Vascular Endothelial Growth Factor ELISA Research Articles

Achillea biebersteinii extracts suppress angiogenesis and enhance sensitivity to 5-fluorouracil of human colon cancer cells via the PTEN/AKT/mTOR pathway in vitro

Objective: To investigate the antiproliferative, anti-angiogenic, and apoptotic effects of extracts of Achillea biebersteinii (ABE) and combined treatments of ABE with 5-fluorouracil (5-FU) on HT-29 cells. Methods: The effects of ABE, 5-FU, and combined treatments on the viability of HT-29 cells were determined by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Isobologram analysis was used to determine synergism between ABE and 5-FU. The apoptotic and anti-angiogenic effects were determined by cell death detection and human vascular endothelial growth factor ELISA method, respectively. Transcriptional and translational expressions of p53, Bax, Bcl-2, p38 MAPK, Akt, PTEN, and mTOR were also evaluated by real-time PCR and Western blotting analysis. Results: ABE decreased the viability of HT-29 cells in a dose-dependent manner. Combined treatment of hexane, chloroform, and methanol extracts of Achillea biebersteinii with 5-FU at IC50 doses decreased the cell viability to 26.0%, 19.1%, and 14.9%, respectively (P Conclusions: Our findings indicate that ABE shows synergism with 5-FU and inhibits the proliferation of HT-29 cells by inducing apoptosis and suppressing angiogenesis, which may provide biological evidence for further use of ABE in the treatment of colorectal cancer.

Ciclopirox olamine promotes the angiogenic response of endothelial cells and mesenchymal stem cells.

Prolyl hydroxylase inhibitors (PHIs) are promising compounds to promote angiogenesis by stabilizing hypoxia-inducible factor-1α (HIF-1α), a master regulator of angiogenesis. Increased HIF-1α presence induces expression of proangiogenic genes such as vascular endothelial growth factor (VEGF). We investigated the pharmacological induction of hypoxia via the PHI ciclopirox olamine (CPX) as angiogenesis strategy on human dermal microvascular endothelial cell (hd-mvEC) spheroids directly and indirectly via activating human mesenchymal stem cells (hMSCs). HMSCs were isolated from bone marrow and hd-mvECs from foreskin biopsies. MSC-conditioned medium after CPX stimulation (MSC-CM CPX) was analyzed by VEGF ELISA and Proteome Profiler™ Human Angiogenesis Array. Direct stimulation with CPX and indirect stimulation via MSC-CM CPX were compared in sprouting assays of hd-mvEC spheroids. Direct stimulation with CPX significantly increased sprouting of hd-mvEC spheroids. MSC-CM CPX also induced sprouting from hd-mvEC spheroids, which was mediated by angiogenic VEGF and other proangiogenic factors that had been produced by stimulated hMSCs. The stimulation with CPX increased the proangiogenic response of hd-mvECs and hMSCs. The direct stimulation of hd-mvECs with CPX has the potential to replace external VEGF supplementation. Thus, CPX can induce angiogenesis in ECs even in the absence of auxiliary cells demonstrating a promising proangiogenic approach.

