SSeCKS, a Src-suppressed protein kinase C substrate with metastasis suppressor activity, is the rodent orthologue of human gravin/AKAP12, a scaffolding protein for protein kinase A and protein kinase C. We show here that the tetracycline-regulated reexpression of SSeCKS in MatLyLu (MLL) prostate cancer cells suppressed formation of macroscopic lung metastases in both spontaneous and experimental models of in vivo metastasis while having minimal inhibitory effects on the <em>growth</em> of primary-site s.c. tumors. SSeCKS decreased angiogenesis in vitro and in vivo by suppressing <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) expression in MLL tumor cells as well as in stromal cells. The forced reexpression of VEGF(165) and VEGF(<em>121</em>) isoforms was sufficient to reverse aspects of SSeCKS metastasis-suppressor activity in both the experimental and spontaneous models. SSeCKS reexpression in MLL cells resulted in the down-regulation of proangiogenic genes, such as osteopontin, tenascin C, KGF, angiopoietin, HIF-1alpha, and PDGFRbeta, and the up-regulation of antiangiogenic genes, such as vasostatin and collagen 18a1, a precursor of endostatin. These results suggest that SSeCKS suppresses formation of metastatic lesions by inhibiting VEGF expression and by inducing soluble antiangiogenic <em>factors</em>.

Primary effusion lymphomas (PELs), which are rare lymphomas associated with Kaposi's sarcoma-associated herpesvirus (or human herpesvirus-8) infection, present as malignant lymphomatous effusions in body cavities. Because PELs prefer liquid <em>growth</em>, we hypothesized that increased <em>vascular</em> permeability would be required for effusions to form. We found that the PEL cell lines BC-1, BCP-1, and BCBL-1 produce high levels of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>/<em>vascular</em> permeability <em>factor</em> (VEGF/VPF). Reverse transcriptase-polymerase chain reaction analysis of RNA from the PEL cell lines amplified the 3 VEGF-secreted isoforms: VEGF/VPF(<em>121</em>), VEGF/VPF(145), and VEGF/VPF(165). Two of the PEL cell lines expressed the VEGF/VPF receptor Flt-1, but VEGF/VPF did not stimulate proliferation in these cells. Most (13/14) control SCID/beige mice inoculated intraperitoneally with BCBL-1 cells and subsequently observed or treated with control antibodies developed effusion lymphoma of human cell origin with prominent bloody ascites. In contrast, none (0/9) of the mice treated with a neutralizing antihuman VEGF/VPF antibody developed ascites and effusion lymphoma. These results demonstrate that VEGF/VPF is critical to BCBL-1 <em>growth</em> as effusion lymphoma in mice and suggest that VEGF/VPF stimulation of <em>vascular</em> permeability may be critical to the pathogenesis of PELs.

<em>Vascular</em> permeability <em>factor</em>, or <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VPF/VEGF) is a disulfide-linked dimeric glycoprotein of about 40 kD that promotes fluid and protein leakage from blood vessels. Various human tumor cell lines and cells including fetal <em>vascular</em> smooth muscle cells produce VPF/VEGF. Since glomerular mesangial cells (MC) are closely related to <em>vascular</em> smooth muscle cells, we examined whether cultured human MC produce VPF/VEGF. Northern blotting analysis revealed that cultured human MC expressed a 3.7 kilobases (kb) VPF/VEGF mRNA. Human peripheral blood mononuclear cells (PBMC) also expressed VPF/VEGF transcripts of 8.6 and 3.8 kb. Although the sizes of the transcripts suggested the existence of unique molecular species of VPF/VEGF mRNA in PBMC, RT-PCR analysis revealed that PBMC as well as human MC expressed <em>121</em>, 165, and 189 amino acid-containing isoforms of VPF/VEGF, implying that there are no unique alternative splicing products of VPF/VEGF mRNA in PBMC. Fetal calf serum and 12-o-tetradecanoyl- phorbol-13-acetate (TPA) transiently enhanced VPF/VEGF mRNA expression in cultured human MC. Transforming <em>growth</em> <em>factor</em>-beta 1 enhanced VPF/VEGF mRNA expression in cultured human MC at least within 24 hours. Dexamethasone (DEX) inhibited the TPA-induced increase in VPF/VEGF mRNA expression, whereas DEX did not change the basal level. The DEX depressed the TPA-induced increase in VPF/VEGF mRNA expression is therefore probably a result of transcriptional control. VPF/VEGF protein was detected in cultured human MC with immunoperoxidase staining using anti-VPF/VEGF antibody. TPA increased VPF/VEGF protein levels as well as those of VPF/VEGF mRNA in cultured human MC.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoxia is one of the major signals that induces angiogenesis. Hypoxic conditions lead to reduced extracellular pH. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) binding to <em>endothelial</em> cells and the extracellular matrix (ECM) increases at acidic pH (7.0-5.5). These interactions are dependent on heparan sulfate proteoglycans, but do not depend on the presence of VEGF receptors. Here we report that VEGF(165) and VEGF(<em>121</em>) binding to fibronectin also increased at acidic pH, and that these interactions are further enhanced by the addition of heparin. These results reveal that the accepted non-heparin-binding isoform of VEGF (VEGF(<em>121</em>)) is converted into a heparin-binding <em>growth</em> <em>factor</em> under acidic conditions. Interestingly, we did not observe increased binding of VEGF to collagen type I at acidic pH in the presence or absence of heparin, indicating that this effect is not a general property of all heparin-binding ECM proteins. The high level of VEGF binding at acidic pH was also rapidly reversed as demonstrated by increased rates of VEGF dissociation from fibronectin and fibronectin-heparin matrices as the pH was raised. The VEGF released from fibronectin retained its ability to stimulate the activation of extracellular-regulated kinase 1/2 in <em>endothelial</em> cells. These results suggest that VEGF may be stored in the extracellular matrix via interactions with fibronectin and heparan sulfate in tissues that are in need of vascularization so that it can aid in directing the dynamic process of <em>growth</em> and migration of new blood vessels.

