Soluble VEGFR-1 Research Articles

Comparison of serum VEGF and its soluble receptor sVEGFR1 with serum cell-free DNA in patients with breast tumor

Background: The observed elevated levels of vascular endothelial growth factor (VEGF), its soluble receptor (sVEGFR1) and cell-free DNA (cfDNA) in the serum of patients with various cancers have attracted much attention to the possibility of using these agents as biomarkers for cancers. We wanted to find out whether VEGF, VEGFR1 or cfDNA is a biomarker for breast cancer. Methods: In this study, we used enzyme-linked immunosorbent assay (ELISA) to examine the levels of serum VEGF and VEGFR1 in 23 patients with benign tumors, 19 patients with breast cancer and 32 age-matched healthy females. The levels of circulating cell-free DNA were measured using TaqMan real-time PCR analysis for the glyceraldehydes 3 phosphate dehydrogenase gene (GAPDH), We compared the serum levels of VEGF and its soluble receptors with those of the cell-free serum DNA. Results: In terms of serum levels of either VEGF or its soluble receptors VEGFR1, we found no significant difference between healthy individuals and women with benign breast tumors and breast cancer. However, there was a significant increase in circulating cell-free DNA in women with both benign and malignant breast tumors when compared with the corresponding healthy control group. We also found a significant negative correlation between VEGF and its soluble receptor VEGFR1 ( r = −0.328 and p = 0.004), and a significant positive correlation between VEGF and cell-free serum DNA ( r = 0.241 and p = 0.033). Conclusions: We can conclude that quantitative analysis of circulating cell-free DNA in serum may provide molecular biological understanding of breast tumors in women. It would also suggest that serum levels of VEGF and VEGFR1 have less significance.

Effects of Brief, Clustered Versus Dispersed Hypoxic Episodes on Systemic and Ocular Growth Factors in a Rat Model of Oxygen-Induced Retinopathy

Oxygen fluctuation patterns in preterm infants who develop retinopathy of prematurity (ROP) are varied and poorly represented in animal models. We examined the hypothesis that clustered (CL) episodes of hypoxia during hyperoxia results in a more severe form of oxygen-induced retinopathy (OIR) than dispersed episodes. Rat pups were exposed to alternating cycles of 1) 50% O2 with three CL episodes of 12% O2 every 6 h; or 2) 50% O2 with one episode of 12% O2 every 2 h, for 7 (P7) or 14 (P14) days postnatal age. Pups were killed after hyperoxia, or placed in room air (RA) until P21. RA littermates were killed at P7, P14, and P21. Systemic and ocular vascular endothelial growth factor (VEGF), soluble VEGFR-1 (sVEGFR-1), insulin-like growth factor I (IGF-I), and growth hormone were examined. All hyperoxia-exposed retinas had evidence of neovascularization. Animals in the CL group had a more severe form of OIR at P21 evidenced by vascular tufts, leaky vessels, retinal hemorrhage, and vascular overgrowth. These characteristics were associated with low body weight; high systemic and ocular VEGF; and low systemic and high ocular sVEGFR-1 and IGF-I. These data suggest that preterm infants who experience CL fluctuations in Pao2 during supplemental O2 therapy are at a higher risk for severe ROP.

The pharmacodynamic effect of TKI258 on plasma biomarkers of angiogenesis in patients with AML and advanced melanoma

