Loss-of-function activin receptor-like kinase 1 gene mutation (ALK1+/-) is associated with brain arteriovenous malformations (AVM) in hereditary hemorrhagic telangiectasia type 2. Other determinants of the lesional phenotype are unknown. In the present study, we investigated the influence of high <em>vascular</em> flow rates on ALK1+/- mice by manipulating cerebral blood flow (CBF) using vasodilators. Adult male ALK1+/- mice underwent adeno-associated viral-mediated <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (AAVVEGF) or lacZ (AAVlacZ as a control) gene transfer into the brain. Two weeks after vector injection, hydralazine or nicardipine was infused intraventricularly for another 14 days. CBF was measured to evaluate relative tissue perfusion. We analyzed the number and morphology of capillaries. Results demonstrated that hydralazine or nicardipine infusion increased focal brain perfusion in all mice. It was noted that focal CBF increased most in AAVVEGF-injected ALK1+/- mice following hydralazine or nicardipine infusion (<em>145</em>+/-23% or 150+/-11%; P<0.05). There were more detectable dilated and dysplastic capillaries (2.4+/-0.3 or 2.0+/-0.4 dysplasia index; P<0.01) in the brains of ALK1+/- mice treated with AAVVEGF and hydralazine or nicardipine compared with the mice treated with them individually. We concluded that increased focal tissue perfusion and angiogenic <em>factor</em> VEGF stimulation could have a synergistic effect to promote capillary dysplasia in a genetic deficit animal model, which may have relevance to further studies of AVMs.

A new family of angiogenic <em>factors</em> named endocrine-gland-derived <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factors</em> (EG-VEGF)/prokineticins (PK) have been recently described as predominantly expressed in steroidogenic tissues. Whether the normal and malignant epithelial prostate cells and tissues express EG-VEGF/PK1 and PK2 and their receptors is still unknown. We studied the expression of EG-VEGF/PK1 and PK2 and their receptors (PK-R1 and PK-R2) in human prostate and their involvement in cancer. Using immunohistochemistry, Western blot, and RT-PCR, we determined the expression of EG-VEGF/PK1 in normal prostate (NP) and malignant prostate tissues (PCa), in epithelial cell primary cultures from normal prostate (NPEC) and malignant prostate (CPEC) and in a panel of prostate cell lines. In NPEC, CPEC, and in EPN, a nontransformed human prostate epithelial cell line, EG-VEGF/PK1, PK2, PK-R1, and PK-R2 mRNA levels were evaluated by quantitative RT-PCR. EG-VEGF/PK1 transcript was found in PCa, in CPEC, in EPN, and in LNCaP, whereas it was detected at low level in NP and in NPEC. EG-VEGF/PK1 was absent in androgen-independent PC3 and DU-<em>145</em> cell lines. Immunochemistry confirmed that EG-VEGF/PK1 protein expression was restricted to hyperplastic and malignant prostate tissues, localized in the glandular epithelial cells, and progressively increased with the prostate cancer Gleason score advancement. EG-VEGF/PK1 and PK2 were weakly expressed in NPEC and EPN. On the other hand, their transcripts were highly detected in CPEC. PK-R1 and PK-R2 were found in NPEC, EPN, and CPEC. Interestingly, CPEC showed a significantly (P < 0.05) higher expression of EG-VEGF/PK1, PK2, PK-R1, and PK-R2 compared with NPEC and EPN. We demonstrated that PKs and their receptors are expressed in human prostate and that their levels increased with prostate malignancy. It may imply that EG-VEGF/PK1 could be involved in prostate carcinogenesis, probably regulating angiogenesis. Thus, the level of EG-VEGF/PK1 could be useful for prostate cancer outcome evaluation and as a target for prostate cancer treatment in the future.

Linomide is a p.o. active antiangiogenic agent that has been demonstrated to be effective in suppressing the in vivo <em>growth</em> of rat and human prostatic cancer xenografts. The present studies were conducted to determine whether the angiogenic molecules, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>/<em>vascular</em> permeability <em>factor</em> (VEGF/VPF) and basic fibroblast <em>growth</em> <em>factor</em> (bFGF) are expressed in vitro by DU-<em>145</em>, PC-3, TSU-PR1, and LnCaP human prostate cancer cell lines and whether Linomide inhibits the secretion of these angiogenic molecules. Additionally, two different androgen-responsive human prostatic cancer xenograft models (i.e., PC-82 and A-2) were used to determine whether androgen ablation-induced reduction in tumor <em>growth</em> is associated with a reduction in tumor VEGF and/or bFGF levels. These studies demonstrated that both VEGF and bFGF proteins are expressed to different degrees in the human prostatic cancer cell lines. The secretion of VEGF but not bFGF is up-regulated by hypoxia. Linomide is unable to inhibit either basal or hypoxia-induced secretion of VEGF. Linomide also has no effect on secreted bFGF levels. Castration inhibited tumor VEGF but had no effect on bFGF levels in both the androgen-responsive PC-82 and A-2 human prostatic cancers when grown in severe combined immunodeficient mice. When given in combination, castration potentiated the inhibition of tumor <em>growth</em> induced by Linomide alone. This potentiation is not due to a further inhibition in tumor VEGF levels induced by castration. Although both castration and Linomide inhibit angiogenesis, the former accomplishes it by inhibiting VEGF secretion, whereas the latter has multiple effects at several steps in the angiogenic process other than VEGF secretion. Based on their different but complementary mechanisms of action, simultaneous combination of androgen ablation with Linomide enhances the anti-prostatic cancer efficacy compared to either monotherapies alone and warrants testing in humans.

