Decreased Vascular Endothelial Growth Factor Expression Research Articles

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.

Increased expression and activity of 12-lipoxygenase in oxygen-induced ischemic retinopathy and proliferative diabetic retinopathy: implications in retinal neovascularization.

OBJECTIVEArachidonic acid is metabolized by 12-lipoxygenase (12-LOX) to 12-hydroxyeicosatetraenoic acid (12-HETE) and has an important role in the regulation of angiogenesis and endothelial cell proliferation and migration. The goal of this study was to investigate whether 12-LOX plays a role in retinal neovascularization (NV).RESEARCH DESIGN AND METHODSExperiments were performed using retinas from a murine model of oxygen-induced ischemic retinopathy (OIR) that was treated with and without the LOX pathway inhibitor, baicalein, or lacking 12-LOX. We also analyzed vitreous samples from patients with and without proliferative diabetic retinopathy (PDR). Western blotting and RT-PCR were used to assess the expression of 12-LOX, vascular endothelial growth factor (VEGF), and pigment epithelium–derived factor (PEDF). Liquid chromatography–mass spectrometry was used to assess the amounts of HETEs in the murine retina and human vitreous samples. The effects of 12-HETE on VEGF and PEDF expression were evaluated in Müller cells (rMCs), primary mouse retinal pigment epithelial cells, and astrocytes.RESULTSRetinal NV during OIR was associated with increased 12-LOX expression and 12-, 15-, and 5-HETE production. The amounts of HETEs also were significantly higher in the vitreous of diabetic patients with PDR. Retinal NV was markedly abrogated in mice treated with baicalein or mice lacking 12-LOX. This was associated with decreased VEGF expression and restoration of PEDF levels. PEDF expression was reduced in 12-HETE–treated rMCs, astrocytes, and the retinal pigment epithelium. Only rMCs and astrocytes showed increased VEGF expression by 12-HETE.CONCLUSIONS12-LOX and its product HETE are important regulators of retinal NV through modulation of VEGF and PEDF expression and could provide a new therapeutic target to prevent and treat ischemic retinopathy.

PI3K/AKT/mTOR Pathway in Angiogenesis

The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is activated in the majority of human cancers. This pathway is known to play a key role in numerous cellular functions including proliferation, adhesion, migration, invasion, metabolism, and survival, but in the current review we focus on its role in angiogenesis. PI3K activation may occur via RAS mutation, loss of phosphatase and tensin homolog (PTEN), or by increased expression of growth factor receptors such as epidermal growth factor receptor. There is a connection between the PI3K pathway and angiogenesis. Hypoxia leads to HIF-1α stabilization and is a major stimulus for increased vascular endothelial growth factor (VEGF) production by tumor cells. However, activation of the PI3K/AKT pathway in tumor cells can also increase VEGF secretion, both by hypoxia-inducible factor 1 (HIF-1) dependent and independent mechanisms. The PI3K/AKT pathway also modulates the expression of other angiogenic factors such as nitric oxide and angiopoietins. Numerous inhibitors targeting the PI3K/AKT/mTOR pathway have been developed, and these agents have been shown to decrease VEGF secretion and angiogenesis. The effect of these inhibitors on tumor vasculature can be difficult to predict. The vasculature of tumors is aberrant, leading to sluggish bloodflow and elevated interstitial blood pressure, which can be perpetuated by the high levels of VEGF. Hence, decreasing VEGF expression can paradoxically lead to vascular normalization and improved bloodflow in some tumors. In addition to its importance in cancer, the PI3K pathway also plays an essential role in the formation of normal blood vessels during development. Embryos with kinase-dead p110α catalytic subunit of PI3K develop vascular defects. Stimulation of endothelial cells by VEGF leads to activation of the PI3K pathway within these cells, which is important for cell migration. Sustained endothelial activation of AKT1 has been shown to induce the formation of structurally abnormal blood vessels that recapitulate the aberrations of tumor vessels. Hence, the PI3K pathway plays an important role in regulating angiogenesis both in normal tissues and in cancers.