14680 Background: TKI258 (dovitinib lactate), is a multi-tyrosine kinase inhibitor of VEGF receptors-1,2,3, FGF receptors-1, 2, 3, PDGFR-β, c-KIT and FLT3. To evaluate the pharmacodynamic effect of TKI258 in patients, a panel of plasma angiogenesis biomarkers were evaluated in two Phase I clinical trials - acute myeloid leukemia (AML) and locally advanced or metastatic melanoma. Methods: Plasma samples were analyzed from selected dosing groups of patients during the dose escalation stages of the two Phase I trials. 19 AML patients received TKI258 at doses ranging 200 - 600 mg/day, with an initial 14 day cycle (7 days on/7 days off), followed by a continuous daily dosing schedule (28 day cycle). 16 melanoma patients received 200 - 500 mg/day on once daily dosing (28 day cycle). Plasma VEGF, placental growth factor (PLGF), basic FGF (bFGF), and soluble VEGFR1 and VEGFR2 (sVEGFR1 and 2) were measured by multiplex assays using Meso-Scale Discovery platform. Plasma levels of each marker were frequently monitored during the course of treatment. Results: Average baseline VEGF levels were higher in the melanoma patients (234 pg/ml, n=16) than in the AML patients (68 pg/ml, n=19). In AML patients, dose- dependent inductions of VEGF (3–11 fold) and PLGF (3–8 fold) at 400 and 600 mg dosing were observed at Cycle 1 Days 3 and 7, and returned to near-baseline levels at the end of Cycle 1 at Day 14 after 1 week off drug treatment 1. In melanoma patients, inductions of VEGF (∼3 fold) and PLGF (2–9 fold) as well as decrease of sVEGFR2 (30–50%) in patients treated with 400 and 500 mg TKI258 were observed at the end of Cycle 1. Significant induction of bFGF (6-fold) was observed at 500 mg TKI258 treatment group in melanoma patients at the end of Cycle 1, but not at other doses or in the AML patients. Conclusions: PlasmaVEGF and PLGF induction and sVEGFR2 decrease could serve as pharmacodynamic biomarkers of VEGFR inhibition by TKI258. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Novartis Novartis Novartis Novartis

Sunitinib in combination with gefitinib in patients (pts) with metastatic renal cell carcinoma (mRCC): Updated results from a phase I/II study

16014 Background: Sunitinib malate is an oral, multitargeted tyrosine kinase inhibitor of VEGFRs and PDGFRs that has shown substantial efficacy in pts with mRCC (JAMA 2006;295:2516; NEJM 2007;356:115). Here, we present updated safety and efficacy data from the phase II part of a study of sunitinib in combination with gefitinib, an EGFR inhibitor. Methods: Eligibility included mRCC with a clear cell component, measurable disease, and ECOG performance status (PS) of 0 or 1. In phase II, pts received sunitinib 37.5 mg (the maximum tolerated dose [MTD] determined in phase I) orally, once-daily in 6-wk cycles (4 wks on treatment, 2 wks off) plus gefitinib 250 mg/day. The primary endpoint of phase II was RECIST-defined objective response rate (ORR). Sample size was determined using a one-sided, single-stage phase II design with a 10% alpha level and 85% power. Results are reported for all pts treated at MTD (N=35) from phase II (n=31) and phase I (n=4). An exploratory analysis of VEGF pathway biomarkers was performed for phase II. Results: Median age was 62 yrs (range: 29–78); 24 pts (69%) had ECOG PS of 0; and 24 pts (69%) had prior immunotherapy. The ORR was 34% (95% CI: 19–52), and 11 pts (31%) had stable disease. Median response duration was 18 mo (95% CI: 18-NR). Median progression-free survival was 11 mo (95% CI: 6-NR), and median overall survival had not been reached; 1-year survival probability was 82% (95% CI: 71–90). The most common grade 3 treatment-related AEs included diarrhea (14%) and gastrointestinal hemorrhage (6%); 3 pts were withdrawn due to treatment- related AEs (ejection fraction decline, cardiac arrhythmia, and asymptomatic ventricular tachycardia). Increases in plasma VEGF-A levels and decreases in plasma VEGF-C and soluble VEGFR-2 and VEGFR-3 levels were observed in patterns generally consistent with those for sunitinib monotherapy. In a preliminary analysis, no statistically significant correlations between these protein levels and efficacy were noted. Conclusions: Updated results from a study of sunitinib plus gefitinib further demonstrate the feasibility of this combination, with similar efficacy and tolerability to that observed with sunitinib monotherapy, along with similar patterns of biomarker modulation. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Pfizer Pfizer Pfizer Pfizer Pfizer

Phase I study of BAY 73–4506, an inhibitor of oncogenic and angiogenic kinases, in patients with advanced solid tumors: Final results of a dose-escalation study