Abstract 160: Role of EMMPRIN in osteosarcoma metastases

Abstract Introduction: Although the survival rate of localized osteosarcoma patients has increased, patients with metastasis carry a poor prognosis. Thus, identification of factors contributing to metastatic progression is required. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell-surface glycoprotein which plays multiple roles in physiologic and pathologic conditions. EMMPRIN stimulates adjacent stromal cells or tumor cells to produce matrix metalloproteinase (MMP) and is highly expressed in multiple cancer types. EMMPRIN also stimulates expression of vascular endothelial growth factor (VEGF) which leads to angiogenesis. However, the expression and role of EMMRPIN in osteosarcoma have never been studied. This study was performed 1) to investigate the expression of EMMPRIN in osteosarcoma and 2) to examine if EMMPRIN, via tumor-stroma interaction, is associated with metastatic potential in osteosarcoma. Methods: Level of EMMPRIN mRNA expression was evaluated by RT-PCR in 6 tumor-derived osteosarcoma cell lines and by immunohistochemistry prechemotherapy biopsies from 52 patients. Clinical data of these patients were reviewed to examine the association of EMMPRIN expression and clinical outcome. Transfection of EMMPRIN-targeting siRNA in SaOS-2 cell line was performed to examine the role of EMMPRIN in osteosarcoma progression. To study the role of EMMPRIN in tumor-stromal interaction, co-culture of SaOS-2 with osteoblast (hFOB) was experimented. Functional in vitro assays that reflect metastatic potential of osteosarcoma cells were performed. MMP production, VEGF production, cell invasion and proliferation were studied by gelatin zymography, VEGF ELISA, Matrigel invasion assay and WST-1 assay, respectively. Results: EMMPRIN was expressed in 90% by immunohistochemistry with strong accentuation along the cell membrane. Level of EMMRIN mRNA expression was significantly higher in 5 of 6 tumor-derived cell lines compared to MG63. Patients with high EMMPRIN expression had significantly worse metastasis-free survival. EMMPRIN mRNA levels was significantly down-regulated by siRNA transfection compared to control-siRNA transfected cell. Co-culture of osteoblast and SaOS-2 enhanced stimulation of pro-MMP2. This stimulation was reversed by transfection of SaOS-2 with EMMPRIN-targeting siRNA. Number of cells crossing the chamber was statistically lower in EMMPRIN-siRNA transfected cells. When osteoblast and SaOS-2 were co-cultured, VEGF expression was increased compared to SaOS-2 only culture. EMMPRIN-targeting siRNA transfection resulted in decrease of VEGF expression. SaOS-2 cells transfected with EMMPRIN-siRNA showed decreased proliferation potential compared to control-siRNA transfected cells. Conclusion: Our data suggest that highly expressed EMMPRIN plays an important role in metastatic progression of osteosarcoma. EMMPRIN could serve as a potential therapeutic target in osteosarcoma. Citation Format: Ilkyu Han, Ha Jung Kim, Han-Soo Kim. Role of EMMPRIN in osteosarcoma metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 160.

Prostaglandin F-2α Stimulates The Secretion of Vascular Endothelial Growth Factor and Induces Cell Proliferation and Migration of Adipose Tissue Derived Mesenchymal Stem Cells.

Objective Tissue engineering today uses factors that can induce differentiation of mesenchymal stem cells (MSCs) into other cell types. However, the problem of angiogenesis in this differentiated tissue remains an unresolved area of research interest. The aim of this study was to investigate the effects of prostaglandin F-2α (PGF-2α) on the expression of vascular endothelial growth factor (VEGF) in human adipose tissue derived MSCs. Materials and Methods In this experimental research, human adipose tissue was digested using collagenase. The isolated MSCs cells were treated with PGF-2α (up to 5 μg/ml) and incubated for 96 hours. Cell proliferation, secretion of VEGF and cell migration were spontaneously assayed by MTT, BrdU, ELISA, RT-PCR and scratching methods. Results Cell growth at 1.0, 2.5, 5 µg/ml of PGF-2α was not significantly reduced compared to control cells, suggesting that these concentrations of PGF-2α are not toxic to cell growth. The results of the BrdU incorporation assay indicated that, in comparison to untreated cells, BrdU incorporation was respectively 1.08, 1.96, 2.0 and 1.8 fold among cells treated with 0.1, 1.0, 2.5 and 5.0 µg/ml of PGF-2α. The scratching test also demonstrated a positive influence on cell proliferation and migration. Cells treated with 1.0 µg/ml of PGF-2α for 12 hours showed the highest relative migration and coverage in comparison to untreated cells. Quantitative VEGF ELISA and RT- PCR results indicated an increase in VEGF expression and secretion in the presence of PGF-2α. The amount of VEGF produced in response to 0.1, 1.0, 2.5 and 5.0 µg/ml of PGF-2α was 62.4 ± 3.2 , 66.3 ± 3.7, 53.1 ± 2.6 and 49.0 ± 2.3 pg/ml, respectively, compared to the 35.2 ± 2.1 pg/ml produced by untreated cells. ConclusionStimulation of VEGF secretion by PGF-2α treated MSCs could be useful for the induction of angiogenesis in tissue engineering in vitro.

Protective Effect of Combined Caffeic Acid Phenethyl Ester and Bevacizumab Against Hydrogen Peroxide-Induced Oxidative Stress in Human RPE Cells