BACKGROUND

SU5416 (semaxanib) is a synthetic small molecule inhibitor of the tyrosine kinase domain of vascular endothelial growth factor receptor 2 (VEGFR2). This Phase II study was conducted to determine the safety and efficacy of SU5416 in patients with recurrent or metastatic head and neck cancers.

METHODS

This was an open label, single arm, Phase 2 study for patients who had received no more than 2 cytotoxic regimens. Thirty-five patients received intravenous SU5416 (145 mg/m(2)) twice per week by intravenous catheter. Radiologic imaging for response assessment was planned at the conclusion of each 8 week cycle. Serum VEGF levels and power Doppler ultrasound tumor imaging were explored as potential surrogate markers for SU5416 activity.

RESULTS

Thirty-two patients had received prior radiotherapy, including 18 patients who received prior concurrent chemoradiotherapy. Twelve patients had received prior chemotherapy in the recurrent disease setting. Thirty-one patients were evaluable for response. There was one partial response and one minor response. The median number of 8-week cycles received was 1 (range 1-4). Median overall survival was 6.25 months. The most common>> or = grade 3 toxicity was headache (31%). There was one fatal carotid artery hemorrhage. Fatigue, nausea, and vomiting were common grade 1-2 adverse events. Power Doppler ultrasound demonstrated decreased tumor vascularity in 5 of 7 patients.

CONCLUSIONS

Treatment with SU5416 in patients with head and neck cancers is feasible, but objective responses are rare. Studies evaluating more potent anti-angiogenic agents in this disease are of interest.

OBJECTIVE

Lymph node metastasis (LNM) is a chief cause of morbidity and mortality in patients with hepatocellular carcinoma (HCC) after hepatectomy. The aim of this study was to investigate the relationship between the expression of CXCR4 and vascular endothelial cell growth factor (VEGF)-C and the clinicopathological features of HCC with LNM.

METHODS

Immunohistochemical staining for CXCR4 and VEGF-C was performed on tissue microarrays that were constructed using tumor specimens from patients with HCC with (N = 123) or without (N = 145) LNM. The relationship between the clinicopathological features of HCC and the expression of CXCR4 and VEGF-C was analyzed using the Pearson chi(2) test, logistical regression analysis, and receiver operating characteristic analysis.

RESULTS

Nuclear CXCR4 expression and VEGF-C expression were positively correlated with LNM and poor outcome in HCC. Moreover, nuclear CXCR4 expression was positively correlated with VEGF-C expression (correlation coefficient 0.256). Receiver operating characteristic analysis revealed that both factors were predictive of HCC LNM {CXCR4: area under the curve, 0.695 [95% confidence interval (CI), 0.630-0.759; VEGF-C: area under the curve, 0.629 (95% CI, 0.562-0.695]}. Patients with tumors exhibiting high nuclear CXCR4 expression or high VEGF-C expression had significantly poorer overall survival than those with low tumor expression of the corresponding factors. Multivariate analysis showed that UICC T stage [odds ratio (OR), 1.615, 95% CI, 1.306-1.997], nuclear CXCR4 expression (OR, 3.998; 95% CI, 2.706-5.907), and VEGF-C expression (OR, 1.903; 95% CI, 1.203-3.011) were independent risk factors for developing HCC LNM.

CONCLUSIONS

These findings suggest that nuclear CXCR4 expression and VEGF-C expression can be used to identify patients with HCC at high risk for developing LNM.

OBJECTIVE

To evaluate the long-term efficacy of intravitreal anti-vascular endothelial growth factor (anti-VEGF) therapy as primary treatment for subfoveal myopic choroidal neovascularization (CNV).

METHODS

In all, 37 treatment-naïve eyes of 37 patients with subfoveal myopic CNV who received intravitreal bevacizumab (n=22) or ranibizumab (n=15) injections with at least 2 years of follow-up were reviewed. All eyes received initial three loading doses of anti-VEGF at monthly intervals and retreatment was performed in persistent or recurrent CNV. Multivariate regression analyses were performed to determine the prognostic factors for visual outcome.

RESULTS

The mean age was 57.3 years and the mean refractive error was -11.7 D. For all eyes, the mean logMAR best-corrected visual acuity improved from 0.86 (20/145) at baseline to 0.48 (20/60) at 2 years (P<0.001). The mean visual improvement for the bevacizumab and ranibizumab groups at 2 years was 2.8 and 5.1 lines, respectively (P=0.073). There was no significant difference in the proportion of eyes having visual gain of three or more lines or visual loss of three or more lines between the two groups. The mean number of injections was 3.8 for both bevacizumab and ranibizumab groups. Multivariate analyses showed that eyes with higher myopic refractive error were less likely to have visual gain after treatment (P=0.043), while size of CNV was negatively correlated with mean change in vision (P=0.046).

