Although graphene oxide (GO) has recently been considered as a highly attractive nanomaterial for future cancer imaging and therapy, it is still a major challenge to improve its in vivo tumor active targeting efficiency. Here in this full article, we demonstrated the successful and significantly enhanced in vivo tumor vasculature targeting efficacy of well-functionalized GO nanoconjugates by using <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (VEGF<em>121</em>) as the targeting ligand. As-developed GO nanoconjugate exhibits excellent in vivo stability, specific in vitro and in vivo <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> receptor (VEGFR) targeting, significantly enhanced tumor accumulation (>8 %ID/g) as well as high tumor-to-muscle contrast, showing great potential for future tumor targeted imaging and therapy.

OBJECTIVE

Does vascular endothelial growth factor (VEGF) have important roles during early embryo development and implantation?

CONCLUSIONS

VEGF plays key roles during mouse preimplantation embryo development, with beneficial effects on time to cavitation, blastocyst cell number and outgrowth, as well as implantation rate and fetal limb development.

BACKGROUND

Embryo implantation requires synchronized dialog between maternal cells and those of the conceptus. Following ovulation, secretions from endometrial glands increase and accumulate in the uterine lumen. These secretions contain important mediators that support the conceptus during the peri-implantation phase. Previously, we demonstrated a significant reduction of VEGFA in the uterine cavity of women with unexplained infertility. Functional studies demonstrated that VEGF significantly enhanced endometrial epithelial cell adhesive properties and embryo outgrowth.

METHODS

Human endometrial lavages (n = 6) were obtained from women of proven fertility. Four-week old Swiss mice were superovulated and mated with Swiss males to obtain embryos for treatment with VEGF in vitro. Preimplantation embryo development was assessed prior to embryo transfer (n = 19-30/treatment group/output). Recipient F1 female mice (8-12 weeks of age) were mated with vasectomized males to induce pseudopregnancy and embryos were transferred. On Day 14.5 of pregnancy, uterine horns were collected for analysis of implantation rates as well as placental and fetal development (n = 14-19/treatment).

METHODS

Lavage fluid was assessed by western immunoblot analysis to determine the VEGF isoforms present. Mouse embryos were treated with either recombinant human (rh)VEGF, or VEGF isoforms 121 and 165. Preimplantation embryo development was quantified using time-lapse microscopy. Blastocysts were (i) stained for cell number, (ii) transferred to wells coated with fibronectin to examine trophoblast outgrowth or (iii) transferred to pseudo pregnant recipients to analyze implantation rates, placental and fetal development.

RESULTS

Western blot analysis revealed the presence of VEGF121 and 165 isoforms in human uterine fluid. Time-lapse microscopy analysis revealed that VEGF (n = 22) and VEGF121 (n = 23) treatment significantly reduced the preimplantation mouse embryo time to cavitation (P < 0.05). VEGF and VEGF165 increased both blastocyst cell number (VEGF n = 27; VEGF165 n = 24: P < 0.001) and outgrowth (n = 15/treatment: 66 h, P < 0.001; 74, 90, 98 and 114 h, P < 0.01) on fibronectin compared with control. Furthermore, rhVEGF improved implantation rates and enhanced fetal limb development (P < 0.05).

CONCLUSIONS

Due to the nature of this work, embryo development and implantation was only examined in the mouse.

CONCLUSIONS

The absence or reduction in levels of VEGF during the preimplantation period likely affects key events during embryo development, implantation and placentation. The potential for improvement of clinical IVF outcomes by the addition of VEGF to human embryo culture media needs further investigation.

BACKGROUND

This study was supported by a University of Melbourne Early Career Researcher Grant #601040, the NHMRC (L.A.S., Program grant #494802; Fellowship #1002028; N.J.H., Fellowship # 628927; J.E.; project grant #1047756) and L.A.S., Monash IVF Research and Education Foundation. N.K.B. was supported by an Australian Postgraduate Award. Work at PHI-MIMR Institute was also supported by the Victorian Government's Operational Infrastructure Support Program. There are no conflicts of interest to declare.

BACKGROUND

Vascular endothelial growth factor (VEGF) induces proliferation of endothelial cells (EC) in vitro and angiogenesis in vivo. Furthermore, a role of VEGF in K(+) channel, nitric oxide (NO) and Ca(2+) signaling was reported. We examined whether the K(+) channel blocker margatoxin (MTX) influences VEGF-induced signaling in human EC.

METHODS

Fluorescence imaging was used to analyze changes in the membrane potential (DiBAC), intracellular Ca(2+) (FURA-2) and NO (DAF) levels in cultured human EC derived from human umbilical vein EC (HUVEC). Proliferation of HUVEC was examined by cell counts (CC) and [(3)H]-thymidine incorporation (TI).