Combination of vascular endothelial growth factor antisense oligonucleotide therapy and radiotherapy increases the curative effects against maxillofacial VX2 tumors in rabbits

Purpose To study the effects of combination of vascular endothelial growth factor (VEGF) antisense oligonucleotide therapy and radiotherapy on maxillofacial VX2 tumors in rabbits. Methods We used 24 New Zealand white rabbits as a model to induce maxillofacial VX2 tumor. The rabbits were randomly divided into the following 4 groups: radiotherapy group (group A), treated with 16 Gy of radiotherapy; VEGF antisense oligonucleotide treatment group (group B), treated with an injection of 150 μg of VEGF antisense oligonucleotide into the local tumor; VEGF antisense oligonucleotide combined with radiotherapy group (group C), treated with an injection of 150 μg of VEGF antisense oligonucleotide into the local tumor immediately after 16 Gy of radiotherapy; and control group (group D), treated with an injection of 300 μl 5% aqueous glucose solution into the local tumor. On days 3 and 14 after treatment, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was performed to calculate maximal enhancement ratio (MER), slope of enhancement (SLE), and tumor volume change. Rabbits were killed on day 14 to obtain samples for pathological examination and immunohistochemical staining for VEGF. Results In group C, tumor volume was significantly reduced on day 14 after treatment, and the difference was statistically different as compared to that before treatment, on day 3 after treatment and other groups ( P < 0.01). Values of both MER and SLE after treatment were significantly lower than the values before treatment ( P < 0.05). Pathological specimen revealed tumor cell edema, bleeding, necrosis, vascular wall thickening and occlusion, and decreased VEGF expression. The immunohistochemical score (IHS) of group C was significantly different from groups A and D respectively ( P < 0.05). Conclusion Injecting the tumor with VEGF antisense oligonucleotide immediately after radiotherapy can enhance the curative effect on rabbit maxillofacial VX2 tumor, and DCE-MRI can serve as a reliable technique for in vivo monitoring.

A novel concept in antiangiogenic and antitumoral therapy: multitarget destabilization of short-lived mRNAs by the zinc finger protein ZFP36L1

Angiogenesis inhibitors have shown clinical benefits in patients with advanced cancer, but further therapeutic improvement is needed. We have previously shown that the zinc finger protein 36, C3H type-like 1 (ZFP36L1) enhances vascular endothelial growth factor (VEGF) mRNA decay through its interaction with AU-rich elements within VEGF 3'-untranslated region. In this study, we evaluated the possibility to develop an antiangiogenic and antitumoral strategy using the mRNA-destabilizing activity of ZFP36L1. We engineered a cell-penetrating ZFP36L1, by fusing it to the protein transduction domains (PTDs) TAT derived from HIV, or the polyarginine peptides R7 or R9. PTD-ZFP36L1 fusion proteins were expressed in bacterial cells and affinity-purified to homogeneity. TAT-, R7- and R9-ZFP36L1 were efficiently internalized into living cells and decreased both endogenous VEGF mRNA half-life and VEGF protein levels in vitro. Importantly, a single injection of R9-TIS11b fusion protein into a high-VEGF expressing tissue in vivo (in this study, the mouse adrenal gland) markedly decreased VEGF expression. We further evaluated the effect of R9-ZFP36L1 on tumor growth using Lewis Lung Carcinoma (LL/2) cells implanted subcutaneously into nude mice. Intratumoral injection of R9-ZFP36L1 significantly reduced tumor growth and markedly decreased the expression of multiple angiogenic and inflammatory cytokines, including VEGF, acidic fibroblast growth factor, tumor necrosis factor α, interleukin (IL)-1α and IL-6, with a concomitant obliteration of tumor vascularization. These findings indicate that R9-ZFP36L1 fusion protein may represent a novel antiangiogenic and antitumoral agent, and supports the emerging idea that modulation of mRNA stability represents a promising therapeutic approach to treat cancer.