ABSTRACTPurpose: This study aimed to evaluate the protective effects of caffeic acid phenethyl ester (CAPE) and combined CAPE-bevacizumab against oxidative stress induced by hydrogen peroxide (H2O2) in human retinal pigment epithelium.Methods: ARPE-19 cells were pretreated with 5, 10, and 30 μM CAPE alone and in combination with bevacizumab for 3 h, then exposed to H2O2 for 16 h. Cell viability was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Vascular endothelial growth factor (VEGF) protein levels in the medium were measured using a human VEGF ELISA kit. Total antioxidant status (TAS) and total oxidant status (TOS) were measured in ARPE-19 cells using the test kit from Rel Assay. Expression levels of VEGF, Bax, Bcl-2, cytochrome c, apoptotic protease activating factor-1 (apaf-1), and caspase-3 were determined using reverse transcription polymerase chain reaction.Results: Pretreatment of ARPE-19 cells with 30 μM CAPE and combined CAPE-bevacizumab reduced H2O2 mediated cell death. H2O2-induced oxidative stress increased TOS and VEGF production, which was significantly inhibited by CAPE and the CAPE-bevacizumab combination. VEGF, Bax, cytochrome c, apaf-1, and caspase-3 gene expressions were significantly decreased in cells pretreated with 5, 10, and 30 μM CAPE and combined CAPE-bevacizumab compared to the H2O2 group. In addition, Bcl-2 expression was significantly increased in both the CAPE and CAPE-bevacizumab combination groups compared to the H2O2 group.Conclusions: CAPE has a protective effect on ARPE-19 cells against oxidative stress, and VEGF protein level and expression can be decreased by incubation with different concentrations of CAPE. These results demonstrate that CAPE suppresses the mitochondria-mediated apoptosis in ARPE-19 cells under oxidative stress. In addition, the use of CAPE in combination with bevacizumab has an additive effect.

Cocultivation of Mesenchymal Stem Cells and Endothelial Progenitor Cells Reveals Antiapoptotic and Proangiogenic Effects

Integrating bioartificial tissues into the host vasculature is a prerequisite for tissue engineering applications. Endothelial progenitor cells (EPCs) display a high angiogenic potential and a low donor-site morbidity, making them ideal for tissue engineering applications. In our study we used a murine EPC cell line (T17b) and rat mesenchymal stem cells (MSCs) for cocultivation experiments. MSCs were cocultured with increasing T17b EPC amounts. Furthermore, MSCs in monoculture were treated with conditioned medium (CM) from T17b EPCs and T17b EPCs were treated with CM from MSCs. Proliferation and apoptosis were quantified with a bromodeoxyuridine ELISA and a DNA fragmentation ELISA, respectively. Osteogenic differentiation was detected with an alkaline phosphatase assay and bone morphogenetic protein-2 ELISA. The production of proangiogenic molecules was measured with a matrix metalloproteinase-3 and vascular endothelial growth factor ELISA as well as nitric oxide assay. We could show that T17b EPCs stimulated MSC proliferation but not vice versa. On the other hand, MSCs promoted the cell survival of EPCs. The growth-inducing and antiapoptotic effects were dependent on heterotypic cell contacts and paracrine mediators. Moreover, proangiogenic growth factors were found in the coculture. Collectively, our results indicate that the coapplication of MSCs and T17b EPCs provides new perspectives for tissue engineering applications.

Angiogenic potential of boron-containing bioactive glasses: in vitro study

Boron-containing bioactive glasses (BGs) are being extensively researched for the treatment and regeneration of bone defects because of their osteostimulatory and neovascularization potential. In this study, we report the effects of the ionic dissolution products (IDPs) of different boron-doped, borosilicate, and borate BG scaffolds on mouse bone marrow stromal cells in vitro, using an angiogenesis assay. Five different BG scaffolds of the system SiO2–Na2O–K2O–MgO–CaO–P2O5–B2O3 (with varying amounts of SiO2 and B2O3) were fabricated by the foam replication technique. Bone marrow stromal cells were cultivated in contact with the IDPs of the boron-containing BG scaffolds at different concentrations for 48 h. The expression and secretion of vascular endothelial growth factor (VEGF) from the cultured cells was measured quantitatively using the VEGF ELISA Kit. Cell viability and cell morphology were determined using WST-8 assay and H&E staining, respectively. The cellular response was found to be dependent on boron content and the B release profile from the glasses corresponded to the positive or negative biological activity of the BGs.