RESULTS

VEGF (5--50 ng/ml) caused a dose-dependent hyperpolarization of EC, with a maximum at 30 ng/ml (n=30, p<0.05). This effect was completely blocked by MTX (5 micromol/l). VEGF caused an increase in transmembrane Ca(2+) influx (n=30, p<0.05) that was sensitive to MTX and the blocker of transmembrane Ca(2+) entry 2-aminoethoxydiphenyl borate (APB, 100 micromol/l). VEGF-induced NO production was significantly reduced by MTX, APB and a reduction in extracellular Ca(2+) (n=30, p<0.05). HUVEC proliferation, examined by CC and TI, was significantly increased by VEGF and inhibited by MTX (CC: -58%, TI --121%); APB (CC --99%, TI--187%); N-monomethyl-L-arginine (300 micromol/l: CC: -86%, TI --164%).

CONCLUSIONS

VEGF caused an MTX-sensitive hyperpolarization which results in an increased transmembrane Ca(2+) entry that is responsible for the effects on endothelial proliferation and NO production.

The long-term safety and efficacy of adenoviral delivery of <em>growth</em> <em>factors</em> in patients with peripheral arterial disease (PAD) is unknown. CI-1023 (Ad(GV)VEGF(<em>121</em>.10)) is a replication-deficient adenovirus encoding human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> isoform <em>121</em>. In this phase I trial, we investigated the safety and efficacy of CI-1023 in subjects with advanced claudication symptoms secondary to infra-inguinal disease. Eighteen subjects >35 years of age with a median ankle brachial index (ABI) at rest of 0.525 (interquartile range 0.4) and angiographic disease involving the infra-inguinal vessels underwent intramuscular injection of CI-1023 (4 x10(8) to 4 x10(10) particle units, n = 15) or placebo (n = 3). Eleven of 15 patients (73%) who received CI-1023 and 1 of 3 subjects (33%) who received placebo, completed 1 year of follow-up. Edema and rash were the most common early adverse event. One infra-inguinal bypass procedure occurred in each of the placebo and CI-1023 groups at days 29 and 104, respectively. One death (day 160) and 1 malignancy (day 274) occurred in the CI-1023 group. Conclusions on efficacy could not be made due to the small number of patients. However, there were encouraging trends in ABI at rest and peak walking time at follow-up.

Neuropilin-1 (NRP-1) is present on the cell surface of <em>endothelial</em> cells, or as a soluble truncated variant. Membrane NRP-1 is proposed to enhance angiogenesis by promoting the formation of a signaling complex between <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-A(165) (VEGF-A(165)), VEGF receptor-2 (VEGFR-2) and heparan sulfate, whereas the soluble NRP-1 is thought to act as an antagonist of signaling complex formation. We have analyzed the angiogenic potential of a chimera comprising the entire extracellular NRP-1 region dimerized through an Fc IgG domain and a monomeric truncated NRP-1 variant. Both NRP-1 proteins stimulated tubular morphogenesis and cell migration in HDMECs and HUVECs. Fc rNRP-1 was able to induce VEGFR-2 phosphorylation and expression of the VEGFR-2 specific target, regulator of calcineurin-1 (RCAN1.4). siRNA mediated gene silencing of VEGFR-2 revealed that VEGFR-2 was required for Fc rNRP-1 mediated activation of the intracellular signaling proteins PLC-γ, AKT, and MAPK and tubular morphogenesis. The stimulatory activity was independent of VEGF-A(165). This was evidenced by depleting the cell culture of exogenous VEGF-A(165), and using instead for routine culture VEGF-A(<em>121</em>), which does not interact with NRP-1, and by the inability of VEGF-A sequestering antibodies to inhibit the angiogenic activity of the NRP proteins. Analysis of angiogenesis over a period of 6 days in an in vitro fibroblast/<em>endothelial</em> co-culture model revealed that Fc rNRP-1 could induce <em>endothelial</em> cell tubular morphogenesis. Thus, we conclude that soluble Fc rNRP-1 is a VEGF-A(165)-independent agonist of VEGFR-2 and stimulates angiogenesis in <em>endothelial</em> cells.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is an important regulator of angiogenesis. In vivo expression of four different VEGF isoforms, consisting of <em>121</em>, 165, 189 or 206 amino acids, has been found in the human organism, with all isoforms arising from a single gene by alternative mRNA splicing. We developed an assay for simultaneous quantitation of VEGF isoform expression using competitive polymerase chain reaction (PCR). RNA was isolated from cells, reverse transcribed to cDNA and coamplified with a synthetical competitor DNA using VEGF specific primers. Amplification products were analyzed electrophoretically and concentration of VEGF transcripts calculated. Concentration of housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts was quantitated as above, VEGF gene activity is presented as ratio VEGF mRNA to GAPDH mRNA. Using this assay, we were able to detect and quantitate in vivo expression of VEGF<em>121</em>, VEGF165 and VEGF189 by human peripheral blood mononuclear cells (PBMC). These are the first quantitative data of in vivo VEGF expression by PBMC, suggesting a role for them in the maintenance of the vasculature.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) promotes the repair of injured vessels by stimulating angiogenesis. VEGF/VPF reportedly has cytoprotective activity but no study has shown the protective effect of VEGF/VPF on glomerular endothelial cells. We examined whether recombinant VEGF/VPF121 and VEGF/VPF165 isoforms could prevent injury of glomerular endothelial cells. Mild glomerular injury was induced in rats by an intravenous-injection of a limited dose of anti-Thy-1.1 antibody to obtain lesions similar to those found in the human disease. Recombinant VEGF/VPF165, VEGF/VPF121 or BSA was administered 4 h before the injection of the antibody, and once daily for 3 days. In the BSA-injected rats, mesangial cell lysis and endothelial cell injury in dilated capillary tufts were evident without endothelial cell apoptosis on days 1-4. Thereafter, cell proliferation and repair began and remodeling of the glomeruli was completed by day 28. Macrophages but not polymorphonuclear leukocytes accumulated significantly in the glomeruli on days 1-4. Treatment with VEGF/VPF isoform protected endothelial cells but not mesangial cells from destruction on day 1, and accelerated the repair of both types of cells, which was completed by day 18, 10 days earlier than that of the control animals. The results indicate that VEGF/VPF121 or VEGF/VPF165 can protect glomerular endothelial cells against injury, independent of apoptosis-inhibition activity, thereby promoting reconstruction of glomeruli. The protective effect of VEGF/VPF on endothelial cells suggests that it could provide therapeutic benefit for certain kidney diseases.