The <em>121</em>-amino acid form of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF<em>121</em>) and the 165-amino acid form (VEGF165) are mitogenic for <em>vascular</em> <em>endothelial</em> cells and induce angiogenesis in vivo. VEGF165 possesses a heparin binding ability and in the absence of heparin-like molecules does not bind efficiently to the VEGF receptors of <em>vascular</em> <em>endothelial</em> cells. The binding of 125I-VEGF165 to the VEGF receptors of <em>endothelial</em> cells, and the heparin-dependent binding of 125I-VEGF165 to a soluble extracellular domain of the VEGF receptor KDR/flk-1, were inhibited by the angiogenesis inhibitor platelet <em>factor</em>-4 (PF4). In contrast, PF4 was not able to inhibit the binding of VEGF<em>121</em>, a VEGF isoform which lacks a heparin binding capacity, to the VEGF receptors of the cells or to KDR/flk-1. These results indicate that PF4 may inhibit VEGF165 binding to VEGF receptors by disrupting the interaction of VEGF165 with cell surface heparan sulfates. Since PF4 mutants lacking a heparin binding ability retain their anti-angiogenic activity, alternative inhibitory mechanisms were also examined. 125I-PF4 bound with high affinity (Kd 5 x 10(-9) M) to VEGF165-coated wells. The binding of 125I-PF4 to the VEGF165-coated wells was inhibited by several types of heparin binding proteins, including unlabeled PF4 and unlabeled VEGF165. The binding was not inhibited by proteins which lack a heparin binding capacity, nor was it inhibited by VEGF<em>121</em>. Heparinase did not inhibit the binding of 125I-PF4 to VEGF165, indicating that heparin-like molecules are not required. These experiments suggest that PF4 can bind to heparin binding proteins such as VEGF165 leading to an inhibition of their receptor binding ability. In agreement with these results, we have observed that PF4 inhibits efficiently the VEGF165 induced proliferation of <em>vascular</em> <em>endothelial</em> cells. Unexpectedly, PF4 also inhibited efficiently the VEGF<em>121</em>-induced proliferation of the cells, indicating that PF4 can disrupt VEGF receptor mediated signal transduction using an unknown mechanism which does not interfere with VEGF<em>121</em> binding.

Overexpression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> receptors (VEGFRs) has been reported in a variety of tumor types. Here we find that 11 out of the 14 bladder tumor cell lines examined express one or more VEGF receptors. Analysis of the T24 bladder tumor cell line reveals a functional autocrine loop involving VEGF and the Flk-1 receptor. Blocking VEGF expression in T24 cells results in a decrease in DNA synthesis. The Flk-1 receptor in T24 cells is phosphorylated in response to VEGF-<em>121</em> or VEGF-165, and an Flk-1 inhibitor blocks VEGF to ERK signaling. We report that VEGF stimulation of T24 cells results in activation of H- and N-Ras and this is dependent on cellular sphingosine kinase 1 (SPK1) activity. Previously, we found VEGF-induced activation of Ras appears to be independent of a Ras-guanine nucleotide exchange <em>factors</em> (GEFs). Here we report that sphingosine can stimulate Ras-GTPase activating protein (GAP) activity in vitro, and sphingosine-1-phosphate (SPP) can block the stimulatory effects of sphingosine. We present a model where the balance between sphingosine and SPP regulates Ras-GAP activity such that stimulation of SPK1 favors downregulation of Ras-GAP and thereby the activation of Ras proteins. These data highlight a VEGF pathway that may be involved in the survival and proliferation of bladder tumor cells as well as other tumor cell types.

Angiogenesis is an indispensable process in the chronic proliferative synovitis and pannus formation of rheumatoid arthritis (RA). This study examined the expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) isoforms and VEGF receptors, Flt-1, KDR and neuropilin-1, in RA and osteoarthritis (OA) synovia, and studied the relationship between their expression and the synovial angiogenesis. By RT-PCR analysis, the isoform VEGF(<em>121</em>) was constitutively expressed in all the RA (17/17 patients) and OA (8/8 patients) synovia. In contrast, the expression of the isoform VEGF(165) was observed in 41% of the RA synovia (7/17 patients), but was undetectable in the OA samples (0/8 patients). The receptor Flt-1 was almost constitutively expressed in RA (15/17 patients) and OA (8/8 patients) synovia, while the expression of KDR was detected in the synovia of six RA patients (6/17 patients; 35%) but none of the OA patients (0/8 patients). The expression of neuropilin-1, an isoform-specific receptor for VEGF(165) which enhances the binding of VEGF(165) to KDR, was also up-regulated in the same RA synovia that expressed KDR. Furthermore, there was a close correlation between the expression of isoform VEGF(165) and that of its receptors KDR and neuropilin-1. Morphometric analysis demonstrated that the <em>vascular</em> density is significantly higher in the RA synovial tissues with expression of VEGF(165), KDR, and neuropilin-1 than in those without their expression (p<0.01). In situ hybridization and immunohistochemical studies indicated that the cells expressing VEGF are macrophage-like synovial lining cells and spindle-shaped cells in the sublining cell layer. These results suggest that the selective up-regulation of the isoform VEGF(165) and its signalling via KDR and neuropilin-1 play an important role in the synovial angiogenesis which occurs in RA.