The effects of STAT3 inhibitors on endometriotic cyst stromal cells

Objective: The Signal Transducer and Activator of Transcription 3 (STAT3) protein regulatesmany aspects of cellular functions, such as proliferation, apoptosis, and angiogenesis. Enhanced expression of STAT3 protein has been demonstrated in the endometriotic tissues, suggesting the involvement of this molecule in the pathogenesis of endometriosi.Wedesigned the present study to evaluate the efficacy of STAT3 inhibitors as promising drug for the treatment of endometriosis. Materials and methods: Ovarian endometriotic cyst tissues were obtained from patients who had undergone a salpingooophorectomy or evisceration for the treatment of ovarian endometriotic cysts. This study was approved by the institutional review board of the Faculty of Medicine, Oita University, and written informed consent was obtained from all patients. Ovarian endometriotic cyst stromal cells (ECSCs) were isolated by collagenase digestion and cultured in the presence of STAT3 inhibitors (WP1066, S3I-201, and Cryptotanshinone). The effects of these STAT3 inhibitors on the proliferation, apoptosis, and vascular endothelial growth factor (VEGF) expression were investigated by cell death detection ELISA, caspase3/7 assay, modified MTT assay, and VEGF ELISA, respectively. Results: All of three STAT3 inhibitors inhibited the cell proliferation and VEGF production in ECSCs, and induced the apoptosis of these cells. Discussion: STAT3 inhibitors are promising candidates for the treatment of endometriosis. At present, STAT3 inhibitors are clinically used as the anti-cancer drugs. The clinical application of these agents for endometriosis is expected in the future.

Dimethyl sulfoxide-caused changes in pro- and anti-angiogenic factor levels could contribute to an anti-angiogenic response in HeLa cells

Dimethyl sulfoxide (DMSO) is widely used in biological research as a general solvent. While it has been previously demonstrated that DMSO possesses a wide range of pharmacological effects, there is no published work regarding the effects of DMSO on pro-angiogenic factor levels. This study was designed to investigate the possible effects of DMSO on the levels of three pro-angiogenic factors released from HeLa cells in vitro. Cells were treated with two different and previously determined concentrations of DMSO. The cytotoxic effects of DMSO concentrations on HeLa cells were determined via MTT. Survival rates of DMSO-treated cells were determined by Invitrogen live/dead viability/cytotoxicity kit and trypan blue exclusion assay.Changes in the pro-angiogenic levels in media were evaluated by Cayman's Substance P Enzyme Immunoassay ELISA kit. Vascular endothelial growth factor ELISA kit and interferon gamma ELISA kit for substance P, VEGF and IFNγ respectively. Changes in substance P levels were corrected by standard western blotting. Changes in VEGF and IFNγ levels were corrected both by western blot and real time PCR.Treatment with 1.4μM DMSO caused a time-dependent inhibition of cell proliferation at 24, 48 and 72h. 1.4μM DMSO caused a significant reduction in VEGF levels at 72h of incubation and sharp increases in IFNγ levels at both 48 and 72h of incubation. According to real time PCR analyses, DMSO (1.4μM) exhibited an inhibitory effect on VEGF but acted as an augmenter of IFNγ release on HeLa cells in vitro.This is the first report showing that the general solvent DMSO suppressed HeLa cell proliferation, decreased the levels of two pro-angiogenic factors (substance P and VEGF) and increased the release of an anti-angiogenic factor IFNγ in vitro.

The Competence of 7,8-Diacetoxy-4-Methylcoumarinand Other Polyphenolic Acetates in Mitigating the Oxidative Stress and their Role in Angiogenesis

The potential role of polyphenolic acetate (PA) in causing diverse biological and pharmacological actions has been well studied in our laboratory. Our investigations, for the first time, established the role of calreticulin transacetylase (CRTAase) in catalyzing the acetylation of nitric oxide synthase (NOS) by Pas leading to robust activation of NOS. 7, 8-Diacetoxy-4-methylcoumarin (DAMC) and other acetoxycoumarins augmented the expression of thioredoxin (TRX) and vascular endothelial growth factor (VEGF) in human peripheral blood mononuclear cells (PBMCs). These findings substantiated our earlier observations that DAMC was a superb inducer of angiogenesis. The enhanced expression of thioredoxin reductase (TRXR) and diminished expression of thioredoxin interacting protein (TRXIP) leading to increased expression and activity of TRX in PBMCs due to the action of DAMC was revealed by real time RT-PCR analysis. The possible activation of TRX due to acetylation was confirmed by the fact that TRX activity of PBMCs was enhanced by variousacetoxycoumarins in tune with their affinities to CRTAase as substrates. DAMC caused enhanced production of NO by way of acetylation of NOS as mentioned above and thereby acted as an inducer of VEGF. Real time RT-PCR and VEGF ELISA results also revealed the overexpression of TRX. DAMC and other PAs were found to reduce the oxidative stress in cells as proved by significant reduction of intracellular ROS levels. Thus, the crucial role of TRX in DAMC-induced angiogenesis with the involvement of VEGF was established.