2558 Background: BAY 73–4506 is a potent inhibitor of the receptors of tyrosine kinases (VEGFR, KIT, RET, FGFR and PDGFR) and serine/threonine kinases (RAF and p38MAPK). In tumor xenograft models, BAY 73–4506 demonstrated a broad spectrum antitumor activity. Methods: This phase I study was a dose-escalation trial investigating the safety, pharmacokinetic (PK), and pharmacodynamic (PD) profile of BAY 73–4506, given orally in 21 days on/7 days off cycles, until discontinuation due to toxicity or tumor progression. PK was assessed on days 1 and 21 of cycle 1. PD markers including DCE-MRI, soluble VEGFR-2 (sVEGFR-2) and VEGF plasma levels were assessed at each cycle. Tumor response was evaluated as per RECIST. Results: 52 patients (pts) with solid tumors and progressive disease were enrolled and treated with doses of 10 to 220 mg once daily. Frequent tumor types included CRC (31%), malignant melanoma (10%), and ovarian cancer (10%). The median treatment duration was 49.5 days (min. 3, max. 609). Drug-related adverse events (AEs) of all grades reported in >20% of pts were hoarseness (54%), dermatological toxicities (50%; CTC 3–4: 13%), mucositis (35%), diarrhea (25%; CTC 3: 2%), fatigue (23%; CTC 3: 2%), and hypertension (23%; CTC 3: 6%). Treatment-related AEs leading to dose reduction, interruption or discontinuation were hand foot skin reaction (15%), diarrhea (8%), and thrombopenia (6%). BAY 73–4506 showed dose-dependent increase in exposure up to 160 mg where it reached a plateau. The major metabolites (active in vitro and in vivo) also showed dose-dependent increases in exposure up to 220 mg and reached similar AUC(0–24)ss to the parent drug at 220 mg. Of the 33 evaluable pts, 9% achieved a partial response (PR), 64% had stable disease (SD), at least 7 weeks after start of treatment, and 48% had SD or PR for more than 11 weeks. Decrease in sVEGFR-2 level or iAUC60s of Gd-DTPA by DCE-MRI correlated with drug exposure. Conclusions: The recommended phase II dose for BAY 73–4506 is 160 mg daily, using the 21 days on/7 days off treatment schedule. Clinical activity (PR+SD) has been demonstrated in 73% of the evaluable pts. An extension cohort (dose level 160 mg) has been started. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Bayer Bayer Bayer Bayer Bayer Bayer

Analyses of plasma cytokine/angiogenic factors (C/AFs) profile during preoperative treatment with pazopanib (GW786034) in early-stage non-small cell lung cancer

7568 Background: Pazopanib is an oral angiogenesis inhibitor targeting VEGFR, PDGFR and c-kit. In a single-arm phase II trial (NCT00367679; VEG105290), patients received pazopanib 800 mg QD for 2–6 weeks before scheduled surgery. Tumor volume measurements were performed using high resolution CT images before and after treatment. An exploratory analysis was planned to describe the modulation of C/AFs during pazopanib treatment and identify potential predictive plasma markers. Methods: Plasma samples were collected pre- treatment and on the last day of pazopanib dosing. Levels of 52 C/AFs were measured by suspension bead multiplex assays (BioRad) or ELISA. Results were correlated with change in tumor volume. Statistical analyses included Wilcoxon tests and Pearson correlation tests. Results: 19 paired samples have been analyzed. Pazopanib treatment was associated with a significant modulation of 13 C/AFs, including a decrease in soluble VEGFR-2 (p<0.001) and increases in PlGF, IFN-α2 and HGF; chemokines/monokines: SDF1-α, CTACK, CXCL-10, CXCL-9, RANTES; and further factors: IL-12; M-CSF and TRAIL (all with p<0.05). PlGF showed the highest up-regulation during treatment with an average 15.9 fold increase, whereas changes compared to baseline for the remaining factors varied between 5 and 60%. The exploratory correlation of C/AF changes with tumor changes indicated an association with 6 factors (sVEGFR-2 (r = 0.77), VEGF (r = - 0.68), IL-16, IL-1RA, MCP-3, and IL-4; all with p<0.05). A significant correlation between baseline levels of C/AFs and tumor reduction was observed for 8 factors (PlGF, IL-2R, IL-12, IL-16, TRAIL, SCF, IL-3 and CTACK). Conclusions: Pazopanib treatment was associated with decreases in sVEGFR2 and increases in an additional 12 C/Afs, with PlGF demonstrating a particularly striking increase. The exploratory analysis identifies that baseline levels of several C/Afs may have predictive value for pazopanib treatment. Moreover a strong correlation between sVEGFR-2 changes during pazopanib treatment and tumor shrinkage was observed making this factor a potentially attractive marker of response. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration GlaxoSmithKline GlaxoSmithKline, Pneum Rx, General Electric GlaxoSmithKline, Pneum Rx GlaxoSmithKline GlaxoSmithKline