Neuropilin-1 (Npn-1) is a receptor shared by class 3 semaphorins and heparin-binding forms of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), protein families that regulate <em>endothelial</em> and neuronal-cell function. Ligand interaction with Npn-1 dictates the choice of signal transducer; plexins transduce semaphorin signals, and VEGF receptors transduce VEGF signals. It is not clear how class 3 semaphorins affect <em>endothelial</em>-cell function and how the shared receptor Npn-1 selects its ligand. We report that semaphorin3A (Sema3A) inhibits <em>endothelial</em>-cell lamellipodia formation, adhesion, survival, proliferation, and cord formation. VEGF(165), but not VEGF(<em>121</em>), could block all these effects of Sema3A. VEGF(165) competed with Sema3A for binding to <em>endothelial</em> cells, effectively reduced cell-surface Npn-1, and promoted its internalization. Use of soluble forms of Npn-1 or VEGF receptor-1 to block VEGF(165) binding to Npn-1 or to VEGF receptors provided evidence that surface Npn-1 and VEGF receptors are required for VEGF(165)-induced Npn-1 internalization. Sema3A also reduced cell-surface Npn-1 in <em>endothelial</em> cells and promoted its internalization, but required a higher concentration than VEGF(165). These results demonstrate that preferential receptor binding and internalization by a ligand are mechanisms by which the common receptor Npn-1 can play an essential role in prioritizing conflicting signals.

BACKGROUND

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-<em>121</em> (VEGF<em>121</em>), an angiogenic protein secreted in response to hypoxic stress, binds to VEGF receptors (VEGFRs) overexpressed on vessels of ischemic tissue. The purpose of this study was to evaluate 64Cu-VEGF<em>121</em> positron emission tomography for noninvasive spatial, temporal, and quantitative monitoring of VEGFR2 expression in a murine model of hindlimb ischemia with and without treadmill exercise training.

RESULTS

64Cu-labeled VEGF<em>121</em> and a VEGF mutant were tested for VEGFR2 binding specificity in cell culture. Mice (n=58) underwent unilateral ligation of the femoral artery, and postoperative tissue ischemia was assessed with laser Doppler imaging. Longitudinal VEGFR2 expression in exercised and nonexercised mice was quantified with 64Cu-VEGF<em>121</em> positron emission tomography at postoperative day 8, 15, 22, and 29 and correlated with postmortem gamma-counting. Hindlimbs were excised for immunohistochemistry, Western blotting, and microvessel density measurements. Compared with the VEGF mutant, VEGF<em>121</em> showed specific binding to VEGFR2. Perfusion in ischemic hindlimbs fell to 9% of contralateral hindlimb on postoperative day 1 and recovered to 82% on day 29. 64Cu-VEGF<em>121</em> uptake in ischemic hindlimbs increased significantly (P < 0.001) from a control level of 0.61+/-0.17% ID/g (percentage of injected dose per gram) to 1.62+/-0.35% ID/g at postoperative day 8, gradually decreased over the following 3 weeks (0.59+/-0.14% ID/g at day 29), and correlated with gamma-counting (R2 = 0.99). Compared with nonexercised mice, 64Cu-VEGF<em>121</em> uptake was increased significantly (P < or = 0.0001) in exercised mice (at day 15, 22, and 29) and correlated with VEGFR2 levels as obtained by Western blotting (R2 = 0.76). Ischemic hindlimb tissue stained positively for VEGFR2. In exercised mice, microvessel density was increased significantly (P<0.001) compared with nonexercised mice.

CONCLUSIONS

64Cu-VEGF<em>121</em> positron emission tomography allows longitudinal spatial and quantitative monitoring of VEGFR2 expression in murine hindlimb ischemia and indirectly visualizes enhanced angiogenesis stimulated by treadmill exercise training.