Abstract 263: Ultrasound Enhancement of Bevacizumab Release from Echogenic Liposomes for Inhibition of Atheroma Progression

Background: Vascular endothelial growth factor (VEGF) is a signal protein that induces vasculogenesis and angiogenesis, which are important to atheroma progression. Bevacizumab is a humanized monoclonal antibody specific for VEGF that causes regression of existing microvessels and has been shown to inhibit atheroma progression. Previous studies demonstrated the inhibitory efficacy of bevacizumab and bevacizumab-loaded echogenic liposomes (BEV-ELIP) for human umbilical vein cell (HUVEC) proliferation and VEGF expression (assayed by VEGF ELISA). Hypothesis: Bevacizumab activity can be enhanced by pulsed Doppler ultrasound treatment of BEV-ELIP. Methods: Bevacizumab (1 μg/ml) was added to 18-hour HUVEC cultures in 96-well plates, followed by 10 nM phorbol 12-myristate 13-acetate (PMA) 4 hours later and incubated for 24 hours. Cell proliferation was measured by the tetrazolium dye MTT. BEV-ELIP in a latex condom was submerged in a water bath and exposed to 6 MHz color Doppler ultrasound (MI = 0.4 ) for 5 min for bevacizumab release. Results: BEV-ELIP treated with US inhibited VEGF expression by 90% relative to non-treated controls and by 70% relative to BEV-ELIP without US (Fig. 1). Also, BEV-ELIP inhibited cell proliferation by 64% relative to untreated controls and by 45% relative to BEV-ELIP without US (Fig. 2). Conclusions: Ultrasound treatment of BEV-ELIP enhanced the agent’s ability to inhibit cell proliferation in the MTT assay and VEGF expression in the VEGF ELISA, consistent with US-triggered BEV release observed in previous studies. This BEV-ELIP formulation can now be translated to in vivo studies for atheroma inhibition.

Co-Transplantation of VEGF-Expressing Human Embryonic Stem Cell Derived Mesenchymal Stem Cells to Enhance Islet Revascularization in Diabetic Nude Mice

Background: Pancreatic islet transplantation has emerged as a promising treatment for type I diabetes. However, its efficacy is severely hampered due to poor islet engraftment and revascularization, which have been resulted to partially loss of transplanted islets. It has been shown that local delivery of vascular endothelial growth factor (VEGF) could accelerate transplanted islet revascularization, although permanent high level of VEGF may lead to undesirable side effects. In this study we investigated the effects of conditional cell-based delivery of VEGF through collagen-fibrin hydrogel on transplanted islet function and revascularization. Material and Methods: RH6 human embryonic stem cell derived mesenchymal stem cells (ES-MSCs) have been generated, characterized and transduced by two lentiviruses containing rtTA and VEGF-A. Tet-on expression of VEGF from these cells was shown by tube formation assay and was confirmed through VEGF ELISA. After co-transplantation of these cells and mouse isolated islets through collagen-fibrin hydrogel in the omental pouch of diabetic nude mice, the blood glucose, body weight, glucose tolerance and serum C-peptide was measured after 28 days. As control groups, islets were transplanted alone and with non-transgenic ES-MSCs. For measurement of revascularization, immunohistoflourscence examination was done and micro vessel density (MVD) and vessel size have been measured. Results: Receiving the same number of islets (300 IEQ) in all transplanted groups, the average blood glucose concentrations of mice transplanted with islets/hESC-MSC:VEGF were significantly lower than mice transplanted with islets or islet/hESC-MSC at different time points. This notable difference in blood sugar of transplant groups, was also reflected in their weight gain. Serum C-peptide level was higher in islet/hESC-MSC:VEGF (349.8±51.2 pM) compared with islet/hESC-MSC group and islet group but it was not markedly different from C-peptide levels in normal mice. In intraperitoneal glucose tolerance test (IP-GTT) to examine glucose responsiveness of the islet graft, mice in islet/hESC-MSC:VEGF group returned to normoglycemic state at the same rate as normal mice, while mice in islet/hESC-MSC and islet groups responded more slowly and did not return to the normoglycemic state. The results showed improved islet functionality and micro-vessel density in the group of mice received islets with VEGF expressing hESC-MSC, compared with control groups. Conclusion: We conclude that conditional expression of VEGF from hESC-MSCs during islet transplantation could enhance islet functionality and revascularization. This result can be used to improve the outcome of clinical islet transplantation. Keywords:Co-transplantation, Embryonic stem, Mesenchymal stem cells.

