0.001). Also, we showed that MNC of CLL and IL-6 individually stimulate neovascularization in co-culture with HMEC-1, without a cumulative effect. We demonstrated elevated angiogenic factors in CLL, while VEGF and IL-6 independently stimulated HIF1α. VEGF stimulation of HIF1α was mostly mTOR dependent, while IL-6 stimulation was NF-κB dependent.

Stimulated expression of ELR+ chemokines, VEGFA and TNF-AIP3 promote mycobacterial dissemination in extrapulmonary tuberculosis patients and Cavia porcellus model of tuberculosis

Pathogenic mycobacteria induce and accelerate blood vessel formation driven by extensive inflammation during granuloma formation, which is a central feature of mycobacterial pathogenesis. Tumor necrosis factor-alpha (TNF-α) enhances the expression of vascular endothelial growth factor (VEGF) and glutamic acid-leucine-arginine (ELR+) chemokines, which are potent inducers of vascularization. Most of the reported research work contends that VEGF growth factor induces neovascularization in human tuberculosis (TB) patients, but the evidence is inconclusive. Considerable ambiguity exists concerning the factors responsible for miliary tuberculosis. To identify such factors, we proposed an alternative explanation that could be found in miliary tuberculosis (MTB) cases. We performed a comparative analysis of angiogenic factors TNF-α, VEGF, and angiogenic ELR+ CXC and CC chemokine ligands in extrapulmonary tuberculosis (EPTB) and pulmonary tuberculosis (PTB) patients. To observe the relationship of these factors with the severity of bacterial burden, guinea pigs were infected with Mycobacterium tuberculosis (M.tb) and levels of the angiogenic factors were examined at different time intervals. Expression of these factors also exhibited a significant positive correlation with bacterial burden in other organs like the spleen, liver, and lymph nodes. We demonstrated statistical data on bacterial burden at different time points following the dissemination of infection in guinea pigs. In this study, we observed that there was a stimulated increase in the expression of ELR+ chemokines and VEGF in EPTB patients as compared to PTB patients. Following increased dissemination, the host immune response clears bacteria from the lungs during disease progression in guinea pigs.

Use of Chicken Embryos as an Angiogenesis Model for Central Nervous System Malignant Tumor Research.

To demonstrate the usability of chicken chorioallantoic membrane (CAM) as an angiogenesis model for the development and treatment of malignant tumors of the central nervous system. A fresh tumor tissue piece taken from Glioblastoma patients, a malignant tumor of the central nervous system, was transferred to the CAM of chicken embryos and left to incubate in the incubator and their development was monitored. After examining the results of the study macroscopically, CAM tissue samples were evaluated both histochemically and immunohistochemically in terms of angiogenic factors VEGF (Vascular Endothelial Growth Factor), bFGF (basic Fibroblast Growth Factor) and PDGF (Platelet Derived Growth Factor). According to histochemical findings obtained from our study when compared with control embryos, blood vessels, fibroblast count and inflammatory infiltration were observed more in the tumor transplanted groups, especially in the tumordeveloping CAM region. There was also intense pleomorphism and marked hypercellularity in the cells. In our immunohistochemical findings, it was determined that bFGF, PDGF, VEGF staining intensities were higher in tumor transplanted groups compared to control groups, and this elevation was more pronounced in the tumor-developing region. As a result, it has been shown that the chicken embryo CAM model may be a suitable in vivo model for cancer angiogenesis studies. The protocol we created in this study will be a source for projects related to the use of therapeutic agents in cancer angiogenesis.

PHD2 attenuates high-glucose-induced blood retinal barrier breakdown in human retinal microvascular endothelial cells by regulating the Hif-1α/VEGF pathway.