Tumors arise initially as a<em>vascular</em> masses in which central hypoxia induces expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-A (VEGF-A) and subsequently tumor <em>vascular</em>ization. However, VEGF-A can also be constitutively expressed as a result of genetic events. VEGF-A is alternatively spliced to yield at least 6 different isoforms. Of these, VEGF-A(<em>121</em>) is freely diffusible whereas basically charged domains in the larger isoforms confer affinity for cell surface or extracellular matrix components. We previously reported that in a mouse brain metastasis model of human melanoma, VEGF-A(<em>121</em>) induced a qualitatively different tumor <em>vascular</em> phenotype than VEGF-A(165) and VEGF-A(189): in contrast to the latter ones, and VEGF-A(<em>121</em>) did not induce a neo<em>vascular</em> bed but rather led to leakage and dilatation of preexistent brain vessels. Here, we correlate <em>vascular</em> phenotypes with spatial VEGF-A expression profiles in clinical brain tumors (low grade gliomas; n = 6, melanoma metastases; n = 4, adenocarcinoma metastases; n = 4, glioblastoma multiforme; n = 3, sarcoma metastasis; n = 1, renal cell carcinoma metastasis; n = 1). We show that tumors that constitutively express VEGF-A present with different <em>vascular</em> beds than tumors in which VEGF-A is expressed as a response to central hypoxia. This phenotypic difference is consistent with a model where in tumors with constitutive VEGF-A expression, all isoforms exert their effects on vasculature, resulting in a classical angiogenic phenotype. In tumors where only central parts express hypoxia-induced VEGF-A, the larger angiogenic isoforms are retained by extracellular matrix, leaving only freely diffusible VEGF-A(<em>121</em>) to exert its dilatation effects on distant vessels.

OBJECTIVE

There is evidence that transient receptor potential canonical type 3 (TRPC3) cation channels are involved in the regulation of blood pressure, but this has not been studied using human renal tissue. We tested the hypothesis that the expression of TRPC3 in human renal tissue is associated with blood pressure in patients.

METHODS

TRPC3 was detected in cultured human <em>endothelial</em> cells and in <em>vascular</em> endothelium cells from human renal tissue by immunoblotting, immunohistochemistry, and quantitative real-time reverse transcriptase-PCR. The changes of TRPC3 and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> receptor type 2 expression in cultured human <em>endothelial</em> cells were measured after administration of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> isoform <em>121</em>.

RESULTS

In cultured human <em>endothelial</em> cells, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> isoform <em>121</em> significantly reduced TRPC3 expression by 57% and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> receptor type 2 by 70%. This reduction was partly blocked by phosphatidylinositol 3-kinase inhibitors, wortmannin, or LY294002. Downregulation of TRPC3 channel expression was associated with reduced calcium influx. The changes of calcium influx could be abolished by the inhibitor of TRPC channels, 2-aminoethoxydiphenylborane, pointing to their functional importance. TRPC3 expression was significantly higher in patients with SBP more than 140 mmHg compared with patients with SBP of 140 mmHg or less (0.00181 +/- 0.00059 versus 0.00037 +/- 0.00012 arbitrary units; P < 0.01).