Axitinib (AG-013736) in patients with metastatic melanoma: A phase II study

9006 Background: No treatment has extended survival in stage IV melanoma. Melanomas are highly angiogenic and metastatic, express high levels of vascular endothelial growth factor receptors (VEGFRs) 2 and 3 and form vascular-like structures in vivo that may be exploited therapeutically. Axitinib is an oral, potent and selective inhibitor of VEGFRs 1, 2, 3 with preliminary evidence of clinical activity as monotherapy against similarly aggressive pro-angiogenic malignancies, such as metastatic renal cell carcinoma. Methods: 32 patients with ≤1 prior treatment for metastatic disease and ECOG PS 0–1 received single-agent axitinib at a starting dose of 5 mg BID. The primary endpoint was objective response rate (ORR) by RECIST criteria. BP was monitored regularly. Blood samples were also collected before and after therapy to explore pharmacodynamic (PD) and potential PK/PD relationships between clinical response and plasma soluble proteins. Results: The most common treatment-related adverse events (AEs) were fatigue (62.5%), hypertension (43.8%), hoarseness (34.4%), and diarrhea (31.3%). One patient experienced grade 5 bowel perforation. ORR was 15.6% (95% CI: 5.3, 32.8%) per investigator assessment with response duration ranging from 2.3 to >10.2 months. Median PFS was 2.3 months (95% CI: 1.8, 5.7). Median OS was 6.8 months (95% CI: 5.2, 10.4; range 0.8->22.6 months). The median OS of patients experiencing dBP ≥ 90 mm Hg on axitinib therapy was 13.0 months (n=13) vs 6.2 months for those with dBP < 90 mm Hg (n= 9). Furthermore, axitinib therapy selectively decreased soluble VEGFR2 and VEGFR3 (vs sKIT), and increased VEGF in the blood, demonstrating PD activity against the targeted VEGF receptors. The study of PK/PD relationships is ongoing. Conclusions: Axitinib has demonstrable single-agent activity in melanoma. Preliminary analyses indicate that patients who achieve elevated dBP ≥ 90 mm Hg on therapy have longer survival associated with potent and selective inhibition of targeted VEGFRs. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Pfizer Oncology Bayer, Genentech™ BioOncology, Novartis, Pfizer Oncology, Wyeth Pfizer Oncology Novartis, Pfizer Oncology Bayer, Bristol-Myers Squibb, Genentech™ BioOncology, Novartis, Pfizer Oncology, Wyeth

Continuous daily dosing (CDD) of sunitinib (SU) in pts with advanced GIST: Updated efficacy, safety, PK and pharmacodynamic analysis