Diabetic macular edema (DME) is one of the most frequent causes of severe vision loss. The pathogenesis of DME is still not fully understood; however, it is hypothesized to result from breakdown of the blood-retinal barrier (BRB) due to retinal inflammation by vascular endothelial growth factor (VEGF) secretion under hyperglycemic conditions. In this investigation, we discovered that Prolyl-4-hydroxylase 2 (PHD2), an upstream regulator of hypoxia-inducible factor 1 (HIF-1) modulates VEGF expression and thus preserves BRB function in the mouse retina. Primary human retinal microvascular endothelial cells (hRMECs) were cultured in human endothelial serum-free growth medium and exposed to hyperglycemia. Changes in cell viability were investigated by an MTT assay. BRB function in each group was revealed by a paracellular permeability assay and trans-endothelial electrical resistance (TEER). Morphological changes in the BRB were investigated by immunofluorescence staining of occludin and zonula occludens-1 (ZO-1). The mRNA and protein levels of the tight junction proteins, PHD2, HIF-1α, and VEGF were measured by reverse transcription-quantitative PCR (RT-qPCR), western blot analysis and ELISA. Under hyperglycemic conditions, the viability of hRMECs was decreased, and PHD2 expression was downregulated, accompanied by increased paracellular permeability and decreased trans-endothelial electrical resistance. Additionally, HIF-1α and VEGF expression levels were increased, whereas the expression levels of tight junction proteins, including occludin and ZO-1, were decreased and BRB function was compromised. The PHD2 activator R59949 (diacylglycerol kinase inhibitor II), altered these pathological changes, and the PHD2 inhibitor dimethyloxalylglycine (DMOG) resulted in the opposite effects. These results demonstrated that PHD2 inhibited HIF-1 activity by inhibiting HIF-1α expression in hRMECs under hyperglycemic conditions, which led to the downregulation of the expression of the angiogenic factor VEGF, and thus helped to maintain the functions of hRMECs. Therefore, it is reasonable to propose that PHD2 could be a potential novel target for the treatment of DME or other diseases with a similar pathogenesis.

Sanhuang Decoction Controls Tumor Microenvironment by Ameliorating Chronic Stress in Breast Cancer: A Report of Ninety Cases.

Long-term endocrine treatment which results in estrogen deprivation causes chronic stress associated with a series of uncomfortable symptoms leading not only to a decrease in quality of life but also to cancer recurrence, which may be mediated primarily through the enhanced expression of angiogenic factors, as well as a series of inflammatory microenvironmental changes that favor tumor progression. In this study, we designed a clinical trial and aimed to explore the effects of Sanhuang Decoction (SHD) treatment on chronic stress, inflammatory factors, and breast cancer recovery. A total of 90 patients with breast cancer who met the inclusion/exclusion criteria were randomly allocated to a treatment or control group. The treatment group received the standard endocrine treatment and the traditional Chinese medicine decoction known as SHD. The control group received the standard endocrine treatment only. The treatment period was 6 months. The modified Kupperman Menopausal Index, the self-rating anxiety scale, and the self-rating depression scale were evaluated once per month. The body microenvironment plasma indices related to chronic stress, such as oxidative and antioxidative stress markers, inflammatory factors, hemorheology, coagulation, lipid and D-dimer, immunologic functions, tumor biomarkers, and angiogenic factors of the vascular endothelial growth factor (VEGF) were measured before and after 6 months of treatment. After treatment for 5 months, the scores in the treatment group decreased to nearly normal levels and the control group showed no significant improvement. After treatment for 6 months, all indices related to the body microenvironment, as well as the tumor biomarkers and carcinoembryonic antigen, carbohydrate antigen 153, and angiogenic factor VEGF levels improved significantly to normal levels in the treatment group. Our primary research showed that treatment with SHD effectively improved the quality of life of breast cancer patients by facilitating a change in the body microenvironment that controlled tumor growth and prevented drug resistance.Clinical Trial RegistrationChinese Clinical Trial Registry, identifier ChiCTR-IIR-2000041413. Date of registration: 2017-06-07 (retrospective registration).