CONCLUSIONS

The data support the hypothesis that TRPC3 expression in human renal tissue including vascular endothelium is associated with blood pressure regulation in humans.

OBJECTIVE

To evaluate the molecular responses of vascular endothelial growth factor (VEGF) and its receptors to dexamethasone (Dex) and celecoxib (Cel) during hyperoxia and during hyperoxia followed by recovery in room air, in newborn rabbit retinas.

METHODS

Newborn rabbits at 3 days postnatal age (n = 96) received room air or oxygen (80%-100%) for 4 days, during which they were administered saline (Sal), Dex, vehicle (Veh), or Cel (n = 12/treatment group). Six animals from each group were killed immediately after hyperoxia (or room air) and the remainder exposed to room air for 5 days. Retinal mRNA expression of VEGF(121), VEGF(165), VEGF receptor-1 (VEGFR-1, or Flt-1), and VEGFR-2 (or KDR/Flk-1) was determined.

RESULTS

Hyperoxia resulted in increased retinal expression of mRNA of the VEGF splice variants in the groups treated with Sal, Dex, and Veh, whereas a decrease in VEGF(121) was noted in the Cel-treated group. In contrast, retinal Flt-1 receptor mRNA was markedly increased in the Cel-treated group only, whereas retinal VEGFR-2 (KDR/Flk-1) receptor mRNA was suppressed in all the treatment groups. Hyperoxia followed by recovery in room air resulted in a minimal decrease in expression of retinal Flt-1 mRNA in the Sal and Dex groups. Cel treatment abolished its expression.

CONCLUSIONS

The findings of increased retinal expression of VEGF mRNA in the newborn rabbit in response to hyperoxia are most likely due to species differences. Selective targeting of VEGF(121) and Flt-1 mRNA by Cel may represent one regulatory pathway for their anti-inflammatory effects. Further studies are needed to evaluate the therapeutic benefits of cyclooxygenase (COX)-2 inhibitors for the treatment and/or prevention of diseases associated with neovascularization.

BACKGROUND

Therapeutic angiogenesis is a new approach to treating ischemic heart disease, and the optimal method for assessing its efficacy is unclear. We used myocardial contrast echocardiography (MCE) to evaluate the therapeutic response to the angiogenic agent, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-<em>121</em> (VEGF<em>121</em>).

RESULTS

After placement of an ameroid constrictor (day 0) around the left anterior descending artery (LAD), dogs were given intracoronary VEGF<em>121</em> protein (108 microg, n=6) or placebo (n=6) on days 7 and 21, and subcutaneous VEGF<em>121</em> (1 mg) or placebo on days 8 to 20 and 22 to 27. On day 48, MCE was performed during rest and dobutamine stress. Videointensity (y) and pulsing interval (t) were fit to an exponential model (y=A[1-e(-beta(t))]) used to derive indices of red cell velocity (beta) and capillary area (A), and parameters were compared with radiolabeled microsphere flow data. VEGF(<em>121</em>) treatment resulted in higher resting left anterior descending artery/left circumflex flow ratio compared with placebo (P<0.03) and improved collateral flow reserve. Beta was 0.94+/-0.37 in VEGF<em>121</em> dogs versus 0.38+/-0.31 in controls (P<0.02), with the greatest difference in the endocardium. The parameter A was comparable in both groups, suggesting that micro<em>vascular</em> changes did not alter capillary cross-sectional area, and histology indicated a trend toward higher arteriolar density in VEGF<em>121</em>-treated animals.

CONCLUSIONS

VEGF<em>121</em> protein improves collateral flow and reserve. MCE can evaluate the transmural location and structural and functional responses of the microvasculature to angiogenic interventions.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), also known as <em>vascular</em> permeability <em>factor</em> (VPF), is an angiogenic <em>factor</em> that plays important roles in tumor <em>growth</em>. Angiogenesis studies on VEGF deal with various types of malignant tumors, but very little is known about the role or significance of VEGF in human thyroid neoplasms. Therefore, in the current study, we determined whether VEGF is found in normal and neoplastic thyroids and whether its expression is altered in different histological types of thyroid neoplasms. Reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that all specimens of thyroid tumors expressed bands corresponding to <em>121</em>-, 165-, and 189-amino acid forms of VEGF. Northern blot analysis showed an increase in VEGF mRNA levels in neoplastic tissues in comparison with normal thyroid samples. By nonisotopic in situ hybridization, most of the tumor cells in follicular adenomas expressed VEGF mRNA, whereas VEGF mRNA expression was identified only in epithelium of isolated follicles in normal thyroid tissues. In papillary thyroid carcinomas, an intense labeling with VEGF probe was often found in overlying tumor cells of neoplastic papillae. VEGF expression was distinctly intensified in undifferentiated carcinoma cells that were immediately adjacent to necrotic foci. The immunohistochemical localizations of VEGF protein were comparable to the localization of VEGF mRNA. In conclusion, our results suggest that the histological types of thyroid tumor may determine the <em>vascular</em> pattern through a paracrine mechanism involving VEGF.