MMP‐2 alters VEGF expression via αVβ3 integrin‐mediated PI3K/AKT signaling in A549 lung cancer cells

Vascular endothelial growth factor (VEGF) is one of the most important angiogenic growth factors for tumor angiogenesis. Here, we sought to explore whether RNA interference (RNAi) targeting matrix metalloproteinase-2 (MMP-2) could disrupt VEGF-mediated angiogenesis in lung cancer. MMP-2 siRNA inhibited lung cancer cell-induced tube formation of endothelial cells in vitro; addition of recombinant human-MMP-2 restored angiogenesis. MMP-2 transcriptional suppression decreased VEGF, phosphatidylinositol 3-kinase (PI3K) protein levels and AKT phosphorylation in lung cancer cells. In addition, MMP-2 suppression decreased hypoxia inducible factor-1alpha (HIF-1alpha), a transcription factor for VEGF, as determined by electrophoretic mobility shift assay (EMSA). We also show that MMP-2 suppression disrupted PI3K dependent VEGF expression; ectopic expression of myr-AKT restored VEGF inhibition. Further, MMP-2 suppression decreased the interaction of integrin-alphaVbeta3 and MMP-2 as confirmed by immunoprecipitation analyses. Studies with either function blocking integrin-alphaVbeta3 antibody or MMP-2 specific inhibitor (ARP-100) indicate that suppression of MMP-2 decreased integrin-alphaVbeta3-mediated induction of PI3K/AKT leading to decreased VEGF expression. Moreover, A549 xenograft tissue sections from mice that treated with MMP-2 siRNA showed reduced expression of VEGF and the angiogenic marker, factor-VIII. The inhibition of tumor angiogenesis in MMP-2 suppressed tumor sections was associated with decreased co-localization of integrin-alphaVbeta3 and MMP-2. In summary, these data provide new insights into the mechanisms underlying MMP-2-mediated VEGF expression in lung tumor angiogenesis.

Q39, a quinoxaline 1,4-Di-N-oxide derivative, inhibits hypoxia-inducible factor-1α expression and the Akt/mTOR/4E-BP1 signaling pathway in human hepatoma cells

Cumulative evidence has established that hypoxia-inducible factor-1α (HIF-1α) and its downstream target, vascular endothelial growth factor (VEGF), play a critical role in hepatocellular carcinoma angiogenesis, invasiveness and metastasis. 3-(4-bromophenyl)-2-(ethylsulfonyl)-6-methylquinoxaline 1,4-dioxide (Q39) has recently shown great antiproliferative activity in extensive cell lines in normoxia and hypoxia. In this study, Q39 exhibited high antiproliferative activity against hepatoma both in vitro and in vivo, mainly by inducing apoptosis. In addition, suppression of HIF-1α by Q39 resulted in a drastic decrease in VEGF expression. These results indicate that Q39 is an effective inhibitor of HIF-1α and provide new perspectives into the mechanism of its anticancer activity. Interestingly, neither the HIF-1α degradation rate nor the HIF-1α steady-state mRNA level was affected by Q39. Instead, suppression of HIF-1α accumulation by Q39 correlated with prominent dephosphorylation of mTOR and 4E-BP1, a pathway known to regulate protein expression at the translational level.

Effects of Antenatal Betamethasone and Dexamethasone on the Lung Expression of Vascular Endothelial Growth Factor and Alveolarization in Newborn Rats Exposed to Acute Hypoxia and Recovered in Normoxia or Hyperoxia

The use of antenatal corticosteroids is widespread and it is important to know their effect(s) on vascular endothelial growth factor (VEGF), which plays a crucial role in pulmonary development. The purpose of this study was to compare pulmonary VEGF expression in newborn rats that were exposed to antenatal betamethasone versus dexamethasone under normoxia, hypoxia and oxidative stress, and to evaluate its impact on alveolarization. Betamethasone, dexamethasone or equivalent saline solution (control group) was administered to pregnant rats. Newborn rats were randomized to room air, hypoxia followed by hyperoxia, or hypoxia followed by air. Pulmonary VEGF protein, VEGF mRNA, and alveolarization were evaluated at 4 days of life. Betamethasone and dexamethasone were observed to have different actions on VEGF expression with a correlation with alveolarization. Antenatal dexamethasone decreased VEGF expression, and dexamethasone and hyperoxia had an additive effect on the inhibition of VEGF with a reduction in alveolar development. Betamethasone appeared to have an effect on the induction of the expression of VEGF, and it seemed to inhibit the negative action of hyperoxia on VEGF. Moreover, betamethasone did not produce a decrease in alveolarization. Our results support the notion that betamethasone could be better than dexamethasone for antenatal lung maturation.