Methodological differences account for inconsistencies in reported free VEGF concentrations in pregnant rats

Free vascular endothelial growth factor (VEGF) is undetectable in plasma during human pregnancy. However, studies examining pregnant rats have reported both low (8-29 pg/ml) and high (527-1,030 pg/ml) free VEGF. These discrepancies cast uncertainty over the use of rat models to study angiogenic factors in pregnancy and preeclampsia. This study investigates methodological factors that may explain these discrepancies. Plasma VEGF in nonpregnant, day 7 pregnant, and day 19 pregnant rats was measured using rat and mouse ELISAs (R&D Systems). The rat ELISA detected VEGF in plasma from nonpregnant rats but not in plasma from day 19 pregnant rats. The mouse ELISA detected higher VEGF concentrations than the rat ELISA in every sample tested. This discrepancy was greater in day 19 pregnant rats (median: 2,273 vs. 0 pg/ml) than in nonpregnant (97 vs. 20 pg/ml) and day 7 pregnant (66 vs. 2 pg/ml) rats. Recovery of recombinant rat VEGF (rrVEGF) spiked into plasma from nonpregnant and day 7 pregnant rats was high for the rat ELISA (82-105%) but low for the mouse ELISA (17-22%). The rat ELISA did not recover rrVEGF in plasma from day 19 pregnant rats, suggesting that this ELISA measures free VEGF. The use of the rat versus mouse ELISA likely explains the differences in reported VEGF concentrations in pregnant rats. While the rat ELISA appears to measure free VEGF, plasma concentrations in nonpregnant and pregnant rats are below the assay sensitivity limit. As most previous studies of pregnant rats used the mouse VEGF ELISA, these data should be interpreted cautiously.

VEGF levels in diagnosis of vasculitic neuropathy.

Vascular endothelial growth factor (VEGF) is secreted from endothelial cells and pericytes in response to hypoxia. It induces angiogenesis and microvascular hyperpermeability. We observe serum VEGF concentrations in some patients with vasculitic neuropathy. Plasma VEGF was measured using GenWay's human VEGF ELISA Kit, which is based on standard sandwich enzyme-linked immune-sorbent assay technology. Human VEGF specific-specific monoclonal antibodies are precoated onto 96-well plates. The human specific detection polyclonal antibodies are biotinylated. The test samples and biotinylated detection antibodies were added to the wells subsequently and then followed by washing with PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex was added and unbound conjugates were washed away with PBS or TBS buffer. HRP substrate TMB was used to visualize HRP enzymatic reaction. The VEGF levels were measured in 5 patients with vasculitic neuropathy and 8 healthy controls. Plasma VEGF was higher in subjects with vasculitic neuropathy as compared to controls. In the control group, we obtained VEGF levels from 9.8 pg/mL up to 15 pg/mL with an average of 11.6 pg/mL for males and 8.9 pg/mL up to 14.5 pg/mL with average of 11.2 pg/mL for females. In female patients with vasculitic neuropathy the plasma VEGF levels were between 79.9 pg/mL and 111.2 pg/mL, with an average of 92.375 pg/mL. We had one case with vasculitic neuropathy in men, in which the plasma VEGF level was 102.9 pg/mL. The results from our study indicate that plasma VEGF levels are significantly associated with vasculitic neuropathy and may be used to predict this disease.

Abstract 326: Granzyme B Releases Vascular Endothelial Growth Factor from Extracellular Matrix Stores and Induces Vascular Permeability in vivo

Introduction The formation of unstable and leaky neovessels underlies the pathogenesis of a large number of chronic inflammatory diseases. Granzyme B (GZMB) is a serine protease that is expressed and released by a variety of immune cells and accumulates in the extracellular matrix (ECM) during chronic inflammation where it cleaves a number of ECM proteins, including fibronectin (FN). Vascular endothelial growth factor (VEGF) is a potent vascular permeabilizing agent that is sequestered in the ECM by binding FN in both normal and diseased tissue. We hypothesize that GZMB cleavage of FN will release VEGF from its extracellular stores and promote vascular permeability as a mechanism that contributes to neovessel leakage during chronic inflammation. Methods GZMB-mediated VEGF release from either FN coated wells or endogenously produce endothelial cell (EC) matrix was measured by VEGF ELISA. VEGF-release supernatants were used to treat EC and VEGF receptor 2 (VEGFR2) activation was evaluated by immunoblotting for phosphorylated VEGFR2. Evan’s blue was injected intravenously to CD1 mice followed by ear injection of either mouse GZMB, saline control, GZMB + neutralizing mouse VEGF antibody or GZMB+ IgG control (n=5 for each experimental group). Vascular leakage was evaluated by Evan’s blue dye extraction. Results GZMB effectively releases VEGF from both FN and from EC matrix, while inhibition of GZMB prevented VEGF release. GZMB-mediated VEGF release resulted in significant activation of VEGFR2 in EC monolayer signified by increased VEGFR2 phosphorylation. GZMB ear injection resulted in a significant increase in vascular permeability in vivo. Importantly, co-injection of GZMB and neutralizing mouse VEGF antibody significantly reduced vascular leakage compared to co-injection of GZMB and matching IgG control. Conclusions and Impact GZMB increases VEGF bioavailability by releasing it from the ECM leading to VEGFR2 activation and increased vascular permeability in vivo. These findings present a novel role for GZMB as a modulator of vascular response during chronic inflammation.