FOXP3 facilitates the invasion and metastasis of non‑small cell lung cancer cells through regulating VEGF, EMT and the Notch1/Hes1 pathway

Forkhead box P3 (FOXP3) is a specific marker of regulatory T cells (Tregs) that is also expressed in tumour cells. Previous studies have revealed that FOXP3 can promote metastasis in several types of cancer, including non-small cell lung cancer (NSCLC); however, the underlying mechanism of FOXP3 remains unclear. The aim of the present study was to investigate the effect of FOXP3 on vascular endothelial growth factor (VEGF), epithelial-to-mesenchymal transition (EMT) and the Notch1/Hes1 pathway in NSCLC. After FOXP3 small interfering RNA (siRNAs) were transfected into A549 cells, the expression of FOXP3 mRNA and protein was determined by reverse transcription-quantitative PCR and western blotting. Cell migration and invasion were analyzed by Transwell assays. The concentrations of matrix metalloproteinase (MMP)-2, MMP-9 and VEGF in the cell supernatant were evaluated by ELISA. The expression of relevant proteins involved in EMT and Notch1/Hes1 pathway were assessed via western blotting. Additionally, the expression of FOXP3, CD31 and E-cadherin was detected by immunohistochemical (IHC) staining of 55 human NSCLC tissue samples. The results demonstrated that FOXP3 knockdown significantly inhibited the cell migratory and invasive abilities, decreased the concentrations of MMP-2, MMP-9 and VEGF, downregulated the protein expression of vimentin, N-cadherin, Notch1 and Hes family BHLH transcription factor 1 (Hes1), and upregulated the protein expression of E-cadherin. Furthermore, FOXP3 expression was positively associated with CD31+ vascular endothelial cells and negatively correlated with E-cadherin in NSCLC tissues. In addition, the Notch1/Hes1 pathway inhibitor DAPT significantly downregulated the expression of FOXP3 in a dose-dependent manner. Taken together, these findings demonstrated that FOXP3 may facilitate the invasive and migratory abilities of NSCLC cells via regulating the angiogenic factor VEGF, the EMT and the Notch1/Hes1 pathway.

Microvascularization and Expression of Fibroblast Growth Factor and Vascular Endothelial Growth Factor and Their Receptors in the Mare Oviduct.

Simple SummaryThe oviduct provides the ideal conditions for fertilization and early embryonic development. Adequate vascularization is essential for proper oviduct physiological function. In this work on the mare oviduct, differences in the oviductal artery and arterioles and their ramifications in the infundibulum, ampulla and isthmus were examined. Locally, vascularization is modulated by the action of angiogenic factors, mediated by their specific receptors. In the present study, the isthmus presented the largest vascular area and the highest number of vascular structures in the follicular phase. We have also shown that the relative abundance of angiogenic transcripts and proteins, such as fibroblast growth factor 1 (FGF1) and 2 (FGF2) and vascular endothelial growth factor (VEGF), and their respective receptors (FGFR1, FGFR2, VEGFR2 = KDR), were present in all portions of the oviduct throughout the estrous cycle. There was an increase in the transcripts of angiogenic receptors FGF1 and FGFR1 in the ampulla and isthmus, and of FGF2 and KDR in the isthmus. This was also observed in the isthmus, where the relative abundance of proteins FGFR1 and KDR was the highest. This study shows that the equine oviduct presents differences in microvascular density in its portions. The angiogenic factors VEGF, FGF1, FGF2 and their respective receptors are expressed in all studied regions of the mare oviduct, in agreement with microvascular patterns.The oviduct presents the ideal conditions for fertilization and early embryonic development. In this study, (i) vascularization pattern; (ii) microvascular density; (iii) transcripts of angiogenic factors (FGF1, FGF2, VEGF) and their receptors—FGFR1, FGFR2, KDR, respectively, and (iv) the relative protein abundance of those receptors were assessed in cyclic mares’ oviducts. The oviductal artery, arterioles and their ramifications, viewed by means of vascular injection-corrosion, differed in the infundibulum, ampulla and isthmus. The isthmus, immunostained with CD31, presented the largest vascular area and the highest number of vascular structures in the follicular phase. Transcripts (qPCR) and relative protein abundance (Western blot) of angiogenic factors fibroblast growth factor 1 (FGF1) and 2 (FGF2) and vascular endothelial growth factor (VEGF), and their respective receptors (FGFR1, FGFR2, VEGFR2 = KDR), were present in all oviduct portions throughout the estrous cycle. Upregulation of the transcripts of angiogenic receptors FGF1 and FGFR1 in the ampulla and isthmus and of FGF2 and KDR in the isthmus were noted. Furthermore, in the isthmus, the relative protein abundance of FGFR1 and KDR was the highest. This study shows that the equine oviduct presents differences in microvascular density in its three portions. The angiogenic factors VEGF, FGF1, FGF2 and their respective receptors are expressed in all studied regions of the mare oviduct, in agreement with microvascular patterns.