CONCLUSIONS

Intravitreal anti-VEGF therapy resulted in long-term visual improvement in myopic CNV. The treatment efficacy in terms of visual gain and number of retreatment appeared to be similar between bevacizumab and ranibizumab.

SU5416 is a small molecule antiangiogenic agent that inhibits <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) stimulation of the KDR tyrosine kinase receptor. In this Phase I dose escalation trial, a weekly dose schedule of SU5416 was tested whereby an initial 5-day loading dose was followed by weekly maintenance infusions. The start dose was 20 mg/m(2) for the loading dose followed by 65 mg/m(2) for the weekly infusions. Dose escalations occurred at 33% until a final dose of 65 mg/m(2) (loading dose) and 190 mg/m(2) (weekly infusion) was obtained. Twenty-two patients were treated at five dose levels; tumor types included gastrointestinal (8), breast (3), lung (4), sarcoma (2), and other (5). The most common serious drug-related toxicity was headache, often associated with nausea and vomiting. Grade 1 and 2 toxicities included headache, nausea, vomiting, asthenia, pain at the infusion site, phlebitis, change in voice, and fevers. Of 19 evaluable patients, 4 obtained clinical benefit as defined by tumor regression (1) or disease stabilization for at least 12 weeks (3). Pharmacokinetic data revealed that the weekly infusion schedule prevented the reported 50-60% induction in SU5416 clearance observed with either daily or twice weekly dosing. Higher baseline levels of urine VEGF were observed in the 4 patients who gained clinical benefit, suggesting this may be a useful marker for predicting response to anti-VEGF therapies. Our results suggest that a weekly schedule of SU5416 shows signs of biological activity and is well tolerated at doses up to <em>145</em> mg/m(2).

OBJECTIVE

Hypoxia-inducible factors HIF-1alpha and HIF-2alpha are considered to be potential targets for antineoplastic therapy because they regulate the expression of genes that contribute to tumor cell survival, aggressiveness, and angiogenesis. Nonsteroidal anti-inflammatory drugs (NSAIDs) have gained considerable interest as anticancer agents because of their cytotoxic and antiangiogenic properties. The aim of this study was to investigate whether NSAIDs inhibit HIFs and HIF-regulated gene expression in prostate cancer cells.

METHODS

PC3 and DU-145 cells were treated with ibuprofen (Ibu) and other NSAIDs under normoxic and hypoxic (95% N(2), 5% CO(2); <10 ppm O(2)) conditions. The effect of NSAIDs on HIF proteins was analyzed by Western blot analysis. HIF-regulated proteins, vascular endothelial growth factor (VEGF) and glucose transporter-1 (Glut-1), were analyzed by ELISA and Western blot analysis, respectively.

RESULTS

Exposure of PC3 and DU-145 cells to hypoxic condition up-regulated HIF-1alpha and HIF-2alpha proteins. Treatment with Ibu under normoxic and hypoxic conditions reduced the level of HIF-1alpha and HIF-2alpha. Ibu-mediated down-regulation of HIFs was associated with down-regulation of HIF-regulated proteins VEGF and Glut-1 in cells exposed to hypoxia. Other nonspecific NSAIDs, diclofenac and ketorolac, also inhibited HIF-1alpha and HIF-2alpha. The reduction in HIFs was observed in PC3 cells that expressed cyclooxygenase-2 (COX-2) protein as well as in DU-145 cells, which did not express COX-2 protein. COX-2-specific inhibitor NS-398 did not inhibit HIF-1alpha or VEGF and GLUT-1.

CONCLUSIONS

These data indicate that one of the effects of NSAIDs is to reduce HIF protein levels. The inhibition of HIFs by NSAIDs was COX-2 independent.

We demonstrate here that epithelial carcinoembryonic antigen (CEA)-related cell adhesion molecule-1 (CEACAM1) downregulation in prostate intraepithelial neoplasia (PIN) is inversely correlated with its upregulation in adjacent blood vessels. CEACAM1 silencing in prostate cancer cell line DU-<em>145</em> via small interfering ribonucleic acid (siRNA) increased but its overexpression suppressed the expression of angiogenic/lymphangiogenic <em>factors</em> such as <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)-A, -C and -D, and angiogenic inhibitor collagen 18/endostatin. Furthermore, CEACAM1 overexpression in DU-<em>145</em> cells increased but CEACAM1 silencing reduced angiopoietin-1 expression. Inverse relation was found for angiopoietin-2. Supernatant of CEACAM1-overexpressing DU-<em>145</em> suppressed but that of CEACAM1-silenced increased the VEGF-induced <em>endothelial</em> tubes. Electron microscopically the majority of PIN-associated blood vessels was structurally destabilized exhibiting <em>endothelial</em> fenestration, trans- and inter-<em>endothelial</em> gaps. In some PIN areas, invasion of single tumor cells into the destabilized blood vessels was observed. These data show that disappearance of epithelial CEACAM1 in PIN is accompanied by its upregulation in adjacent vasculature which apparently correlates with <em>vascular</em> destabilization and increased <em>vascular</em>ization of prostate cancer. Strategies to either conserve the epithelial CEACAM1 or to target <em>endothelial</em> CEACAM1 might be useful for an anti-angiogenic therapy of prostate cancer.