The production of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factors</em> (VEGF), a major cause of neoangiogenesis, is a prerequisite for tumor <em>growth</em> and invasion. VEGF have also been shown to be important for the formation of osteoclasts. Because giant cell tumors of bone (GCT) are frequently hyper<em>vascular</em> and have the ability to recruit macrophages and multinucleated osteoclast-like giant cells, we evaluated the levels of VEGF gene transcript in several of these tumors using Northern blot analyses, semiquantitative reverse transcription polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), and immunohistochemistry. Our results showed that three major isoforms of VEGF (<em>121</em>, 165, and 189) were expressed in all cases of GCT investigated, with isoform <em>121</em> transcripts the most abundant. By both FISH and immunohistochemistry, we have shown that VEGF was present in spindle-shaped stromal-like tumor cells, round macrophage-like cells, and osteoclast-like multinucleate giant cells. Moreover, we have shown that the levels of VEGF gene expression but not microvessel density correlated with Enneking's clinical stage of GCT. There were higher levels of VEGF gene expression in stage III GCT than in stage I/II GCT (P < .0357). In conclusion, our results indicate that overexpression of VEGF may be associated with the advanced stage of the neoplasm.

Angiogenesis is stimulated by <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) acting via <em>endothelial</em> cell-specific receptors, such as VEGFR-2, that are overexpressed at the sites of angiogenesis. If VEGF retains activity as a fusion protein with a large N-terminal extension, it would facilitate development of VEGF-based vehicles for receptor-mediated delivery of therapeutic and diagnostic agents to the sites of angiogenesis. We have constructed, expressed in Escherichia coli, and purified VEGF fusion proteins containing a 158-amino acid N-terminal extension fused to human VEGF(<em>121</em>), VEGF(165), and VEGF(189). We report here that VEGF fusion proteins induce tyrosine autophosphorylation of VEGFR-2 and its downstream targets, as well as cell contraction in cells overexpressing VEGFR-2. Although N-terminal extensions decrease the affinity of VEGF fusion proteins to VEGFR-2, at saturating concentrations these proteins are as efficient as correct size VEGF(165). We hypothesize that VEGF fusion proteins may be employed for targeting <em>endothelial</em> cells at the sites of angiogenesis.

Angiogenesis plays an important role in bone development and postnatal bone fracture repair. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> receptors (VEGFRs) are primarily involved in angiogenesis. This study investigated the expression of VEGF isoforms, VEGFR-1, and VEGFR-2 during the osteoblastic differentiation of cultured human periosteal-derived cells. In addition, the effect of exogenous VEGF on the osteoblastic differentiation of cultured human periosteal-derived cells was also examined. The expression of the VEGF isoforms (VEGF(<em>121</em>), VEGF(165), VEGF(189), and VEGF(206)), VEGFR-1, and VEGFR-2 was observed in the periosteal-derived cells. Administration of KRN633, a VEGFR-1 and VEGFR-2 inhibitor, decreased the alkaline phosphatase (ALP) activity during the osteoblastic differentiation of cultured human periosteal-derived cells. However, the administration of VEGFR2 Kinase Inhibitor IV, a VEGFR-2 inhibitor, did not affect the ALP activity. The addition of recombinant human VEGF(165) elevated the ALP activity and increased the calcium content in the periosteal-derived cells. Treating the periosteal-derived cells with recombinant human VEGF(165) resulted in an increase in Runx2 transactivation in the periosteal-derived cells. These results suggest that exogenous VEGF stimulates the osteoblastic differentiation of cultured human periosteal-derived cells and VEGF might act as an autocrine <em>growth</em> <em>factor</em> for the osteoblastic differentiation of cultured human periosteal-derived cells.