Prevention of ovarian hyperstimulation syndrome in a rat model: efficacy comparison between cabergoline and meloxicam

To compare the efficacy of cabergoline (Cb2) and meloxicam in curbing vascular endothelial growth factor (VEGF) expression and preventing ovarian hyperstimulation syndrome (OHSS). Randomized controlled, animal study. Academic facility. We used a total of 50 immature Wistar female rats randomly to create an experimental OHSS model. Ten rats each formed the control group and mild OHSS group. The remaining 30 were separated into three equal groups of severe OHSS. Mild and severe OHSS were induced through ovarian stimulation with gonadotropins. One group with severe OHSS was administered a low-dose 100 microg/kg Cb2 therapy; another group with severe OHSS received 600 microg/kg meloxicam. Body weight, vascular permeability (VP), VEGF expression, ovary weight, and diameter were then compared. The efficacy of Cb2 and meloxicam for preventing OHSS. Comparison of the severe OHSS groups with the controls and mild OHSS group revealed significant increases in VEGF expression, VP, ovary weight, and diameter. The increase in VEGF expression was demonstrated to be dependent on human chorionic gonadotropin doses. However, low-dose Cb2 and meloxicam therapies were shown to be ineffective in decreasing VEGF expression and VP, ovary weight, and ovary diameter in severe OHSS. VEGF elevation played a critical part in OHSS pathogenesis, but the therapies administered failed to curb VEGF expression.

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.

Abstract LB-371: Altered vascular endothelial growth factor mRNA stability may lead to cancer development following immunosuppressive therapy

Abstract The development of cancer is an increasing and major problem in immunosuppressed patients, particularly after solid organ transplantation. In part, the increased cancer risk is associated with the use of immunosuppressive agents, especially calcineurin inhibitors (CNIs). The CNIs are used to prevent inflammatory diseases and allograft rejection. However, little is known about the mechanism(s) underlying their ability to promote the development and recurrence of cancer. We have recently demonstrated that CNI can mediate the transcriptional activation of the angiogenic cytokine vascular endothelial growth factor (VEGF) and promote a rapid progression of human renal cancer. In contrast, the mTOR inhibitor rapamycin (RAPA), which is thought to have anti-cancer effects, has been reported to decrease VEGF expression. Here, we investigated whether the CNI cyclosporine (CsA) and RAPA could alter the mRNA stability of VEGF in 786-0 and Caki-1 renal cancer cells. By using actinomycin-D, we observed that CsA increased, while RAPA decreased the VEGF mRNA stability as observed by real time PCR. It is established that the mRNA-binding protein HuR may play a critical role in VEGF mRNA stability. By using HuR-siRNA, we found that the knockdown of HuR significantly decreased the CNI-induced VEGF mRNA stability. By Western blot analysis, it has been observed that CNI treatment induced the translocation of HuR from the nucleus to the cytoplasm; CNI also induced the association between HuR and protein kinase C (PKC)-δ, and promoted the phosphorylation of HuR. Interestingly, there was no significant change in the cytoplasmic translocation of HuR following RAPA treatment. Finally, we found that the inhibition of PKC-δ using a dominant-negative plasmid significantly decreased the CsA-induced cytoplasmic translocation of HuR and the VEGF mRNA stability. Together, we suggest that targeting the pathways that promote the transcription as well as the mRNA stability (post-transcriptional modification) of VEGF in response to CNI might serve as novel therapeutics for the prevention and treatment of cancer in immunosuppressed patients. 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 LB-371.

Adiponectin deficiency exacerbates cardiac dysfunction following pressure overload through disruption of an AMPK-dependent angiogenic response