Neo<em>vascular</em>ization is involved in beneficial and detrimental processes of tendon pathology. We investigated the influence of repetitive motion on the expression of the most important angiogenic <em>factor</em>, the <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) in the 3T3 NIH fibroblast cell line and in cultures of rat Achilles tendon fibroblasts. Monolayers of subconfluently grown cells were stretched in rectangular silicone dishes with cyclic uniaxial movement. Strain was applied over 24 h varying the frequency (0.5-1 Hz). Fibroblasts (3T3 fibroblasts and rat Achilles tendon cultures) cultivated without the application of cyclic strain released measurable VEGF amounts into their culture supernatants. Cyclic stretching of the cells with a frequency of 1 Hz resulted in an increased expression of VEGF. A low frequency (0.5 Hz) reduced VEGF expression to control levels. RT PCR revealed VEGF <em>121</em> and VEGF 165 as the only splice forms that were induced by cyclic stretching. Western blot experiments could further show that cyclic stretching induced activation of the transcription <em>factor</em> HIF-1alpha. These results demonstrate that mechanical <em>factors</em> are involved in the regulation of VEGF expression in tendon tissue.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), through its activation of cell surface receptor tyrosine kinases including VEGFR1 and VEGFR2, is a vital regulator of stimulatory and inhibitory processes that keep angiogenesis--new capillary <em>growth</em> from existing microvasculature--at a dynamic balance in normal physiology. Soluble VEGF receptor-1 (sVEGFR1)--a naturally-occurring truncated version of VEGFR1 lacking the transmembrane and intracellular signaling domains--has been postulated to exert inhibitory effects on angiogenic signaling via two mechanisms: direct sequestration of angiogenic ligands such as VEGF; or dominant-negative heterodimerization with surface VEGFRs. In pre-clinical studies, sVEGFR1 gene and protein therapy have demonstrated efficacy in inhibiting tumor angiogenesis; while in clinical studies, sVEGFR1 has shown utility as a diagnostic or prognostic marker in a widening array of angiogenesis-dependent diseases. Here we developed a novel computational multi-tissue model for recapitulating the dynamic systemic distributions of VEGF and sVEGFR1. Model features included: physiologically-based multi-scale compartmentalization of the human body; inter-compartmental macromolecular biotransport processes (<em>vascular</em> permeability, lymphatic drainage); and molecularly-detailed binding interactions between the ligand isoforms VEGF(<em>121</em>) and VEGF(165), signaling receptors VEGFR1 and VEGFR2, non-signaling co-receptor neuropilin-1 (NRP1), as well as sVEGFR1. The model was parameterized to represent a healthy human subject, whereupon we investigated the effects of sVEGFR1 on the distribution and activation of VEGF ligands and receptors. We assessed the healthy baseline stability of circulating VEGF and sVEGFR1 levels in plasma, as well as their reliability in indicating tissue-level angiogenic signaling potential. Unexpectedly, simulated results showed that sVEGFR1 - acting as a diffusible VEGF sink alone, i.e., without sVEGFR1-VEGFR heterodimerization--did not significantly lower interstitial VEGF, nor inhibit signaling potential in tissues. Additionally, the sensitivity of plasma VEGF and sVEGFR1 to physiological fluctuations in transport rates may partially account for the heterogeneity in clinical measurements of these circulating angiogenic markers, potentially hindering their diagnostic reliability for diseases.

The products of the neuropilin-1 (Np-1) and neuropilin-2 (Np-2) genes are receptors for <em>factors</em> belonging to the class 3 semaphorin family and participate in the guidance of <em>growing</em> axons to their targets. In the presence of heparin-like molecules, both receptors also function as receptors for the heparin-binding 165-amino acid isoform of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF(165)). Both receptors are unable to bind to the <em>121</em>-amino acid isoform of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF(<em>121</em>)), which lacks a heparin-binding domain. Interestingly, complexes corresponding in size to (125)I-VEGF(<em>121</em>).neuropilin complexes are formed when (125)I-VEGF(<em>121</em>) is bound and cross-linked to porcine aortic <em>endothelial</em> cells co-expressing VEGFR-1 and either Np-1 or Np-2. These complexes do not seem to represent complexes of (125)I-VEGF(<em>121</em>) with a truncated form of VEGFR-1, presumably formed as a result of the presence of Np-1 or Np-2 in the cells, because such truncated forms could not be detected with anti-VEGFR-1 antibodies. Antibodies directed against VEGFR-1 co-immunoprecipitated the (125)I-VEGF(<em>121</em>).Np-2 sized cross-linked complex along with (125)I-VEGF(<em>121</em>).VEGFR-1 complexes from cells expressing both VEGFR-1 and Np-2 but not from control cells, indicating that VEGFR-1 and Np-2 associate with each other. To perform the reciprocal experiment we have expressed in porcine aortic <em>endothelial</em> cells a Np-2 receptor containing an in-frame myc epitope at the C terminus. Surprisingly, the myc-tagged Np-2 receptor lost most of its VEGF(165) binding capacity but not its semaphorin-3F binding ability. Nevertheless, when Np-2myc was co-expressed in cells with VEGFR-1, it partially regained its VEGF(165) binding ability. Antibodies directed against the myc epitope co-immunoprecipitated (125)I-VEGF(165).Np-2myc and (125)I- VEGF(165).VEGFR-1 complexes from cells co-expressing VEGFR-1 and Np-2myc, indicating again that VEGFR-1 associates with Np-2. Our experiments therefore indicate that Np-2, and possibly also Np-1, associate with VEGFR-1 and that such complexes may be part of a cell membrane-associated signaling complex.

OBJECTIVE

The expression of vascular endothelial growth factor (VEGF) and its receptors KDR and Flt-1 by gastric carcinoma tissues and different gastric carcinoma cell lines was detected to elucidate the molecular mechanism of this growth factor in promoting tumor growth.