<em>Vascular</em> permeability <em>factor</em>, also known as <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VPF/VEGF), is a disulfide-linked dimeric glycoprotein of about 40 kDa that enhances <em>vascular</em> permeability, induces chemotaxis and activation of monocytes/macrophages, and promotes <em>growth</em> of <em>vascular</em> <em>endothelial</em> cells. It has been reported that several tumor cell lines and normal cells produce VPF/VEGF. To examine the possibility of VPF/VEGF mRNA expression in human peripheral T cells and its mechanism(s) of regulation, we developed a non-radioisotopic semiquantitative reverse transcription-polymerase chain reaction (RT-PCR; VPF/VEGF, GAPDH co-amplification) assay which detects all species of VPF/VEGF mRNA alternative splicing products. T cells expressed negligible VPF/VEGF mRNA in the resting state. However, TPA markedly enhanced the expression of <em>121</em>-, 165- and 189-amino-acid-containing isoforms of VPF/VEGF mRNA in T cells. Both CD4+ and CD8+ T cells expressed VPF/VEGF mRNA in response to TPA treatment. Moreover, T cell receptor stimulation with monoclonal anti-CD3 antibody with or without IL-1 beta enhanced VPF/VEGF mRNA expression in T cells. These findings suggest that T cell activation induces VPF/VEGF expression in the cells, resulting in induction of its biological activities.

Vasoactive intestinal peptide (VIP) upregulates the expression of <em>vascular</em> <em>endothelial</em> cell <em>growth</em> <em>factor</em> (VEGF(189), VEGF(165) and VEGF(<em>121</em>)) mRNAs in human prostate cancer LNCaP cells, as shown by reverse transcriptase-polymerase chain reaction (RT-PCR). Real-time RT-PCR indicated that the effect was maximal by 1-2 h and must be accounted for increased transcription since VIP decreased VEGF(165) mRNA stability. VIP stimulated VEGF(165) protein synthesis as measured by ELISA. VIP regulation of VEGF expression was mediated by VPAC(1) receptor and was cAMP/protein kinase A (PKA) dependent. Phosphoinositide 3-kinase (PI3-K) and mitogen-activated protein kinase MEK1/2 systems may also be involved as shown with specific kinase inhibitors. These actions together with the observation of VIP-induced neuroendocrine differentiation in LNCaP cells suggest a proangiogenic potential of VIP in prostate cancer.

BACKGROUND

Adrenomedullin is a potent vasodilatory and hypotensive peptide as well as an endogenous immunomodulatory factor with predominantly anti-inflammatory effects. The purpose of the present study was to evaluate the therapeutic effects of adrenomedullin in rabbits with antigen-induced arthritis, an experimental model of rheumatoid arthritis.

METHODS

Following the induction of arthritis in both knee joints by ovalbumin injection into the joint spaces of pre-immunized rabbits, increasing daily doses of adrenomedullin were injected into the knee joint spaces or saline was injected into the contralateral knee joint spaces as the control. For time-course experiments, adrenomedullin and saline were injected into the knee joint spaces daily for 7 days and 20 days. The degree of joint swelling and the histological change in the knee joints injected with adrenomedullin were compared with the control knee joints. Histological evaluation of the infrapatellar fat pads and synovial tissue was performed. TNFalpha, IL-6, vascular endothelial growth factor and transforming growth factor-beta mRNA levels in the synovial tissue were measured using real-time quantitative PCR.

RESULTS

Daily injections of adrenomedullin into the knee joint spaces of rabbits with antigen-induced arthritis decreased joint swelling. Histological examination revealed that adrenomedullin reduced edematous changes and the infiltration of inflammatory cells in the synovial tissues. Analysis of mRNA levels showed that adrenomedullin significantly reduced TNFalpha mRNA expression by 21% to 49% in a dose-dependent manner, and dose-dependently increased IL-6 mRNA expression by 45% to 121%.

CONCLUSIONS

These results suggest that daily injections of adrenomedullin into the knee joint spaces of rabbits with antigen-induced arthritis ameliorated the inflammatory response in arthritic joints. Adrenomedullin may thus be useful as a treatment for rheumatoid arthritis; however, the effect of adrenomedullin on IL-6 production in the synovial tissue may be an undesirable adverse effect in rheumatoid arthritis therapy.

OBJECTIVE

Tissue regeneration requires both <em>growth</em> <em>factor</em> and extracellular matrix such as collagen, serving as a scaffold for cell <em>growth</em>. We established FNCBD-VEGF<em>121</em>, consisting of the fibronectin collagen-binding domain (FNCBD) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) <em>121</em>, and investigated its properties.

RESULTS

FNCBD-VEGF<em>121</em> specifically bound to gelatin and type I, II, III, IV, and V collagen. This collagen-bound FNCBD-VEGF<em>121</em> captured soluble VEGF receptor 2 (VEGFR-2)/Fc chimeric protein. Cell <em>growth</em>-promoting activity of FNCBD-VEGF<em>121</em> was almost identical to that of VEGF<em>121</em>. The VEGF fusion protein significantly enhanced the expression of VEGFR-2 (71.6+/-0.8%) on <em>endothelial</em> progenitor cells (EPCs) derived from umbilical cord blood. Expectably, the collagen-bound VEGF fusion protein not only promoted the <em>growth</em> of <em>endothelial</em> cells (ECs) but also induced the expression of VEGFR-2 (63.7+/-0.8%) on non-adherent cells expanded from bone marrow CD34+ cells. Moreover, the VEGF fusion protein enhanced sprout formation of ECs in a matrigel model. In vivo experiments revealed that FNCBD-VEGF<em>121</em> had local effects but not systemic effect on EPC mobilization.