Although increasing evidence indicates that an adipokine adiponectin exerts protective actions on heart, its effects on coronary angiogenesis following pressure overload have not been examined previously. Because disruption of angiogenesis during heart growth leads to contractile dysfunction and heart failure, we hypothesized that adiponectin modulates cardiac remodeling in response to pressure overload through its ability to regulate adaptive angiogenesis. Adiponectin-knockout (APN-KO) and wild-type (WT) mice were subjected to pressure overload caused by transverse aortic constriction (TAC). APN-KO mice exhibited greater cardiac hypertrophy, pulmonary congestion, left ventricular (LV) interstitial fibrosis and LV systolic dysfunction after TAC surgery compared with WT mice. APN-KO mice also displayed reduced capillary density in the myocardium after TAC, which was accompanied by a significant decrease in expression of vascular endothelial growth factor (VEGF) and phosphorylation of AMP-activated protein kinase (AMPK). Inhibition of AMPK in WT mice resulted in aggravated LV systolic function, attenuated myocardial capillary density and decreased VEGF expression in response to TAC. The adverse effects of AMPK inhibition on cardiac function and angiogenic response following TAC were diminished in APN-KO mice relative to WT mice. Moreover, adenovirus-mediated VEGF delivery reversed the TAC-induced deficiencies in cardiac microvessel formation and ventricular function observed in the APN-KO mice. In cultured cardiac myocytes, adiponectin treatment stimulated VEGF production, which was inhibited by inactivation of AMPK signaling pathway. Collectively, these data show that adiponectin deficiency can accelerate the transition from cardiac hypertrophy to heart failure during pressure overload through disruption of AMPK-dependent angiogenic regulatory axis.

The Synergistic Effect of Recombinant Human Endostatin (YH-16) Combined with Oxaliplatin on Human Colorectal Carcinoma

This study was designed to investigate the effect of recombinant human endostatin (YH-16) combined with oxaliplatin (L-OHP) on the growth of orthotopically-implanted human colorectal carcinoma in nude mice. Tumour volumes and weights were measured after therapy. Tumour cell morphology was observed by light and electron microscopy. Apoptosis was detected using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) assay, and vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) were determined by immunohistochemical staining. Tumour volumes in all treatment groups were significantly reduced compared with controls. YH-16 and L-OHP either alone or in combination caused tumour cell apoptosis, except in the YH-16 low-dose group. MVD was strongly inhibited in the treatment groups compared with the controls, although only YH-16 combined with L-OHP or L-OHP alone decreased VEGF expression. No obvious change in side-effects occurred. In conclusion, YH-16 combined with L-OHP inhibited growth and induced apoptosis in orthotopically-transplanted human colorectal carcinoma in nude mice without increasing side-effects.

Treatment with Nesiritide, A Recombinant B-Type Natriuretic Peptide, Reduces Vascular Remodeling Following Balloon-Induced Endothelial Injuries in Rabbits

Re-stenosis or remodeling of coronary and peripheral arteries remains a major complication following balloon-angioplasty or stenting. This study was designed to investigate the effect of nesiritide, a recombinant B-type natriuretic peptide (BNP), on vascular remodeling following balloon-induced endothelial injuries. Twenty-eight male New Zealand rabbits were divided into nesiritide-treated (0.1 mg/kg/day, sc, for 4 weeks, n = 10), saline-treated control (n = 10) and sham-operated groups (n = 8). In the nesiritide and control groups, a balloon catheter was inserted to the right iliac artery to induce injuries. Vascular endothelial growth factor (VEGF) was measured by immunohistochemistry. The area under internal elastic membrane of the arterial wall (643.2 ± 134.1 vs 493.7 ± 139.3 µm(2), p < 0.05) and the area under external elastic membrane (1495.1 ± 204.9 vs 1265.9 ± 232.6 µm(2), p < 0.05) in the nesiritide group were greater than those in the control group, but were smaller than those in the sham-operated group (p < 0.05). The stenosis ratio was lower in the nesiritide group than in the control group (18.7 ± 7.7% vs 38.0 ± 8.3%, p < 0.01). Importantly, the VEGF expression rate was significantly lower in the nesiritide group than in the control group (42.2 ± 8.8% vs 56.1 ± 13.1%, p < 0.05), while there were no signs of VEGF expression in the non-injured arteries of the three groups. In conclusion, nesiritide treatment reduces the stenosis of the rabbit iliac artery following balloon-induced endothelial injuries probably by decreasing VEGF expression.