METHODS

The expression of VEGF, Flt-1 and KDR was determined by reverse transcription-polymerase chain reaction (RT-PCR) in gastric cancer cell lines RF-1, RF-48, AGS-1, NCI-N87, NCI-SNU-1, NCI-SNU-5, NCI-SNU-16 and KATO-III. The expression of Flt-1 and KDR in paraffin-embedded specimens of gastric cancer was determined by immunohistochemistry. The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to assess the role of VEGF in tumor cell proliferation.

RESULTS

All 8 gastric cancer cell lines analyzed expressed VEGF(121) and VEGF(165) and six of them expressed both Flt-1 and KDR, while cell line NCI-SNU-5 expressed Flt-1 only and cell line KATOIII expressed neither Flt-1 nor KDR. The gastric carcinoma tissues expressed Flt-1 and KDR widely, with the positive rate of expression of Flt-1 and KDR being 84.6 % and 70 % respectively. The exogenous VEGF stimulated the growth of KDR-positive cell lines NCI-N87 and AGS-1 in a dose-dependent manner but exhibited no effect on the growth of KDR-negative cell line NCI-N87.

CONCLUSIONS

VEGF and its receptors KDR and Flt-1 were expressed widely in gastric carcinoma cells and the VEGF stimulated KDR-positive tumor cell growth directly. These results suggest that VEGF may play a role in promoting tumor growth and metastasis by participating in both paracrine and autocrine pathways.

Pancreatic cancer has a poor prognosis, and treatment strategies based on preclinical research have not succeeded in significantly extending patient survival. This failure likely stems from the general lack of information on pancreatic tumor physiology, attributable to the difficulties in developing relevant, orthotopic models that accurately reflect pancreatic cancer in the clinic. To overcome this limitation, we developed abdominal wall windows suitable for intravital microscopy that allowed us to monitor angiogenesis and micro<em>vascular</em> function noninvasively during tumor <em>growth</em> in vivo. We used two complementary tumor models in mice: orthotopic (human ductal pancreatic adenocarcinoma, PANC-1, grown in the pancreas), and ectopic (PANC-1 grown in the abdominal wall). We found that orthotopic PANC-1 tumors grew faster than the ectopic tumors and exhibited metastatic spread in the late stage similar to advanced pancreatic cancer in the clinic. Orthotopic PANC-1 tumors expressed <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)(<em>121</em>) and VEGF(165), contained higher levels of tumor cell-derived VEGF protein, and maintained <em>vascular</em> density and hyperpermeability during exponential tumor <em>growth</em>. Orthotopic PANC-1 tumors showed lower leukocyte-<em>endothelial</em> interactions in the early stage of <em>growth</em>. In addition, both VEGF(<em>121</em>) and VEGF(165) promoted the <em>growth</em> of PANC-1 cells in vitro. Finally, Anti-VEGF neutralizing antibody inhibited angiogenesis and tumor <em>growth</em> of PANC-1 tumors in both sites. We conclude that the orthotopic pancreas microenvironment enhances VEGF expression, which stimulates <em>growth</em> of PANC-1 tumors (compared with ectopic tumors). The mechanism is autocrine and/or paracrine and also is involved in the maintenance of blood vessels. This comparative system of orthotopic and ectopic pancreatic cancer will provide the rigorous understanding of pancreatic tumor pathophysiology needed for development of novel therapeutic strategies.

OBJECTIVE

Vascular endothelial growth factor (VEGF), which is produced by tumor cells, is a potent endothelial cell mitogen. The aim of the present study was to evaluate the response of orthotopic prostate cancer xenografts and prostate cancer bone metastasis to anti-VEGF receptor (flk-1) antibody (DC101) treatment.

METHODS

Orthotopic prostate cancer models (PC-3M-MM2 and LNCaP-LN3 prostate carcinoma cells) and a prostate cancer bone metastasis model (PC-3M-MM2) were used for these experiments. Early and established tumors were treated with saline, paclitaxel, DC101, or a DC101-plus-paclitaxel combination for 5 weeks (PC-3M-MM2) and 12 weeks (LNCaP-LN3). At the end of therapy, tumors were removed and weighed. Apoptosis, tumor cell proliferation, and angiogenesis- and metastasis-related gene expression were evaluated using immunohistochemistry, in situ hybridization, and terminal deoxynucleotidyl transferase-ediated nick end labeling (TUNEL).

RESULTS

After treatment of early tumors (PC-3M-MM2), median prostate tumor weights (+/-SE) were 1230 +/- 210 mg in untreated controls, 482 +/- 121 mg in mice treated with paclitaxel (P = 0.009), 148 +/- 27 mg in mice treated with DC101 (P < 0.001), and 48 +/- 10 mg in mice treated with the combination of DC101 and paclitaxel (P < 0.001). Lymph node metastasis occurred in 7 of the 9 control mice, 5 of the 9 paclitaxel-treated animals, 5 of the 12 DC101-treated animals, and 2 of the 11 animals in the combination therapy group. Treatment with DC101 alone or in combination with paclitaxel reduced tumor-induced neovascularity measured by microvessel density and tumor cell proliferation (by proliferating cell nuclear antigen) and enhanced apoptosis (measured by TUNEL) in tumor cells and endothelial cells compared with controls. In the tibial prostate cancer metastasis model, significant inhibition of tumor growth was observed. In the LNCaP-LN3 orthotopic prostate cancer model, tumors occurred in 7 of the 10 control mice, 4 of the 10 paclitaxel-treated animals, 5 of the 10 DC101-treated animals, and 2 of the 11 animals in the combination therapy group (P < 0.05). The efficacy of DC101 was much greater in the treatment of early tumors, which suggests that tumor burden may be a critical factor in determining the response to DC101. In vitro and in vivo analysis of endothelial cell function showed reduced matrix metalloproteinase type 9 production in endothelial cells treated with DC101.