Effect of Chemically Induced Hypoxia on Osteogenic and Angiogenic Differentiation of Bone Marrow Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells in Direct Coculture.

Bone is an active tissue where bone mineralization and resorption occur simultaneously. In the case of fracture, there are numerous factors required to facilitate bone healing including precursor cells and blood vessels. To evaluate the interaction between bone marrow-derived mesenchymal stem cells (BMSC)—the precursor cells able to differentiate into bone-forming cells and human umbilical vein endothelial cells (HUVEC)—a cell source widely used for the study of blood vessels. We performed direct coculture of BMSC and HUVEC in normoxia and chemically induced hypoxia using Cobalt(II) chloride and Dimethyloxaloylglycine and in the condition where oxygen level was maintained at 1% as well. Cell proliferation was analyzed by crystal violet staining. Osteogenesis was examined by Alizarin Red and Collagen type I staining. Expression of angiogenic factor-vascular endothelial growth factor (VEGF) and endothelial marker-von Willebrand factor (VWF) were demonstrated by immunohistochemistry and enzyme-linked immunosorbent assay. The quantitative polymerase chain reaction was also used to evaluate gene expression. The results showed that coculture in normoxia could retain both osteogenic differentiation and endothelial markers while hypoxic condition limits cell proliferation and osteogenesis but favors the angiogenic function even after 1 of day treatment.

Macrophage Smad3 Protects the Infarcted Heart, Stimulating Phagocytosis and Regulating Inflammation.

TGF (transforming growth factor)-β is critically involved in myocardial injury, repair, and fibrosis, activating both Smad (small mothers against decapentaplegic)-dependent and non-Smad pathways. The in vivo role of TGF-β signaling in regulation of macrophage function is poorly understood. We hypothesized that in the infarcted myocardium, activation of TGF-β/Smad signaling in macrophages may regulate repair and remodeling. To investigate the role of macrophage-specific TGF-β Smad3 signaling in a mouse model of myocardial infarction and to dissect the mechanisms mediating Smad-dependent modulation of macrophage function. TGF-βs markedly activated Smad3 in macrophages, without affecting Smad-independent pathways. Phagocytosis rapidly and directly activated macrophage Smad3, in the absence of active TGF-β release. MyS3KO (myeloid cell-specific Smad3 knockout) mice had no baseline defects but exhibited increased late mortality and accentuated dilative postmyocardial infarction remodeling. Adverse outcome in infarcted MyS3KO mice was associated with perturbations in phagocytic activity, defective transition of macrophages to an anti-inflammatory phenotype, scar expansion, and accentuated apoptosis of border zone cardiomyocytes. In vitro, Smad3 null macrophages exhibited reduced expression of genes associated with eat-me signals, such as Mfge8 (milk fat globule-epidermal growth factor factor 8), and reduced capacity to produce the anti-inflammatory mediators IL (interleukin)-10 and TGF-β1, and the angiogenic growth factor VEGF (vascular endothelial growth factor). Mfge8 partly rescued the phagocytic defect of Smad3 null macrophages, without affecting inflammatory activity. Impaired anti-inflammatory actions of Smad3 null macrophages were associated with marked attenuation of phagocytosis-induced PPAR (peroxisome proliferator-activated receptor) expression. MyS3KO mice had no significant alterations in microvascular density and interstitial fibrosis in remodeling myocardial segments. We demonstrate that Smad3 critically regulates function of infarct macrophages, by mediating acquisition of a phagocytic phenotype and by contributing to anti-inflammatory transition. Smad3-dependent actions in macrophages protect the infarcted heart from adverse remodeling.