Efficient Regulation of VEGF Expression by Promoter-Targeted Lentiviral shRNAs Based on Epigenetic Mechanism

We studied a possibility that shRNAs can lead to transcriptional gene activation at the promoter level via epigenetic mechanism. The purpose of this study was to test the effects on vascular endothelial growth factor (VEGF-A) expression by promoter targeted small hairpin RNAs (shRNAs) in vitro and in experimental animals in vivo using stable local lentiviral gene transfer. One shRNA was identified which strongly increased VEGF-A expression in C166 endothelial cells at mRNA and protein level whereas another shRNA decreased VEGF-A expression. Quantitative chromatin immunoprecipitation analysis revealed that the repressing shRNA caused epigenetic changes, which increased nucleosome density within the promoter and transcription start site and led to repression of VEGF-A expression. Epigenetic changes caused by the activating shRNA were opposite to those caused by the repressing shRNA. These results were confirmed in vivo in an ischemic mouse hindlimb model after local gene transfer where VEGF-A upregulation achieved by promoter-targeted shRNA increased vascularity and blood flow. We show that lentivirus-mediated delivery of shRNA molecules targeted to specific regions in the mVEGF-A promoter either induce or repress VEGF-A expression via epigenetic modulation. Thus, we describe a new approach of gene therapy, epigenetherapy, based on an epigenetic mechanism at the promoter level. Controlling transcription through manipulation of specific epigenetic marks provides a novel approach for the treatment of several diseases.

Targeting of mTOR is associated with decreased growth and decreased VEGF expression in acute myeloid leukaemia cells

The mammalian target of rapamycin (mTOR) has recently been implicated in leukaemic cell growth, tumour-associated angiogenesis and expression of vascular endothelial growth factor (VEGF). We examined whether mTOR plays a role as regulator of growth and VEGF-expression in acute myeloid leukaemia (AML). Three mTOR-targeting drugs, rapamycin, everolimus (RAD001) and CCI-779, were applied. The effects of these drugs on growth, survival, apoptosis and VEGF expression in primary AML cells and various AML cell lines were examined. Growth of AML cells and AML-derived cell lines was assessed by (3)H-thymidine incorporation, survival was examined by light- and electron microscopy, by Tunel assay and by AnnexinV-staining, and the expression of VEGF by Northern blotting, RT-PCR and ELISA. Rapamycin was found to counteract growth in the AML cell lines U937 and KG1a as well as in primary AML cells in 14/18 patients examined. The effects of rapamycin and its derivatives were dose-dependent (IC(50): 10 pM-100 nM). It was also found that exposure to mTOR-targeting drugs resulted in apoptosis and in decreased expression of VEGF in leukaemic cells. mTOR-targeting drugs exert antileukaemic effects on AML cells in vitro through multiple actions, including direct inhibition of proliferation, induction of apoptosis and suppression of VEGF. Based on this study and other studies, mTOR can be regarded as a potential drug target in AML.

Alteration of cochlear pericytes in response to noise trauma and the involvement of HIF‐1α and VEGF

This study explored the effect of acoustic trauma on cochlear pericytes. Transmission electron microscopy revealed that pericytes on the capillaries of the stria vascularis were closely associated with the endothelium in control guinea pigs and mice. Foot processes of the pericytes were tightly positioned adjacent to endothelial cells. Exposure to wide‐band noise at the level of 120 dB for 3 hours per day for two consecutive days produced a significant hearing threshold shift and structurally damaged blood vessels in the stria vascularis. The serum protein, IgG, leaked from capillaries of the stria vascularis. Pericytes lost their tight association with endothelial cells. A pericyte structural protein, desmin, substantially increased after noise exposure in guinea pigs and mice, with a corresponding increase in pericyte coverage of vessels. The increased expression of desmin was associated with the induction of hypoxia‐induced factor (HIF‐1α) and the upregulation of vascular endothelial growth factor (VEGF). Inhibition of HIF‐1αactivity decreased VEGF expression in the vessels of the stria vascularis. Blocking VEGF activity significantly attenuated the expression of desmin in the pericytes. Taken together, these data demonstrate that cochlear pericytes were markedly affected by acoustic trauma and displayed an abnormal morphology. HIF‐1α activation and VEGF upregulation are important factors for the alteration of pericyte structural protein desmin.Supported by NIDCD DC 008888 and NIDCD DC 005983

Differential Effects of EGCG, a Green Tea Antioxidant, on Vascular Endothelial Growth Factor (VEGF) Expression and Proliferation in Cultured ER‐positive and ER‐negative Human Breast Cancer Cells