Radiation up-regulates the expression of VEGF in a canine oral melanoma cell line

To evaluate radiosensitivity and the effects of radiation on the expression of vascular endothelial growth factor (VEGF) and VEGF receptors in the canine oral melanoma cell line, TLM 1, cells were irradiated with doses of 0, 2, 4, 6, 8 and 10 Gray (Gy). Survival rates were then determined by a MTT assay, while vascular endothelial growth factor receptor (VEGFR)-1 and -2 expression was measured by flow cytometry and apoptotic cell death rates were investigated using an Annexin assay. Additionally, a commercially available canine VEGF ELISA kit was used to measure VEGF. Radiosensitivity was detected in TLM 1 cells, and mitotic and apoptotic cell death was found to occur in a radiation dose dependent manner. VEGF was secreted constitutively and significant up-regulation was observed in the 8 and 10 Gy irradiated cells. In addition, a minor portion of TLM 1 cells expressed vascular endothelial growth factor receptor (VEGFR)-1 intracellularly. VEGFR-2 was detected in the cytoplasm and was down-regulated following radiation with increasing dosages. In TLM 1 cells, apoptosis plays an important role in radiation induced cell death. It has also been suggested that the significantly higher VEGF production in the 8 and 10 Gy group could lead to tumour resistance.

Effect of CFSE labeling on proliferation, differentiation and secretion of rabbit bone marrow mesenchymal stem cells

Objective To explore the experimental conditions of labeling rabbit bone marrow mesenchymal stem cells (Rb-MSCs) with 5,6-carboxyfluorescein diacetic succinimidyl ester (CFSE) and to investigate the impact on the biological characteristics of Rb-MSCs in vitro.Methods Rb-MSCs were separated and purified by whole bone marrow adherent culture and then were identified by morphology and surface markers.Rb-MSCs were labeled with CFSE and the labeling effect was measured by flow cytometer.The proliferation capacity of labeled cells was detected with CCK-8.The differentiation capacity of labeled cells was investigated by being induced to osteoblasts and lipoblasts.The capacity of labeled cells to secret vascular endothelial growth factor (VEGF) was detected by VEGF ELISA kits.Results The primary Rb-MSCs adhered in 48 h,being fusiform and colony-like growth.Subculture cells became fibroblast-like cells in order with uniform configuration.Most (above 98％) cultured cells expressed the surface markers CD29 and CD44 except for CD45.Compared with other labeling conditions,10μmol/L final concentration of CFSE and 10 min was the best one with a 100％ labeling rate and high fluorescence intensity.Compared with unlabeled cells,the ability of the labeled cells to proliferate and to secrete VEGF was not significantly decreased (P ＞ 0.05).Moreover,the labeled cells had osteogenic and adipogenic differentiation capacity.Conclusions It was a simple and efficient method to label Rb-MSCs with CFSE,especially in a short-term.The capacity of cell proliferation,differentiation,and secretion were not affected. Key words: Bone marrow cells/cytology ; Mesenchymal stem cells/cytology ; Cells, cultured ; Staining and labeling/methods ; Rabbits

Poly(oligo-D-arginine) With Internal Disulfide Linkages as a Cytoplasm-sensitive Carrier for siRNA Delivery

Small interfering RNA (siRNA) has emerged as a therapeutic strategy for various diseases due to its target-specific gene silencing; however, its relatively high molecular weight, negative charge, and low stability hamper in vitro and in vivo applications. Approaches to overcome those drawbacks have relied on nonviral siRNA carriers based on cationic polymers or peptides. Nevertheless, cationic polymer-based siRNA carriers have yet to resolve intrinsic problems such as cytotoxicity and immunogenicity. An environment-sensitive carrier was recently proposed to enhance siRNA bioactivity and to reduce the carrier safety issues. Only a few studies, however, have shown cytoplasm-sensitive dissociation of the polyplex. In the present study, we clearly demonstrated decondensation of siRNA/poly(oligo-D-arginine) polyplex in the cytoplasm in response to intracellular glutathione (GSH) and the enhanced bioactivity of siRNA against VEGF (siVEGF) used as a model both in vitro and in an animal model. Reducible poly(oligo-D-arginine) (rPOA) rapidly dissociated in the cytoplasm, resulting in fast siRNA release to its target location while maintaining siRNA bioactivity both in vitro and in vivo.