3D Plotted Biphasic Bone Scaffolds for Growth Factor Delivery: Biological Characterization In Vitro and In Vivo.

Bioprinting enables the integration of biological components into scaffolds during fabrication that has the advantage of high loading efficiency and better control of release and/or spatial positioning. In this study, a biphasic scaffold fabricated by extrusion-based 3D multichannel plotting of a calcium phosphate cement (CPC) paste and an alginate/gellan gum (AlgGG) hydrogel paste laden with the angiogenic factor VEGF (vascular endothelial growth factor) is investigated with regard to biological response in vitro and in vivo. Rat mesenchymal stromal cells are able to adhere and grow on both CPC and AlgGG strands, and differentiate toward osteoblasts. A sustained VEGF release is observed, which is able to stimulate endothelial cell proliferation as well as angiogenesis in vitro that indicates maintenance of its biological activity. After implantation into a segmental bone defect in the femur diaphysis of rats, a clear reduction of the defect size by newly formed bone tissue occurs from the distal and proximal ends of the host bone within 12 weeks. The CPC component shows excellent osteoconductivity whereas the local VEGF release from the AlgGG hydrogel gives rise to an enhanced vascularization of the defect region. This work contributes to the development of novel therapeutic concepts for improved bone regeneration which are based on 3D bioprinting.

Inhibition of the signaling pathway of syndecan-1 by synstatin: A promising anti-integrin inhibitor of angiogenesis and proliferation in HCC in rats

Aim of workThe study was conducted for evaluation of the antitumor activity of SSTN92-119 against HCC induced by thioacetamide in rats. MethodsSixty male Sprague-Dawley rats were randomized into four equal groups: Control, SSTN92-119, HCC, and HCC + SSTN92-119. Liver function tests were measured in serum. Liver homogenate was used for determination of: i) integrinαѴβ3 (ITGαѴβ3), insulin like growth factor-1 receptor (IGF-1R), vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and alpha-fetoprotein (AFP) levels by ELISA, ii) syndecan-1 (CD-138), IGF-1R and VEGF genes expressions by qRT-PCR, iii) MDA, NO, GSH concentrations and SOD activity. Histopathological and immunohistochemical examination of liver tissue was performed. ResultsSSTN92-119 decreased HCC-induced elevation in ALT, AST, ALP and GGT activities and reversed HCC-induced reduction in total protein and albumin concentrations significantly. SSTN92-119 significantly elevated hepatic SOD and GSH and reduced both NO and MDA levels. Protein levels of ITGαѴβ3, IGF-1R, VEGF, FGF-2 and AFP were decreased in HCC- SSTN92-119 group as well as gene expression of CD-138, IGF-1R and VEGF compared with HCC group. ConclusionsSSTN92-119 down regulates ITGαѴβ3 receptor and subsequently reduces the activation of angiogenic growth factors VEGF and FGF-2. Therefore, SSTN92-119 is becoming a promising anti-integrin αѴβ3 that inhibits angiogenesis and proliferation in HCC.