10554 Background: SU is an oral multitargeted tyrosine kinase inhibitor of KIT, PDGFRs, VEGFRs, FLT3, CSF-1R and RET, approved multinationally for the treatment of advanced imatinib (IM)-resistant/intolerant GIST on an intermittent dosing schedule comprising 4 wk on 50 mg/d followed by 2 wk off treatment. The safety and efficacy of SU 37.5 mg CDD in pts with GIST after IM failure were evaluated in this phase II study. Methods: In this open-label, multicenter trial, pts were randomized either to morning (AM) or evening SU dosing. The primary endpoint was clinical benefit rate (CBR; % of pts with RECIST-defined CR, PR or SD ≥24 wk). Secondary endpoints included PFS, OS, ORR, safety/tolerability, PK parameters and plasma concentrations of potential biomarkers. Results: 60/61 randomized pts (30/arm) received CDD of SU. As of Dec. 2007, pts had started a median of 11 28-d treatment cycles (range 1–24). 24 pts had completed 1 y of study treatment, and 37 had discontinued. To date, the overall CBR is 55%, including 7 pts (12%) with PRs. Median PFS overall is 32 wk (95% CI 25–48). With 60% of pts still alive, median OS is estimated to be 88 wk (95% CI 68-NA). The most common treatment-emergent (all- causality) non-hematologic Aes of any grade were diarrhea (45%), abdominal pain (42%) and asthenia (38%). Grade 3/4 hematologic laboratory abnormalities included reduced levels of neutrophils (12%), hemoglobin (12%) and platelets (3%). Dose reduction to 25 mg due to Aes occurred in 14 pts (23%). Sunitinib CDD achieved constant drug exposure with no unexpected accumulation. 23 pts on AM dosing had plasma samples taken at ≥2 timepoints during treatment. Analysis of VEGF, soluble (s)VEGFR-2, sVEGFR-3 and sKIT concentrations in these samples supported a persistent pharmacodynamic effect of SU with CDD, without the concentration rebound observed during off-treatment periods with intermittent dosing. Preliminary analyses suggested a correlation between OS and decreases in plasma sKIT after the first 3 cycles, and particularly after cycle 5 (n=17; P=0.007). Conclusions: CDD of SU appears to be a safe and potentially effective dosing strategy for pts with GIST after IM failure. CDD is associated with constant drug exposure and a persistent pharmacodynamic effect. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Pfizer GlaxoSmithKline, Infinity, MedImmune, Novartis, Pfizer, PharmaMar, Roche, sanofi-aventis Pfizer GlaxoSmithKline, Novartis, Pfizer, PharmaMar, Roche, SigmaTau Infinity, Janssen-Cilag, Novartis, Pfizer, SigmaTau Infinity, Novartis, Pfizer Novartis, PharmaMar

Assessment of VEGF-receptor system expression in the porcine endometrial stromal cells in response to insulin-like growth factor-I, relaxin, oxytocin and prostaglandin E2

Several factors participate in regulation of growth and development as well as angiogenesis of the uterus during pregnancy, and hence little is known about the role of hormonal regulation of vascular endothelial growth factor (VEGF)-receptor system expression. This study has examined the effect of insulin-like growth factor-I (IGF-I), relaxin (RLX), oxytocin (OT) and prostaglandin (PG) E(2), on VEGF secretion and VEGF-receptor system mRNA expression in the porcine endometrial stromal cells. IGF-I and RLX were identified as the most effective inducers of VEGF secretion and mRNA expression. Although PGE(2) stimulated VEGF secretion and VEGF164 mRNA expression, OT inhibited both secretion and mRNA expression of VEGF. When tested for VEGF receptors (R), all factors failed to affect their mRNA expression. Media conditioned by stromal cells collected after IGF-I and RLX treatment significantly increased endothelial cell proliferation and this effect was blocked by soluble VEGFR-1. These data suggest that during early pregnancy IGF-I, RLX and PGE(2) can affect VEGF expression in the endometrium and therefore may support uterine and embryo development, implantation and pregnancy.

Successful inhibition of intracranial human glioblastoma multiforme xenograft growth via systemic adenoviral delivery of soluble endostatin and soluble vascular endothelial growth factor receptor-2