CONCLUSIONS

This study confirms the principle of tumor growth inhibition by targeting angiogenesis within tumors and supports the use of anti-VEGF receptor agents.

This study aims to investigate the expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and matrix metalloproteinase-9 (MMP-9) in giant cell tumor of bone (GCT) and other osteolytic lesions in bone. By using semi-quantitative RT-PCR, we showed that three major isoforms of VEGF (<em>121</em>, 165 and 189) were expressed in GCTs, with isoform <em>121</em> being the most abundant. The expression levels of VEGF and MMP-9 mRNA were significantly higher in advanced GCTs (stage II/III) than in stage I GCTs. We further elucidated the cellular localization of VEGF and MMP-9 gene transcripts in GCT and other osteolytic lesions using an in situ hybridization assay. The results showed that stromal tumor cells and osteoclast-like giant cells of GCT, fibrous stromal cells in anuerysmal bone cysts and fibrous dysplasia, and Langerhans-type giant cells as well as histocytes in eosinophillic granuloma, were all strongly positive for VEGF and MMP-9 mRNA expression. In a prospective study, we performed VEGF and MMP-9 immuno-staining on paraffin sections of pathological tissues harvested from 48 patients (14 GCT, 10 anuerysmal bone cysts, 10 eosinophillic granuloma, 4 fibrous dysplasia, 2 simple bone cyst, 2 osteomyelitis and 6 patients with fractured femoral head as control). The results showed that the differences in VEGF and MMP-9 expression between Stage I and other advanced Stages (II, III and recurrent) were highly significant (p<0.001), with advanced stages showing a higher mean expression. The difference between recurrent and Stage II and III lesions, was also statistically significant (p=0.03 for VEGF, and p=0.01 for MMP-9 expression), with recurrent lesions showing a higher mean expression of both VEGF and MMP-9. In conclusion, VEGF and MMP-9 expression in osteolytic lesions of bone co-relates well with the extent of bone destruction and local recurrence. Their expression may therefore provide some prognostic indication of the possible aggressive behavior of the underlying pathology.

We reported previously that <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> isoform A (VEGF-A) expression by Mel57 human melanoma cells led to tumor progression in a murine brain metastasis model in an angiogenesis-independent fashion by dilation of co-opted, pre-existing vessels and concomitant enhanced blood supply (B. Kusters et al., Cancer Res., 62: 341-345, 2002). Here, we compare the activities of the <em>121</em>, 165, and 189 VEGF-A isoforms in this model by transfecting Mel57 cells with the respective cDNAs and by injecting the resulting stably transfected cell lines in the internal carotid arteries of nude mice (n = 10 for each isoform). Although the three isoforms had similar potency to induce <em>endothelial</em> cell proliferation, VEGF(<em>121</em>) expression did not result in sprouting angiogenesis but rather led to extensive vasodilation and increased permeability of pre-existing, predominantly peritumoral vessels. Sometimes, proliferating <em>endothelial</em> cells accumulated in vessel lumina, giving these a micro<em>vascular</em>, glomeruloid, proliferation-like appearance. Expression of VEGF(165) or VEGF(189) was associated with induction of an intratumoral neo<em>vascular</em> bed. In VEGF(165)-expressing tumors, daughter <em>endothelial</em> cells were distributed among newly formed vessels that were extensively dilated. This also occurred in VEGF(189) tumors, but there, vasodilation was less pronounced. Using contrast-enhanced magnetic resonance imaging, the different <em>vascular</em> phenotypes were visualized on characteristic radiological images. VEGF(165) expression was the most unfavorable of the three. Mice carrying VEGF(165) tumors became moribund earlier than those carrying VEGF(<em>121</em>)-expressing tumors (16 +/- 4 days versus 22 +/- 3 days). Our data demonstrate that VEGF-A isoforms differ in angiogenic properties that can be visualized by contrast-enhanced magnetic resonance imaging.