Abstract 1434: EMMPRIN expression is associated with metastatic progression in osteosarcoma

Abstract Introduction: Despite the improvement of survival in localized osteosarcoma patients, patients with metastasis still carry a poor prognosis. Thus, identification of factors contributing to metastasis is needed. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell-surface glycoprotein which plays multiple roles in physiologic and pathologic conditions. EMMPRIN is highly expressed in several cancers and stimulates adjacent stromal cells or tumor cells to produce matrix metalloproteinase (MMP). EMMPRIN also stimulates expression of vascular endothelial growth factor (VEGF) which leads to angiogenesis. These findings have made EMMPRIN an attractive therapeutic target in many cancer types. The goals of this study were; to investigate the expression of EMMPRIN in osteosarcoma; to examine if EMMPRIN, via tumor-stroma interaction, is associated with metastatic potential in osteosarcoma. Methods: Level of EMMPRIN mRNA expression was evaluated by RT-PCR in 6 tumor-derived osteosarcoma cell lines and by immunohistochemistry prechemotherapy biopsies from 52 patients. Clinical data of these patients were reviewed to examine the association of EMMPRIN expression and clinical outcome. Transfection of EMMPRIN-targeting siRNA in SaOS-2 was performed to test the role of EMMPRIN in osteosarcoma progression. To study the role of EMMPRIN in tumor-stromal interaction, co-culture of SaOS-2 with osteoblast (hFOB) was experimented. Functional in vitro assays that reflect metastatic potential of osteosarcoma cells were performed. MMP production, VEGF production, cell invasion and proliferation were studied by gelatin zymography, VEGF ELISA, Matrigel invasion assay and WST-1 assay, respectively. Results: EMMPRIN was expressed in 90% by immunohistochemistry with strong accentuation along the cell membrane. Level of EMMRIN mRNA expression was significantly higher in 5 of 6 tumor-derived cell lines compared to MG63. Patients with high EMMPRIN expression had significantly worse metastasis-free survival. EMMPRIN mRNA levels was significantly down-regulated by siRNA transfection compared to control-siRNA transfected cell. Co-culture of osteoblast and SaOS-2 enhanced stimulation of pro-MMP2. This stimulation was reversed by transfection of SaOS-2 with EMMPRIN-targeting siRNA. Number of cells crossing the chamber was statistically lower in EMMPRIN-siRNA transfected cells. When osteoblast and SaOS-2 were co-cultured, VEGF expression was increased compared to SaOS-2 only culture. EMMPRIN-targeting siRNA transfection resulted in decrease of VEGF expression. SaOS-2 cells transfected with EMMPRIN-siRNA showed decreased proliferation potential compared to control-siRNA transfected cells. Conclusion: Our data suggest that highly expressed EMMPRIN plays an important role in metastatic potential of osteosarcoma. EMMPRIN could serve as a potential therapeutic target in osteosarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1434.

Effect of radiation on vascular endothelial growth factor expression in the C2 canine mastocytoma cell line

To establish the radiosensitivity and effect of irradiation on vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) expression in the canine mastocytoma cell line C2. Canine mastocytoma cell line C2. C2 cells were irradiated with single doses of 2, 4, 6, and 8 Gy. The 3-(4, 5-di-methyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and proliferation assays with (methyl-hydrogen 3) thymidine were used for radiosensitivity experiments. Expression of VEGFR was determined via flow cytometry and apoptotic rate via annexin assay. Human and canine VEGF ELISA kits were evaluated in crossover assay experiments, and the canine kit was used thereafter. C2 cells secreted VEGF constitutively. Radiation did not induce a significant increase in VEGF secretion, regardless of radiation dose. Consistently, radiation did not up-regulate VEGFR. Cell survival rates decreased in a dose-dependent manner. The apoptotic cell fraction had a dose-dependent increase that reached its maximum 24 to 48 hours after radiation. The C2 cell line was radiosensitive, and a fraction (up to 40%) of cells died via apoptosis in a dose-dependent manner. In response to radiation, C2 cells did not upregulate VEGF production or VEGFR. Further studies are needed to determine whether tumor control could be improved by combining radiotherapy with VEGFR inhibitors or apoptosis-modulating agents.