Serum levels of VEGF and MCSF in HER2+ / HER2- breast cancer patients with metronomic neoadjuvant chemotherapy

Metronomic therapy has been gaining importance in the neoadjuvant setting of breast cancer treatment. Its clinical benefits may involve antiangiogenic machinery. Cancer cells induce angiogenesis to support tumor growth by secreting factors, such as vascular endothelial growth factor (VEGF). In breast cancer, Trastuzumab (TZM) based treatment is of key importance and is believed to reduce diameter and volume of blood vessels as well as vascular permeability. Here in we investigated serum levels of angiogenic factors VEGF and MCSF in patients receiving metronomic neoadjuvant therapy with or without TZM. We observed in HER2+ cohort stable levels of MCSF through treatment, whereas VEGF trend was of decreasing levels. In HER2- cohort we observed increasing levels of MCSF and VEGF trend. Overall, HER2+ patients had better pathological response to treatment. These findings suggest that angiogenic pathway may be involved in TZM anti-tumoral effect in the neoadjuvant setting.

Angiogenesis in prostate cancer and benign prostatic hyperplasia assessed by VEGF and CD-34 IHC: A comparative clinico-pathological study

IntroductionProstate carcinoma is still a dreaded disease wanting more effective treatment and definitive early detection for a better prognosis and cure of life. ObjectiveThe present study was planned to investigate the correlation of vascular endothelial growth factor (VEGF) expression level and microvessel density (MVD) between the BPH and prostate cancer subjects to analyze their diagnostic and prognostic value. Subjects and methodsFreshly diagnosed histopathologically confirmed 50 cases of prostate cancer and 50 cases of BPH were included. Expression level of VEGF was measured using Immunohistochemistry (IHC), while MVD was determined via CD34 endothelium-specific antibodies. In the case group, we have also recorded the Gleason's score of prostate cancer and investigated its correlation with angiogenic factor VEGF and MVD CD34. ResultsThe study showed a statistically significant difference value of VEGF expression level between the prostate cancer and BPH group (p<0.001). The mean MVD CD34 in the prostate cancer and BPH groups were 29.66±0.21 and 9.96±0.25, respectively. The difference of MVD CD34 expression between the groups was also found significant (p<0.001). VEGF scoring was significantly correlated with Gleason's scores of prostate cancer (p=0.005). ConclusionsThe present findings may support the assumption that VEGF and CD34 expression level might have an important role in the prediction of prostate cancer as it was significantly differed with BPH. In addition, VEGF expression level showed intense staining in the tissue samples with higher grading of prostate cancer which reveals its importance as prognostic marker.

Secretion of the angiogenic factor VEGF after photodynamic therapy with ALA under hypoxia-like conditions in colon cancer cells

BackgroundPhotodynamic therapy (PDT), eliminates not only the tumor, but also modulates signaling factors release, e.g. vascular endothelial growth factor (VEGF), which plays a crucial role in cancer progression. Assessment of the VEGF-secreting activity of resistant colon cancer cells in different degree of malignancy: SW480 and SW620 under hypoxia-like conditions during δ- aminolevulinic acid (ALA) PDT was the objective of our study. MethodsThe colon cancer cell lines SW480 and SW620 were treated in sublethal doses with ALA PDT in hypoxia- like conditions with cobalt chloride (CoCl2). To assess cell viability, MTT assays were performed and the discrimination of the cell death mode was monitored via fluorescence microscopy. The cells cytotoxicity using LDH test was assessed. Determination of VEGF was carried out using the Bio- Plex Assay Pro™ kit on the Bio- Plex Suspension Array System. ResultsALA PDT used in sublethal doses decreases release of VEGF in more aggressively growing SW620 colon cancer cell line in hypoxia-like conditions. In addition the level of secretion of VEGF in SW620 was much higher than in SW480 cells, which correlates with the grade of aggressive growth of colon cancer cells. ConclusionOur outcomes offer evidence, that in hypoxia mimic condition sublethal ALA-PDT- mediated VEGF inhibition could be clinically relevant.

Adipose Extracellular Matrix/Stromal Vascular Fraction Gel Secretes Angiogenic Factors and Enhances Skin Wound Healing in a Murine Model.