Drug-dependent dissociation or association of cellular receptors represents a potent pharmacologic mode of action for regulating cell fate and function. Transferring the knowledge of pharmacologically triggered protein-protein interactions to materials science will enable novel design concepts for stimuli-sensing smart hydrogels. Here, we show the design and validation of an antibiotic-sensing hydrogel for the trigger-inducible release of human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>. Genetically engineered bacterial gyrase subunit B (GyrB) (ref. 4) coupled to polyacrylamide was dimerized by the addition of the aminocoumarin antibiotic coumermycin, resulting in hydrogel formation. Addition of increasing concentrations of clinically validated novobiocin (Albamycin) dissociated the GyrB subunits, thereby resulting in dissociation of the hydrogel and dose- and time-dependent liberation of the entrapped protein pharmaceutical VEGF(<em>121</em>) for triggering proliferation of human umbilical vein <em>endothelial</em> cells. Pharmacologically controlled hydrogels have the potential to fulfil the promises of stimuli-sensing materials as smart devices for spatiotemporally controlled delivery of drugs within the patient.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGF-A) is a potent secreted mitogen critical for physiological and pathological angiogenesis. Regulation of VEGF-A occurs at multiple levels, including transcription, mRNA stabilization, splicing, translation and differential cellular localization of various isoforms. Recent advances in our understanding of the posttranscriptional regulation of VEGF-A are comprised of the identification of stabilizing mRNA-binding proteins and the discovery of two internal ribosomal entry sites (IRES) as well as two alternative initiation codons in the 5'UTR of the VEGF-A mRNA. We have previously reported that VEGF-A translation initiation at both the AUG and CUG codons is dependent on the exon content of the coding region. In this report, we show that the expression of different VEGF-A isoforms is regulated by a small upstream open reading frame (uORF) located within an internal ribosome entry site, which is translated through a cap-independent mechanism. This uORF acts as a cis-regulatory element that regulates negatively the expression of the VEGF <em>121</em> isoform. Our data provide a framework for understanding how VEGF-A mRNAs are translated, and how the production of the VEGF <em>121</em> isoform is secured under non-hypoxic environmental conditions.

To help define the safety profile of the use of adenovirus (Ad) gene transfer vectors in humans, this report summarizes our experience since April 1993 of the local administration of E1(-)/E3(-) Ad vectors to humans using low (<10(9) particle units) or intermediate (10(9)-10(11) particle units) doses. Included in the study are 90 individuals and 12 controls, with diverse comorbid conditions, including cystic fibrosis, colon cancer metastatic to liver, severe coronary artery disease, and peripheral <em>vascular</em> disease, as well as normals. These individuals received 140 different administrations of vector, with up to seven administrations to a single individual. The vectors used include three different transgenes (human cystic fibrosis transmembrane conductance regulator cDNA, E. coli cytosine deaminase gene, and the human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> cDNA) administered by six different routes (nasal epithelium, bronchial epithelium, percutaneous to solid tumor, intradermal, epicardial injection of the myocardium, and skeletal muscle). The total population was followed for 130.4 patient-years. The study assesses adverse events, common laboratory tests, and long-term follow-up, including incidence of death or development of malignancy. The total group incidence of major adverse events linked to an Ad vector was 0.7%. There were no deaths attributable to the Ad vectors per se, and the incidence of malignancy was within that expected for the population. Overall, the observations are consistent with the concept that local administration of low and intermediate doses of Ad vectors appears to be well tolerated.

Therapeutic induction of angiogenesis is a potential treatment for chronic ischemia. Heparan sulfate proteoglycans are known to play an important role by their interactions with proangiogenic <em>growth</em> <em>factors</em> such as <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF). Low molecular weight fucoidan (LMWF), a sulfated polysaccharide from brown seaweeds that mimic some biological activities of heparin, has been shown recently to promote re<em>vascular</em>ization in rat critical hindlimb ischemia. In this report, we first used cultured human <em>endothelial</em> cells (ECs) to investigate the possible ability of LMWF to enhance the actions of VEGF(165). Data showed that LMWF greatly enhances EC tube formation in <em>growth</em> <em>factor</em> reduced matrigel. LMWF is a strong enhancer of VEGF(165)-induced EC chemotaxis, but not proliferation. In addition, LMWF has no effect on VEGF(<em>121</em>)-induced EC migration, a VEGF isoform that does not bind to heparan sulfate proteoglycans. Then, with binding studies using (125)I-VEGF(165), we observed that LMWF enhances the binding of VEGF(165) to recombinant VEGFR-2 and Neuropilin-1 (NRP1), but not to VEGFR-1. Surface plasmon resonance analysis showed that LMWF binds with high affinity to VEGF(165) (1.2 nm) and its receptors (5-20 nm), but not to VEGF(<em>121</em>). Pre-injection of LMWF on immobilized receptors shows that VEGF(165) has the highest affinity for VEGFR-2 and NRP1, as compared with VEGFR-1. Overall, the effects of LMWF were much more pronounced than those of LMW heparin. These findings suggested an efficient mechanism of action of LMWF by promoting VEGF(165) binding to VEGFR-2 and NRP1 on ECs that could help in stimulating therapeutic re<em>vascular</em>ization.

The gene for the major angiogenic <em>factor</em>, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), encodes several spliced isoforms. We reported previously that overexpression of two VEGF isoforms, VEGF(<em>121</em>) and VEGF(165), by human glioma U87 MG cells induced tumor-associated intracerebral hemorrhage, whereas expression of a third form, VEGF(189), did not cause vessel rupture. Here, we test whether these VEGF isoforms have distinct activities for enhancing <em>vascular</em>ization and <em>growth</em> of gliomas in mice. U87 MG cells that overexpressed VEGF(165) or VEGF(189) grew more rapidly than the parental cells in both s.c. and intracranial (i.c.) locations. However, cells that overexpressed VEGF(<em>121</em>) only showed enhancement of i.c. tumor <em>growth</em> but had a minimal effect on s.c. glioma progression. At both anatomical sties, VEGF(165) and VEGF(189) strongly augmented neo<em>vascular</em>ization, whereas VEGF(<em>121</em>) only increased vessel density in brain tumors. In each type of glioma, expression of VEGF receptors -1 and -2 largely phenocopied the tumor vasculature, because increased VEGF/VEGF receptor-activated microvessel densities were strongly correlated with the angiogenicity and tumorigenicity elicited by the VEGF isoforms at both anatomical sites. One notable difference between the sites was the expression of vitronectin, a prototypic ligand of alpha(v)beta(3) and alpha(v)beta(5) integrins, detected in i.c. but not in s.c., gliomas. <em>Endothelial</em> cell migration stimulated by VEGF(<em>121</em>) was potentiated by vitronectin to a greater extent than that stimulated by VEGF(165). This data demonstrates that VEGF isoforms have distinct activities at different anatomical sites and suggest that the microenvironment of different tissues affects the function of VEGF isoforms.