CONCLUSIONS

These results suggest that FNCBD-VEGF<em>121</em> stably maintains an optimally high and local concentration of VEGF with collagen matrix and stimulates both ECs and EPCs in situ, supplying a <em>vascular</em> regeneration niche.

BACKGROUND

The aim of this study is to evaluate whether the administration of renin-angiotensin system (RAS) inhibitors, angiotensin-I converting enzyme inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs), is associated with treatment outcome in patients with esophageal squamous cell carcinoma.

METHODS

A total of 141 esophageal squamous cell carcinoma patients receiving esophagectomy were identified, and were divided into two groups: an ACEI/ARB group (n=20), and a non-ACEI/ARB group (n=<em>121</em>). The effect of ACEIs or ARBs on cell proliferation and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) secretion of esophageal squamous cell carcinoma cell lines, CE81T/VGH and TE2, were investigated by 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Enzyme-linked immunosorbent assay (ELISA), respectively.

RESULTS

Use of ACEI/ARB (p=0.032), pathologic T stage (p<0.001), pathologic N stage (p=0.012), tumor stage (p=0.006), and tumor location (p=0.032) were significantly associated with superior overall survival. In multivariate comparison, use of ACEI/ARB (p=0.006), tumor stage (p=0.002), and tumor location (p=0.014) represented the independent prognosticators of superior overall survival. In cell lines, ACEIs/ARBs inhibit cell proliferation and VEGF secretion in a dose-dependent manner.

CONCLUSIONS

ACEIs/ARBs administration is independently associated with superior overall survival in patients with esophageal squamous cell carcinoma receiving esophagectomy. Our data support further investigation of the role of RAS inhibitors as a potential therapy in esophageal squamous cell carcinoma.

<em>Vascular</em> participation manifested by a runny nose (rhinorrhea) is a prominent feature of the acute consequences of rhinovirus infection. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is an angiogenic <em>factor</em> that also induces potent increases in <em>vascular</em> permeability; it is a candidate mediator of rhinorrhea in response to rhinovirus infection as well as contributing to enhanced <em>vascular</em> leakage in rhinovirus-linked asthma exacerbations. It has been shown that rhinovirus induces significant increases in both VEGF protein and mRNA in primary airway fibroblasts [Ghildyal et al. (2005): J Med Virol 75:608-615]. The current studies assessed VEGF responses to rhinovirus in primary culture airway epithelium, in epithelial and fibroblast cell lines and in rhinovirus-infected nasal secretions. Epithelial and fibroblast cells were infected with rhinovirus serotype 16 and VEGF protein and isoforms assessed by ELISA and RT-PCR, respectively. VEGF protein was released by both epithelial and fibroblast cell lines and primary airway epithelial cells in culture but was not increased following rhinovirus infection. PCR products coding for four or five of the six known VEGF isoforms were produced (<em>121</em>, 145, 165 and 183, and/or 189 amino acids) in cell lines and primary culture cells, but no specific isoform was linked to rhinovirus infection. Nasal VEGF was also measured in a cohort of asthmatics with verified rhinovirus and respiratory syncytial virus (RSV) infection. VEGF was not raised following rhinovirus infection alone, but was increased significantly if concomitant RSV infection was present. The data suggest that fibroblasts rather than the epithelium may play a key role in VEGF mediated <em>vascular</em> responses after rhinovirus infection. This may aid recruitment of inflammatory cells and contribute to airway inflammation and bronchial obstruction.