Estrogen receptor (ER)‐positive breast cancers have a better prognosis than ER‐negative breast cancers, which are more aggressive. The present study determines whether EGCG, a green tea antioxidant, exerts different effects on VEGF expression and proliferation in cultured ER‐positive (MCF‐7) and ER‐negative (MDA‐MB‐231) human breast cancer cells. MCF‐7 and MDA‐MB‐231 cells were cultured using RPMI 1640 media with 10% FBS. ELISA showed that VEGF and VEGF receptor‐2 were more highly expressed in ER‐negative cells than ER‐positive cells (&gt;8‐fold, P&lt;0.01; n=8). 3H‐thymidine incorporation showed that EGCG at 10, 25, and 50 µg/ml caused a dose‐related inhibition in the proliferation of MCF‐7 cells, by 29, 54, and 85%, compared to the control, respectively (P&lt;0.01). However, the same concentrations of EGCG did not inhibit the proliferation of MDA‐MB‐231 cells. EGCG at 50 µg/ml significantly decreased VEGF expression (1752±49 vs. 2254±91 pg/mg; P&lt;0.01) in cultured MCF‐7 cells, compared to the control. However, EGCG at 10 to 50 µg/ml did not inhibit VEGF expression in cultured MDA‐MB‐231 cells. These results suggest that highly expressed VEGF and its receptor may be involved in the aggressive malignant signaling systems of ER‐negative breast cancers, and that EGCG, a green tea antioxidant, can be used to treat ER‐positive human breast cancers, which are sensitive to EGCG. (HL51971 &amp; AA013821)

Suppression of Vascular Endothelial Growth Factor (VEGF) Expression by Targeting the Bcr-Abl Oncogene and Protein Tyrosine Kinase Activity in Bcr-Abl-positive Leukaemia Cells

Studies have shown that vascular endothelial growth factor (VEGF), a major and potent inducer of angiogenesis, is directly triggered by the disease-related oncogene Bcr-Abl in Bcr-Abl-positive cells. In this study, inhibition of Bcr-Abl tyrosine kinase activity by imatinib significantly decreased VEGF expression in Bcr-Abl-positive K562 cells in vitro. Imatinib treatment in vivo of nude mice xenografted with K562 cells resulted in a significant reduction in tumour size and microvessel density compared with untreated tumours. In addition, interfering with Bcr-Abl oncogene expression with small interfering RNAs (siRNAs) not only induced a specific reduction of Bcr-Abl mRNA and protein expression, but also efficiently inhibited the expression of VEGF in K562 cells. Combined treatment with imatinib and Bcr-Abl-targeting siRNAs resulted in an enhanced effect on VEGF suppression in K562 cells. The combined application of Bcr-Abl-targeting siRNAs and imatinib may provide a potent novel therapeutic approach for chronic myeloid leukaemia.

Oral picropodophyllin (PPP) is well tolerated in vivo and inhibits IGF‐1R expression and growth of uveal melanoma

The cyclolignan picropodophyllin (PPP) efficiently blocks the activity of insulin-like growth factor-1 receptor (IGF-1R) and inhibits growth of uveal melanoma cells in vitro and in vivo. In this study, we aimed to investigate the efficiency of orally administered PPP on growth of uveal melanoma xenografts. Further, we focused on the effect of PPP on vascular endothelial growth factor (VEGF) in vivo and evaluated its effects in combination with other established anti-tumor agents in vitro. Four different uveal melanoma cell lines (OCM-1, OCM-3, OCM-8, 92-1) were treated with PPP alone and in combination with imatinib mesylate, cisplatin, 5-FU and doxorubicin. Cell viability was determined by XTT assay. SCID mice xenografted with uveal melanoma cells were used to determine anti-tumor efficacy of oral PPP in vivo. Tumor samples obtained from the in vivo experiments were analyzed for VEGF and IGF-1R expression by western blotting. PPP was found to be superior to the other anti-tumor agents in killing uveal melanoma cells. Oral PPP inhibited uveal melanoma growth in vivo and was well tolerated by the animals. PPP decreased VEGF expression in the tumors. Oral PPP is well tolerated in vivo and caused total growth inhibition of uveal melanoma xenografts as well as it decreased the levels of VEGF in the tumors.