The aim of the present study was to evaluate the role of angiotensin II (AngII) in regulating both the gene expression and secretion of <em>vascular</em> permeability <em>factor</em>/<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VPF/VEGF) in human mesangial cells (HMC) in culture. Densitometric analysis of Northern blot experiments demonstrated that AngII increases VPF/VEGF mRNA in a dose-dependent manner. The levels of VPF/VEGF mRNA in HMC exposed for 3 h to 10 nM, 100 nM, and 1 microM AngII were, respectively, 1.5-, 2.3-, and 1.6-fold higher than control cells (P < 0.05, P < 0.0001, and P < 0.05, respectively). This effect was blocked by the pretreatment with losartan (1 microM) (P < 0.005), a selective antagonist of the AngII AT1 receptor. Reverse transcription-PCR performed in HMC using oligonucleotide primers specific for all VPF/VEGF mRNA splicing variants detected three bands corresponding to VEGF 189, 165, and <em>121</em>. Exposure of the cells to 100 nM AngII resulted in an increase of all the mRNA transcripts. Furthermore, in situ hybridization experiments showed that the levels of hybridization signals for VPF/VEGF mRNA resulted consistently higher in HMC exposed for 3 h to AngII (100 nM) than in control cells. The effects of AngII on the secretion of VPF/VEGF peptide in the culture medium of HMC were assessed using an enzyme-linked immunosorbent assay method. When different concentrations of AngII were tested in 3-h stimulation periods, the percentage of increase in the levels of released VPF/VEGF was significantly higher than control cells for AngII concentrations of 100 nM (62 +/- 11% mean +/- SD, P < 0.0001) and 1 microM (17.3 +/- 10.9%, P < 0.01). The pretreatment of HMC with losartan (1 microM) prevented the increase of VPF/VEGF secretion induced by AngII (100 nM) (AngII 54.7 +/- 3.9 pg/microg DNA versus AngII + losartan 37.8 +/- 3.6 pg/microg DNA, mean +/- SD, P < 0.005). VPF/VEGF protein was time dependently released in the culture medium under basal, steady-state conditions. Compared with control cells, AngII (100 nM) caused a significant increase in the levels of released VPF/VEGF after 3 and 6 h (control 33.8 +/- 1.7 pg/microg DNA at 3 h, 42.1 +/- 1.1 at 6 h, and 117.7 +/- 10 at 24 h; AngII 54.7 +/- 3.9 at 3 h, P < 0.0001, 61.6 +/- 8.7 at 6 h, P < 0.05, and 144.7 +/- 22.7 at 24 h, NS; mean +/- SD). According to the results obtained from enzyme-linked immunosorbent assay experiments, Western blot analysis showed that the intensity of the 19-kD band corresponding to VPF/VEGF was 1.5-fold higher in AngII (100 nM)-treated HMC than in control cells. Similarly, immunocytochemistry on HMC demonstrated an increase in intracellular VPF/VEGF immunostaining in response to AngII treatment (100 nM) compared with control cells. This study demonstrated that in HMC, AngII augmented the levels of VPF/VEGF gene expression and stimulated the synthesis and secretion of its peptide by activating AT1 receptors. Through these mechanisms, AngII may affect the functions of <em>endothelial</em> cells during the development of renal diseases involving the glomerulus.

A Disintegrin and Metalloprotease (ADAM) are transmembrane proteases displaying multiple functions. ADAM with ThromboSpondin-like motifs (ADAMTS) are secreted proteases characterised by thrombospondin (TS) motifs in their C-terminal domain. The aim of this work was to evaluate the expression pattern of ADAMs and ADAMTS in non small cell lung carcinomas (NSCLC) and to investigate the possible correlation between their expression and cancer progression. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunohistochemical analyses were performed on NSCLC samples and corresponding nondiseased tissue fragments. Among the ADAMs evaluated (ADAM-8, -9, -10, -12, -15, -17, ADAMTS-1, TS-2 and TS-12), a modulation of ADAM-12 and ADAMTS-1 mRNA expression was observed. Amounts of ADAM-12 mRNA transcripts were increased in tumour tissues as compared to the corresponding controls. In sharp contrast, ADAMTS-1 mRNA levels were significantly lower in tumour tissues when compared to corresponding nondiseased lung. These results were corroborated at the protein level by Western blot and immunohistochemistry. A positive correlation was observed between the mRNA levels of ADAM-12 and those of two <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)-A isoforms (VEGF-A(165) and VEGF-A(<em>121</em>)). Taken together, these results providing evidence for an overexpression of ADAM-12 and a lower expression of ADAMTS-1 in non-small-cell lung cancer suggest that these proteases play different functions in cancer progression.