Vascular endothelial growth factor (VEGF) is a glycoprotein that plays an important role in neovascularization and increases vascular permeability. We reported that VEGF is involved in motion pain of patients with rotator cuff disease by causing synovial proliferation in the subacromial bursa (SAB). The present study investigates whether VEGF is also involved in the development of shoulder contracture in diabetics with rotator cuff disease. We examined 67 patients with rotator cuff disease, including 36 with complete cuff tears, 20 with incomplete tears, and 11 without apparent tears (subacromial bursitis). The patients were into groups according to the presence or absence of diabetes (14 type II diabetics and 53 non-diabetics). Specimens of the synovium of the SAB were obtained from all patients during surgery. Expression of the VEGF gene in the synovium of the subacromial bursa was evaluated by using the reverse transcriptase polymerase chain reaction. The VEGF protein was localized by immunohistochemistry, and the number of vessels was evaluated based on CD34 immunoreactivity. The results showed that VEGF mRNA was expressed in significantly more diabetics (100%, 14/14) than in non-diabetics (70%, 37/53) (P=0.0159, Fisher's test). Investigation of VEGF isoform expression revealed VEGF121 in all 14 diabetics and in 37 of the 53 non-diabetics, VEGF165 in 12 of the 14 diabetics and in 21 of the 53 non-diabetics, and VEGF189 in 1 of the 14 diabetics and in 2 of the 53 non-diabetics. No VEGF206 was expressed in either group. VEGF protein was localized in both vascular endothelial cells and synovial lining cells. The mean number of VEGF-positive vessels and the vessel area were also significantly greater in the diabetics (p<0.015, Mann-Whitney U test). Synovial proliferation and shoulder joint contracture were more common in the diabetics (P=0.0329 and P=0.073, respectively; Fisher's test). The mean preoperative range of shoulder motion significantly differed in terms of elevation between two groups: 103.8 degrees in diabetics and 124.9 degrees in no diabetics (p=0.0039 Mann-Whitney U test). In contrast, external rotation did not significantly differ: 44 degrees in diabetics and 49 degrees in non-diabetics (p=0.4957, Mann-Whitney U test). These results suggest that VEGF121 and VEGF165 expression in the SAB is responsible for the development of shoulder joint contracture, especially in elevation, among type II diabetic patients with rotator cuff disease.

The pathophysiology of tumor <em>growth</em> following skeletal metastases and the poor response of this type of lesion to therapeutic intervention remains incompletely understood. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)-A and its receptors play a role in both osteoclastogenesis and tumor <em>growth</em>. Systemic (i.v.) treatment of nude mice bearing intrafemoral prostate (PC-3) tumors with the <em>vascular</em> ablative agent VEGF(<em>121</em>)/recombinant gelonin (rGel) strongly inhibited tumor <em>growth</em>. Fifty percent of treated animals had complete regression of bone tumors with no development of lytic bone lesions. Immunohistochemical analysis showed that VEGF(<em>121</em>)/rGel treatment suppressed tumor-mediated osteoclastogenesis in vivo. In vitro treatment of murine osteoclast precursors, both cell line (RAW264.7) and bone marrow-derived monocytes (BMM), revealed that VEGF(<em>121</em>)/rGel was selectively cytotoxic to osteoclast precursor cells rather than mature osteoclasts. VEGF(<em>121</em>)/rGel cytotoxicity was mediated by Flt-1, which was down-regulated during osteoclast differentiation. Analysis by flow cytometry and reverse transcription-PCR showed that both BMM and RAW264.7 cells display high levels of Flt-1 but low levels of Flk-1. Internalization of VEGF(<em>121</em>)/rGel into osteoclast precursor cells was suppressed by pretreatment with an Flt-1 neutralizing antibody or by placenta <em>growth</em> <em>factor</em> but not with an Flk-1 neutralizing antibody. Thus, VEGF(<em>121</em>)/rGel inhibits osteoclast maturation in vivo and it seems that this process is important in the resulting suppression of skeletal osteolytic lesions. This is a novel and unique mechanism of action for this class of agents and suggests a potentially new approach for treatment or prevention of tumor <em>growth</em> in bone.

BACKGROUND

Polyethylenimine (PEI)-based nonviral gene-delivery systems are commonly employed because of their high transfection efficiency. However, the toxic nature of PEI is a significant obstacle in clinical gene therapy. In this study, we developed biocompatible glycerol triacrylate-spermine (GT-SPE) polyspermine as a nanosized gene carrier for potential lung cancer gene therapy.

METHODS

The GT-SPE was synthesized using the Michael addition reaction between GT and SPE. The molecular weight was characterized using gel permeability chromatography multiangle laser light scattering and the composition of the polymer was analyzed using proton nuclear magnetic resonance.

RESULTS

The GT-SPE successfully protected the DNA from nucleases. The average particle size of the GT-SPE was <em>121</em> nm with a zeta potential of +23.45 mV. The GT-SPE was found to be less toxic than PEI for various cell lines, as well as for a murine model. Finally, our results showed that the GT-SPE/small hairpin Akt1 (shAkt1) complex suppressed lung tumorigenesis in a K-ras(LA1) lung cancer mice model by inducing apoptosis through the Akt signaling pathway and cell cycle arrest. Aerosol delivered GT-SPE/shAkt1, which reduced matrix metalloproteinase-9 activity and suppressed the expression levels of proliferating cell nuclear antigen, as well as <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factors</em> and CD31, which are known proliferation and angiogenesis markers, respectively.

CONCLUSIONS

Our data suggest that GT-SPE may be a candidate for short hairpin-shaped RNA-based aerosol lung cancer gene therapy.