BACKGROUND

Breast cancer is the most common type of cancer among women, and despite improved treatments, it remains a major challenge. However, improved mechanistic insight may lead to novel therapeutic strategies. miR-142-3p belongs to the miR-142 family and is involved in pathogenesis and metastasis of various types of malignancies by targeting several important messenger RNAs (mRNAs) including Bach-1. This is especially true for breast cancer, where Bach-1 is involved in the metastatic spread by deregulation of metastasis-associated genes.

METHODS

In this study, we collected 24 breast cancer tissues with 24 adjusted normal tissues to measure the expression levels of miR-142-3p and Bach-1 mRNA using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and IHC. miR-142-3p targeting of Bach-1 expression in MCF-7 and MDA-MB-468 breast cancer cells was evaluated using bioinformatics, qRT-PCR and western blot analyses. The cellular proliferation, invasion, and migration were assessed by MTT, transwell matrigel and wound healing assay and the EMT-associated proteins C-X-C chemokine receptor type 4 (CXCR-4), matrix metalloproteinase-9 (MMP9), and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> receptor (VEGFR) were analyzed by western blot analysis. Also, the expression levels of tumor suppressors including miR-330, miR-<em>145</em>, and miR-34a were estimated by qRT-PCR.

RESULTS

Analysis of paired specimens of primary malignant and normal tissues showed that miR-142-3p was downregulated, while Bach-1 mRNA and protein both were overexpressed in the breast cancer tumors. This inverse relationship was confirmed by cell line experiments demonstrating that miR-142-3p expression reduced Bach-1 mRNA levels. Furthermore, replacement of miR-142-3p could inhibit the proliferation, invasion, and migration in breast cancer potentially by targeting of Bach-1 mRNA and subsequent inhibition of CXCR4, MMP9, and VEGFR protein expressions. In addition, induction of miR-142-3p could upregulate tumor suppressor miRNAs, including miR-330, miR-<em>145</em>, and miR34a.

CONCLUSIONS

For the first time, our results revealed that miR-142-3p could target Bach-1in breast cancer cells leading to the reduction of EMT-related proteins and reduced cell proliferation, invasion, and migration. The results also demonstrated that miR-142-3p could regulate important tumor suppressor miRNAs in breast cancer cells. In conclusion, our results suggest that miR-142-3p could be a good candidate for the targeted therapy of breast cancer, especially for the invasive type.

OBJECTIVE

Diabetes mellitus (DM) is associated with reduced progression of abdominal aortic aneurysm (AAA) disease. Mechanisms responsible for this negative association remain unknown. We created AAAs in hyperglycemic mice to examine the influence of serum glucose concentration on experimental aneurysm progression.

METHODS

Aortic aneurysms were induced in hyperglycemic (DM) and normoglycemic models by using intra-aortic porcine pancreatic elastase (PPE) infusion in C57BL/6 mice or by systemic infusion of angiotensin II (ANG) in apolipoprotein E-deficient (ApoE(-/-)) mice, respectively. In an additional DM cohort, insulin therapy was initiated after aneurysm induction. Aneurysmal aortic enlargement progression was monitored with serial transabdominal ultrasound measurements. At sacrifice, AAA cellularity and proteolytic activity were evaluated by immunohistochemistry and substrate zymography, respectively. Influences of serum glucose levels on macrophage migration were examined in separate models of thioglycollate-induced murine peritonitis.

RESULTS

At 14 days after PPE infusion, AAA enlargement in hyperglycemic mice (serum glucose ≥ 300 mg/dL) was less than that in euglycemic mice (PPE-DM: 54% ± 19% vs PPE: 84% ± 24%, P < .0001). PPE-DM mice also demonstrated reduced aortic mural macrophage infiltration (<em>145</em> ± 87 vs 253 ± 119 cells/cross-sectional area, P = .0325), elastolysis (% residual elastin: 20% ± 7% vs 12% ± 6%, P = .0209), and neo<em>vascular</em>ization (12 ± 8 vs 20 ± 6 vessels/high powered field, P = .0229) compared with PPE mice. Hyperglycemia limited AAA enlargement after ANG infusion in ApoE(-/-) mice (ANG-DM: 38% ± 12% vs ANG: 61% ± 37% at day 28). Peritoneal macrophage production was reduced in response to thioglycollate stimulation in hyperglycemic mice, with limited augmentation noted in response to <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> administration. Insulin therapy reduced serum glucose levels and was associated with AAA enlargement rates intermediate between euglycemic and hyperglycemic mice (PPE: 1.21 ± 0.14 mm vs PPE-DM: 1.00 ± 0.04 mm vs PPE-DM + insulin: 1.14 ± 0.05 mm).

CONCLUSIONS

Hyperglycemia reduces progression of experimental AAA disease; lowering of serum glucose levels with insulin treatment diminishes this protective effect. Identifying mechanisms of hyperglycemic aneurysm inhibition may accelerate development of novel clinical therapies for AAA disease.

OBJECTIVE

The formation of new blood vessels from the pre-existing vasculature is necessary for support of primary tumor growth and appears coincident with the development of metastasis. In previous studies, inhibition of vascular endothelial growth factor (VEGF), a potent angiogenic factor and mediator of vascular permeability, inhibited tumor neovascularization with consequent inhibition of both primary tumor growth and micrometastases when administered at the time of tumor inoculation. In the present study, we examined the effect of inhibiting VEGF on primary tumor growth and metastases in an in vivo model of established metastatic prostate cancer.