Mesenchymal stem cells are an attractive cell type for cytotherapy in wound healing. The authors recently developed a novel, adipose-tissue-derived, injectable extracellular matrix/stromal vascular fraction gel (ECM/SVF-gel) for stem cell therapy. This study was designed to assess the therapeutic effects of ECM/SVF-gel on wound healing and potential mechanisms. ECM/SVF-gel was prepared for use in nude mouse excisional wound healing model. An SVF cell suspension and phosphate-buffered saline injection served as the control. The expression levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and monocyte chemotactic protein-1 (MCP-1) in ECM/SVF-gel were analyzed at different time points. Angiogenesis (tube formation) assays of ECM/SVF-gel extracts were evaluated, and vessels density in skin was determined. The ECM/SVF-gel extract promoted tube formation in vitro and increased the expression of the angiogenic factors VEGF and bFGF compared with those in the control. The expression of the inflammatory chemoattractant MCP-1 was high in ECM/SVF-gel at the early stage and decreased sharply during the late stage of wound healing. The potent angiogenic effects exerted by ECM/SVF-gel may contribute to the improvement of wound healing, and these effects could be related to the enhanced inflammatory response in ECM/SVF-gel during the early stage of wound healing.

Mechanical strain stimulates vasculogenesis and expression of angiogenesis guidance molecules of embryonic stem cells through elevation of intracellular calcium, reactive oxygen species and nitric oxide generation

ObjectivesDifferentiation of embryonic stem (ES) cells may be regulated by mechanical strain. Herein, signaling molecules underlying mechanical stimulation of vasculogenesis and expression of angiogenesis guidance cues were investigated in ES cell-derived embryoid bodies. Methods and resultsTreatment of embryoid bodies with 10% static mechanical strain using a Flexercell strain system significantly increased CD31-positive vascular structures and the angiogenesis guidance molecules plexinB1, ephrin B2, neuropilin1 (NRP1), semaphorin 4D (sem4D) and robo4 as well as vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-BB (PDGF-BB) as evaluated by Western blot and real time RT-PCR. In contrast ephrin type 4 receptor B (EphB4) expression was down-regulated upon mechanical strain, indicating an arterial-type differentiation. Robo1 protein expression was modestly increased with no change in mRNA expression. Mechanical strain increased intracellular calcium as well as reactive oxygen species (ROS) and nitric oxide (NO). Mechanical strain-induced vasculogenesis was abolished by the NOS inhibitor L-NAME, the NADPH oxidase inhibitor VAS2870, upon chelation of intracellular calcium by BAPTA as well as upon siRNA inactivation of ephrin B2, NRP1 and robo4. BAPTA blunted the strain-induced expression of angiogenic growth factors, the increase in NO and ROS as well as the expression of NRP1, sem4D and plexinB1, whereas ephrin B2, EphB4 as well as robo1 and robo4 expression were not impaired. ConclusionsMechanical strain stimulates vasculogenesis of ES cells by the intracellular messengers ROS, NO and calcium as well as by upregulation of angiogenesis guidance molecules and the angiogenic growth factors VEGF, FGF-2 and PDGF-BB.

Biological effects of plasma rich in growth factors (PRGF) on human endometrial fibroblasts

ObjectivesTo evaluate the biological outcomes of plasma rich in growth factors (PRGF) on human endometrial fibroblasts in culture. Study designPRGF was obtained from three healthy donors and human endometrial fibroblasts (HEF) were isolated from endometrial specimens from five healthy women. The effects of PRGF on cell proliferation and migration, secretion of vascular endothelial growth factor (VEGF), procollagen type I and hyaluronic acid (HA) and contractility of isolated and cultured human endometrial fibroblasts (HEF) were analyzed. Statistical analysis was performed in order to compare the effects of PRGF with respect to control situation (T-test or Mann–Whitney U-test). ResultsWe report a significantly elevated human endometrial fibroblast proliferation and migration after treatment with PRGF. In addition, stimulation of HEF with PRGF induced an increased expression of the angiogenic factor VEGF and favored the endometrial matrix remodeling by the secretion of procollagen type I and HA and endometrial regeneration by elevating the contractility of HEF. These results were obtained for all PRGF donors and each endometrial cell line. ConclusionsThe myriad of growth factors contained in PRGF promoted HEF proliferation, migration and synthesis of paracrine molecules apart from increasing their contractility potential. These preliminary results suggest that PRGF improves the biological activity of HEF in vitro, enhancing the regulation of several cellular processes implied in endometrial regeneration. This innovative treatment deserves further investigation for its potential in “in vivo” endometrial development and especially in human embryo implantation.