METHODS

The human prostate cancer cell line DU-145 was found to secrete VEGF. DU-145.luciferase, a subclone stably transfected with an expression vector encoding the luciferase gene, injected subcutaneously, consistently formed tumors in C.B.-17 scid/scid mice. After 6 weeks, assay of whole lung lysates showed significant luciferase activity, consistent with the presence of micrometastasis.

RESULTS

Twice weekly treatment of the animals with a monoclonal anti-VEGF neutralizing antibody, A4.6.1, not only suppressed primary tumor growth, but inhibited metastatic dissemination to the lung. When treatment was delayed until the primary tumors were well-established, further growth was still inhibited, as was the progression of metastatic disease.

CONCLUSIONS

Inhibition of tumor-secreted VEGF by a neutralizing antibody is sufficient to significantly impair prostate tumor growth and its subsequent metastasis in an in vivo model of established advanced prostate cancer. These data suggest a critical role for VEGF in initiation and maintenance of tumor angiogenesis in prostate cancer. Inhibition of VEGF in patients with VEGF-secreting prostate cancers may prove an effective approach for inhibiting disease progression even after micro-metastatic dissemination has occurred.

The antiproliferative effects of an antagonist of <em>growth</em> hormone-releasing hormone (GHRH) JV-1-38 were evaluated in nude mice bearing s.c. xenografts of LNCaP and MDA-PCa-2b human androgen-sensitive and DU-<em>145</em> androgen-independent prostate cancers. In the androgen-sensitive models, JV-1-38 greatly potentiated the antitumor effect of androgen deprivation induced by surgical castration, but was ineffective when given alone. Thus, in castrated animals bearing MDA-PCa-2b cancers, the administration of JV-1-38 for 35 days virtually arrested tumor <em>growth</em> (94% inhibition vs. intact control, P < 0.01; and 75% vs. castrated control, P < 0.05). The <em>growth</em> of LNCaP tumors was also powerfully suppressed by JV-1-38 combined with castration (83% inhibition vs. intact control, P < 0.01; and 68% vs. castrated control, P < 0.05). However, in androgen-independent DU-<em>145</em> cancers, JV-1-38 alone could inhibit tumor <em>growth</em> by 57% (P < 0.05) after 45 days. In animals bearing MDA-PCa-2b and LNCaP tumors, the reduction in serum prostate-specific antigen levels, after therapy with JV-1-38, paralleled the decrease in tumor volume. Inhibition of MDA-PCa-2b and DU-<em>145</em> cancers was associated with the reduction in the expression of mRNA and protein levels of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>. The mRNA expression for GHRH receptor splice variants was found in all these models of prostate cancer. Our results demonstrate that GHRH antagonists inhibit androgen-independent prostate cancers and, after combination with androgen deprivation, also androgen-sensitive tumors. Thus, the therapy with GHRH antagonist could be considered for the management of both androgen-dependent or -independent prostate cancers.

SU5416 (Z-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone; semaxanib) is a small molecule inhibitor of the <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> receptor (VEGFR2). A Phase I dose escalation study was performed. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used as a pharmacodynamic assessment tool. In all, 27 patients were recruited. SU5416 was administered twice weekly by fixed rate intravenous infusion. Patients were treated in sequential cohorts of three patients at 48, 65, 85 110 and <em>145</em> mg m-2. A further dose level of 190 mg m-2 after a 2-week lead in period at a lower dose was completed; thereafter, the cohort at <em>145</em> mg m-2 was expanded. SU5416 showed linear pharmacokinetics to <em>145</em> mg m-2 with a large volume of distribution and rapid clearance. A significant degree of interpatient variability was seen. SU5416 was well tolerated, by definition a maximum-tolerated dose was not defined. No reproducible changes were seen in DCE-MRI end points. Serial assessments of VEGF in a cohort of patients treated at <em>145</em> mg m-2 did not show a statistically significant treatment-related change. Parallel assessments of the impact of SU5416 on coagulation profiles in six patients showed a transient effect within the fibrinolytic pathway. Clinical experience showed that patients who had breaks of therapy longer than a week could not have treatment reinitiated at a dose of 190 mg m-2 without unacceptable toxicity. The <em>145</em> mg m-2 dose level is thus the recommended dose for future study.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), a key regulatory protein in neoangiogenesis, is strongly expressed in a variety of primary brain tumors, particularly malignant gliomas. In previous studies, high levels of VEGF were also reported in tumor cysts of glioblastomas. Using an ELISA method we measured the concentration of VEGF in matched samples of aspiration fluid from tumor cysts and serum. Samples were collected from 14 patients with primary brain tumors of various histology (six glioblastomas, one protoplasmatic astrocytoma, two pilocytic astrocytomas, one ependymoma, one meningioma, and three craniopharyngiomas) and two patients with solitary cystic brain metastases from adenocarcinomas of the lung. Aspiration fluids of tumor cysts from all patients revealed high VEGF levels ranging between 882 and 1,263,000 pg/ml, which were 2 to more than 2,000 times higher than the corresponding serum levels. Maximum VEGF levels were detectable in cyst fluids from recurrent glioblastoma. Serum VEGF levels ranged between 125 and 716 pg/ml and did not differ from serum levels in <em>145</em> healthy volunteers. In a single patient with metastatic lung cancer the concentration of VEGF in serum and cyst fluid was determined during disease progression. During 60 days of follow-up VEGF concentrations in the cyst fluid collected by puncture of an Ommaya reservoir increased 650-fold, while serum levels remained rather constant. These findings indicate that immunoreactive VEGF is produced at the tumor site and abundantly released into the cyst fluid of primary and metastatic brain tumors. Interestingly, this abundant local release is not reflected in serum VEGF levels, even in the case of very high VEGF concentrations in tumor cysts. Thus, VEGF may be biologically relevant for the formation of tumor cysts in brain tumors and correlates with local disease progression.

Hair follicle <em>vascular</em>ization appears to be closely related to the processes involved in hair cycle regulation, in which <em>growth</em> <em>factors</em>, cytokines and other bioactive molecules are involved. In particular, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), essential for angiogenesis and <em>vascular</em> permeability, may be responsible for maintaining proper vasculature around the hair follicle during the anagen <em>growth</em> phase. The aim of our study was to compare the in vitro angiogenic capacity, i.e. the steady-state expression of the VEGF gene, of different cultured cell types derived from normal human hair follicles, corresponding to different follicular compartments. Human dermal papilla cells (DPC), fibrous sheath fibroblasts, dermal fibroblasts, and follicular and interfollicular keratinocytes were cultured and studied in vitro for VEGF expression at the mRNA level using RT-PCR, and for VEGF protein synthesis by radioimmunoprecipitation and immunocytochemistry. In vivo examination for VEGF expression in human terminal hair follicles was performed using immunohistochemical methods. In the present report the expression of four different VEGF molecular isoforms, differing in their angiogenic capacity, are described in different cultured follicular cell types for the first time. Cultured follicular cells strongly expressed mRNA of four VEGF molecular species identified as the 121-, <em>145</em>-, 165- and 189-amino acid splice variants, the most prominent being the 121-amino acid molecule. DPC, and also other mesenchymal cells such as fibrous sheath fibroblasts and dermal fibroblasts, in vivo and in vitro strongly expressed VEGF mRNA and synthesized a 46-kDa VEGF protein, whereas follicular and interfollicular keratinocytes in vitro expressed lower levels of VEGF mRNA and proteins than mesenchymal cells. As the highest expression of VEGF was found in DPC, we suggest that DPC are mainly responsible for angiogenic processes possibly related to the human hair cycle.

BACKGROUND

Vascular endothelial growth factor (VEGF) is a potent angiogenic and vascular permeability factor, and its polymorphisms are associated with proliferative diabetic retinopathy (PDR) and macular edema.

OBJECTIVE

We investigated the contributions of VEGF gene polymorphisms to nonproliferative diabetic retinopathy (NPDR) as well as PDR.

METHODS

In this study we compared VEGF gene variants in a sample of Korean type 2 diabetes patients with and without diabetic retinopathy (DR) and in healthy controls. Of the diabetes patients, 145 had PDR, 108 had NPDR, and 134 had no retinopathy (noDR). They were all duration matched. Samples were genotyped for rs699947, rs1570360, and rs2010963 polymorphisms.

RESULTS

We found a significant association between the A allele at rs699947 with DR (odds ratio = 1.84 (95% confidence interval = 1.28-2.66); P = 0.001 vs. noDR). Patients with NPDR, as well as PDR, had increased incidence of the A allele. The AGG haplotype was more frequently found in patients with DR than in patients with noDR (odds ratio = 4.79 (95% confidence interval = 1.42-16.16); P = 0.006). PDR and NPDR patients exhibited an increased incidence of the AGG haplotype.

CONCLUSIONS

VEGF polymorphisms might be a useful predictive marker for the development and progression of DR at an earlier stage of diabetes.

C17orf37/MGC14832, a novel gene located on human chromosome 17q12 in the ERBB2 amplicon, is abundantly expressed in breast cancer. C17orf37 expression has been reported to positively correlate with grade and stage of cancer progression; however the functional significance of C17orf37 overexpression in cancer biology is not known. Here, we show that C17orf37 is highly expressed in prostate cancer cell lines and tumors, compared to minimal expression in normal prostate cells and tissues. Cellular localization studies by confocal and total internal reflection fluorescence microscopy revealed predominant expression of C17orf37 in the cytosol with intense staining in the membrane of prostate cancer cells. RNA-interference-mediated downregulation of C17orf37 resulted in decreased migration and invasion of DU-<em>145</em> prostate cancer cells, and suppressed the DNA-binding activity of nuclear <em>factor</em>-kappaB (NF-kappaB) transcription <em>factor</em> resulting in reduced expression of downstream target genes matrix metalloproteinase 9, urokinase plasminogen activator and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>. Phosphorylation of PKB/Akt was also reduced upon C17orf37 downregulation, suggesting C17orf37 acts as a signaling molecule that increases invasive potential of prostate cancer cells by NF-kappaB-mediated downstream target genes. Our data strongly suggest C17orf37 overexpression in prostate cancer functionally enhances migration and invasion of tumor cells, and is an important target for cancer therapy.