Altered Biological Potential and Radioresponse of Murine Tumors in Different Microenvironments.

PurposeThis study was conducted to evaluate the biological features of murine hepatocarcinoma according to different tumor microenvironmental models and to determine the change in molecular and immunologic responses after radiation.Materials and MethodsTumor models were established in the liver (orthotopic) and thigh (heterotopic) of male C3H/HeN mice. Tumor growth and lung metastasis were assessed in these models. To evaluate the radiation effect, the tumors were irradiated with 10 Gy. Factors associated with tumor microenvironment including vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), transforming growth factor beta1 (TGF-β1), CD31, and serum interleukin-6 (IL-6) were evaluated. Tumor-infiltrating regulatory immune cells, regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs) were also analyzed.ResultsA higher number of lung metastases were observed in the orthotopic tumor model than in the heterotopic tumor model. VEGF, CD31, COX-2, and TGF-β1 expression was more prominent in the orthotopic tumor model than in the heterotopic tumor model. Expression of the angiogenic factor VEGF and key regulatory molecules (TGF-β1 and COX-2) decreased following radiation in the orthotopic tumor model, while the serum IL-6 level increased after radiation. In the orthotopic tumor model, the number of both Tregs and MDSCs in the tumor burden decreased after radiation.ConclusionThe orthotopic tumor model showed higher metastatic potential and more aggressive molecular features than the heterotopic tumor model. These findings suggest that the orthotopic tumor mouse model may be more reflective of the tumor microenvironment and suitable for use in the translational research of radiation treatment.

PLASMA EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) AND BASIC FIBROBLAST GROWTH FACTOR (bFGF) IN PATIENTS WITH BRAIN TUMORS

OBJECTIVE: Angiogenesis and angiogenic growth factors are of basic importance in the development and progresion of brain tumours. The purpose of the study was to evaluate the plasma levels of two angiogenic factors Vascular Endothelial Growth Factor (VEGF) and Basic Fibro blast Growth Factor (bFGF) inpatients with brain tumors and potential possibilities to use them with predictive value. MATERIAL – METHOD: In order to determine amount of VEGF and FGFb in plasma were examined 35 patients, (glioblastoma multiforme, GBM, n=7; astrocytoma, n=7; meningioma, n=11; and healthy control group, n=10) were analysed. For determination of plasma concentrations of angiogenic factor, highly specific enzyme-linked immuno sorbent assays (ELISAs) were used. The data underwent regression and correlation analysis for estimation of the eventual interrelations. RESULTS: Median levels of bFGF in glioblastoma patients were higher compared with those with low grade gliomas, meningiomas or healthy patients. Highest levels of VEGF concentrations were detected in plasma derived from patients suffering from LGG. Plasma expression of these angiogenic factors shows dynamic changes pre- and postoperative, but cannot be explained only with tumor resection, grade or type of the tumor, and probably is result of physiological regeneration of the operative wound. No correlation between the survival time of the patients and the plasma levels of the tested growth factors was obtained. CONCLUSIONS: Expression of VEGF and bFGF in plasma are not reliable marker. The detectability of the angiogenic factors VEGF and bFGF in the plasma of patients suffering from various types of brain tumours is described. The plasma detectability of the individual angiopoietic factors seems to depend at least partly on the tumour type as well as on tumour progression. This might be of prognostic and therapeutic relevance.