<em>Vascular</em> permeability <em>factor</em> (VPF) also known as <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), is a dimeric protein that affects <em>endothelial</em> cell (EC) and <em>vascular</em> functions including enhancement of micro<em>vascular</em> permeability and stimulation of EC <em>growth</em>. To investigate the structural features of VPF/VEGF necessary for efficient dimerization, secretion, and biological activities, we employed site-directed mutagenesis with a Cos-1 cell expression system. Several cysteine residues essential for VPF dimerization were identified by mutation analysis of the Cys-25, Cys-56, and Cys-67 residues. Mutant VPF isoforms lacking either of these cysteines were secreted as monomers and were completely inactive in both <em>vascular</em> permeability and <em>endothelial</em> cell mitotic assays. VPF Cys-<em>145</em> mutant protein was efficiently secreted as a glycosylated, dimeric polypeptide, but had a reduction in biological activities. The site of N-linked glycosylation was directly identified as Asn-74, which, when mutated produced an inefficiently secreted dimeric protein without post-translational glycosylation, yet maintained full <em>vascular</em> permeability activity. Finally, we found that one VPF mutant isoform Cys-101 was not secreted and this mutant functioned as a dominant-negative suppressor of wild-type VPF secretion as demonstrated by co-expression assays in Cos-1 cells.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) mediates increased <em>vascular</em> permeability and <em>endothelial</em> mitogenesis, and may orchestrate normal glomerular permselectivity and proteinuria. Distinct isoforms result from differential gene splicing. VEGF binds to two cell surface tyrosine-kinase receptors, KDR (kinase domain region) and Flt-1 (fms-like tyrosine kinase-1). The latter also exists in a soluble form (sFlt), which is inhibitory. We have studied patterns of VEGF-isoform and VEGF-receptor expression in isolated single normal human glomeruli. mRNA from 190 glomeruli (from 20 individuals) was harvested on to magnetic beads, and nested reverse transcription-PCR was performed using primers for the VEGF isoforms and VEGF receptors. Simultaneous nested reverse transcription-PCR for CD45 was conducted in order to exclude leucocyte contamination. Unexpected products were isolated, cloned and sequenced. Multiple patterns of glomerular VEGF mRNA isoform expression were identified. Most frequently (58%), all three common forms were expressed. VEGF(189) (i.e. 189-amino-acid form of VEGF) was expressed in 63%, VEGF(165) in 85% and VEGF(121) in 84% of glomeruli. Two unexpected PCR products were also identified: 18% of glomeruli expressed VEGF(<em>145</em>), and 27% of glomeruli expressed a new truncated VEGF splice variant, VEGF(148), lacking exon 6, the terminal part of exon 7 and exon 8. Multiple patterns of VEGF-receptor expression were also identified, the most common being expression of all three isoforms (28%). Overall, KDR was seen in 59% of glomeruli, Flt-1 in 45% and sFlt in 57%. Thus the expression of VEGF within normal glomeruli is complex and variable, with inter- and intra-individual variation. Furthermore, sFlt appears to be the co-dominant form of VEGF receptor expressed within glomeruli, suggesting that, in healthy individuals, a degree of VEGF autoregulation is the norm. The physiological importance of VEGF(148) remains to be confirmed.

The development of ovarian follicles and subsequent corpus luteum formation is accompanied by very active angiogenesis. Ovarian granulosa cells produce <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), which is a potent <em>endothelial</em> cell mitogen and an angiogenic agent. The complementary DNAs of two other <em>factors</em> structurally related to VEGF, namely VEGF-B and VEGF-C, were recently cloned, but little is known of their regulation in the ovary. We first studied the expression of the messenger RNAs (mRNAs) of the three VEGF isotypes in freshly isolated human granulosa-luteal (GL) cells obtained at oocyte retrieval for in vitro fertilization. The hormonal regulation of these mRNAs was subsequently studied in primary cultures of human GL cells. Analysis of cultured GL cell RNA by reverse transcription-PCR revealed that these cells express the alternatively spliced transcripts representing 121-, <em>145</em>-, and 165-amino acid VEGF isoforms. Northern blot hybridization analyses indicated that transcripts of 4.5 and 3.7 kilobases for VEGF, and 1.4 and 2.4 kilobases for VEGF-B and VEGF-C, respectively, are expressed in human GL cells. The basal VEGF mRNA levels declined steadily, whereas VEGF-B mRNA levels were rather invariant over a 10-day culture period of GL cells. In contrast, VEGF-C mRNA levels increased toward the end of culture. For studying the hormonal regulation of VEGF isotype mRNAs, GL cells were treated with hCG, recombinant human FSH, PGE2, as well as 8-bromo-cAMP and 12-O-tetradecanoylphorbol 13-acetate, which activate protein kinase A- and protein kinase C-dependent signaling pathways, respectively. All test agents stimulated the expression of VEGF mRNA levels in a concentration-dependent manner. Time-course studies indicated that all treatments induced VEGF mRNA levels as early as incubation for 2 h, and the effect was sustained up to 48 h. VEGF-B mRNA levels were not regulated by any of the test agents. However, we found that hCG and 8-bromo-cAMP decreased VEGF-C mRNA levels with a maximal response observed at 24 and 48 h after cellular treatment. We conclude that the mRNAs of VEGF, VEGF-B, and VEGF-C are expressed in human GL cells and that their mRNA steady state levels are regulated in cultured human GL cells in an isotype-specific manner. The differential regulation of VEGF, VEGF-B, and VEGF-C in human GL cells suggests that distinct VEGF isotypes may play different roles during the <em>vascular</em>ization of the human ovarian follicle and corpus luteum.

BACKGROUND

MicroRNA miR145 has been implicated in vascular smooth muscle cell differentiation, but its mechanisms of action and downstream targets have not been fully defined.

OBJECTIVE

Here, we sought to explore and define the mechanisms of miR145 function in smooth muscle cells.

RESULTS

Using a combination of cell culture assays and in vivo mouse models to modulate miR145, we characterized its downstream actions on smooth muscle phenotypes. Our results show that the miR-143/145 gene cluster is induced in smooth muscle cells by coculture with endothelial cells. Endothelial cell-induced expression of miR-143/145 is augmented by Notch signaling and accordingly expression is reduced in Notch receptor-deficient cells. Screens to identify miR145-regulated genes revealed that the transforming growth factor (TGF)-β pathway has a significantly high number of putative target genes, and we show that TGFβ receptor II is a direct target of miR145. Extracellular matrix genes that are regulated by TGFβ receptor II were attenuated by miR145 overexpression, and miR145 mutant mice exhibit an increase in extracellular matrix synthesis. Furthermore, activation of TGFβ signaling via angiotensin II infusion revealed a pronounced fibrotic response in the absence of miR145.

CONCLUSIONS

These data demonstrate a specific role for miR145 in the regulation of matrix gene expression in smooth muscle cells and suggest that miR145 acts to suppress TGFβ-dependent extracellular matrix accumulation and fibrosis, while promoting TGFβ-induced smooth muscle cell differentiation. Our findings offer evidence to explain how TGFβ signaling exhibits distinct downstream actions via its regulation by a specific microRNA.

Inhibitors of the <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) pathway frequently induce hypertension when used to treat patients with advanced renal cell carcinoma (RCC). This analysis characterizes hypertension and hypertension-related events in patients treated with the VEGF pathway inhibitors axitinib or sorafenib in the AXIS trial. AXIS was a randomized phase III study of axitinib versus sorafenib in patients with metastatic RCC following failure of one prior systemic regimen. Patients with uncontrolled hypertension were excluded, but patients with hypertension controlled with antihypertensive medication were allowed to participate. Guidelines for hypertension management included adjustment or addition of antihypertensive medications and/or axitinib or sorafenib dose reductions, interruptions, or discontinuations. Treatment-emergent all-causality hypertension occurred in <em>145</em> (40.4 %) axitinib-treated patients (N = 359) and 103 (29.0 %) sorafenib-treated patients (N = 355), with grade 3 hypertension reported in 55 (15.3 %) and 38 (10.7 %) patients, respectively, and grade 4 hypertension reported in one (0.3 %) patient in each arm. Hypertension-related events led to axitinib dose interruptions (n = 46; 12.8 %), dose reductions (n = 16; 4.5 %), or discontinuations (n = 1; 0.3 %). Approximately 50 % of axitinib-treated patients with grade 3 or 4 hypertension continued treatment for ≥ 9 months. Hypertension-related sequelae occurred in <1 % of axitinib-treated patients. Hypertension was more frequently observed during treatment with axitinib than sorafenib in patients with RCC, but axitinib-induced hypertension rarely led to treatment discontinuation or cardio<em>vascular</em> sequelae. Recommendations for monitoring blood pressure and managing hypertension during axitinib therapy are presented.

Impaired materno-placental perfusion causes two important obstetric complications, fetal <em>growth</em> restriction and preeclampsia. This study investigated whether adenoviral vector-mediated overexpression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) in the uterine arteries (UtAs) increases uterine artery blood flow (UBF). First-generation adenovirus vectors (5 x 10(11) particles) containing the VEGF gene (Ad.VEGF-A or -D) or the beta-galactosidase reporter gene (Ad.lacZ) were injected into the UtAs of pregnant sheep (n=6) at 88-102 days of gestation (term=<em>145</em> days). UBF was measured using Doppler sonography before, and 4-7 days after injection. Mean UBF increased significantly from 233+/-156 (s.d.) ml min(-1) to 753+/-415 ml min(-1) following Ad.VEGF-A injection (P=0.005, n=5); Ad.lacZ infection had no significant effect. Organ bath experiments on uterine arterial sections 4-7 days after injection showed that, compared with Ad.lacZ vessels, Ad.VEGF-A-transduced vessels had a reduced contractile response to phenylephrine (E max 148+/-10.9 vs E max 228.2+/-27.5, P<0.05) but increased relaxation with bradykinin (pD2 (-log EC50) values 9.11+/-0.01 vs 8.65+/-0.11, P<0.05). Injection of Ad.VEGF-A into the UtAs increases UBF by enhancing vasodilatation. This may provide the basis for therapy in pregnancies complicated by uteroplacental insufficiency.