Glioblastoma multiforme (GBM) is characterized by neovascularization, raising the question of whether angiogenic blockade may be a useful therapeutic strategy for this disease. It has been suggested, however, that, to be useful, angiogenic blockade must be persistent and at levels sufficient to overcome proangiogenic signals from tumor cells. In this report, the authors tested the hypothesis that sustained high concentrations of 2 different antiangiogenic proteins, delivered using a systemic gene therapy strategy, could inhibit the growth of established intracranial U87 human GBM xenografts in nude mice. Mice harboring established U87 intracranial tumors received intravenous injections of adenoviral vectors encoding either the extracellular domain of vascular endothelial growth factor receptor-2-Fc fusion protein (Ad-VEGFR2-Fc) alone, soluble endostatin (Ad-ES) alone, a combination of Ad-VEGFR2-Fc and Ad-ES, or immunoglobulin 1-Fc (Ad-Fc) as a control. Three weeks after treatment, magnetic resonance imaging-based determination of tumor volume showed that treatment with Ad-VEGFR2-Fc, Ad-ES, or Ad-VEGFR2-Fc in combination with Ad-ES, produced 69, 59, and 74% growth inhibition, respectively. Bioluminescent monitoring of tumor growth revealed growth inhibition in the same treatment groups to be 62, 74, and 72%, respectively. Staining with proliferating cell nuclear antigen and with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling showed reduced tumor cell proliferation and increased apoptosis in all antiangiogenic treatment groups. These results suggest that systemic delivery and sustained production of endostatin and soluble VEGFR2 can slow intracranial glial tumor growth by both reducing cell proliferation and increasing tumor apoptosis. This work adds further support to the concept of using antiangiogenesis therapy for intracranial GBM.

Serum soluble VEGFR-1 is an independent risk factor for wasting and mortality in prevalent hemodialysis patients

Serum soluble VEGFR-1 is an independent risk factor for wasting and mortality in prevalent hemodialysis patients

A proangiogenic peptide derived from vascular endothelial growth factor receptor-1 acts through α5β1 integrin

AbstractVascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase receptor for growth factors of the VEGF family. Endothelial cells express a membrane-bound and a soluble variant of this protein, the latter being mainly considered as a negative regulator of VEGF-A signaling. We previously reported that the soluble form is deposited in the extracellular matrix produced by endothelial cells in culture and is able to promote cell adhesion and migration through binding to α5β1 integrin. In this study, we demonstrate that the Ig-like domain II of VEGFR-1, which contains the binding determinants for the growth factors, is involved in the interaction with α5β1 integrin. To identify domain regions involved in integrin binding, we designed 12 peptides putatively mimicking the domain II surface and tested their ability to inhibit α5β1-mediated endothelial cell adhesion to soluble VEGFR-1 and directly support cell adhesion. One peptide endowed with both these properties was identified and shown to inhibit endothelial cell migration toward soluble VEGFR-1 as well. This peptide directly binds α5β1 integrin, but not VEGF-A, inducing endothelial cell tubule formation in vitro and neoangiogenesis in vivo. Alanine scanning mutagenesis of the peptide defined which residues were responsible for its biologic activity and integrin binding.

VEGF‐D promotes tumor growth and lymphatic spread in a mouse model of hepatocellular carcinoma

Lymphatic spread is an important clinical determinant for the prognosis of hepatocellular carcinoma (HCC), but little is known about the control of lymphangiogenesis in HCC. We addressed expression and biological role of the pro-(lymph), angiogenic protein VEGF-D in this tumor entity. Using immunohistochemistry and in situ hybridization on specimens of HCC, cirrhotic and normal liver we found abundant expression of VEGF-D exclusively in the tumor cells. The cognate receptor VEGFR-3 was detected on blood and lymphatic vessels. By clinicopathological analysis VEGF-D expression was correlated with pT-stage of the primary, lymph node metastasis and lymphangiosis carcinomatosa. Three out of 4 human HCC cell lines expressed and secreted VEGF-D. To approach its biological function, VEGF-D deficient SKHep-1 cells were stably transfected with VEGF-D cDNA and effects on tumor progression were determined in vivo. Compared to mock-transfected controls, subcutaneous tumors derived from VEGF-D expressing cells were larger and more frequently metastasized to regional lymph nodes. VEGF-D expressing tumors exhibited increased microvessel density and increased abundance of peri- and intratumoral lymphatics, as assessed by immunostaining for CD31 and for LYVE-1 and/or podoplanin, respectively. Furthermore, coexpression of the soluble extracellular VEGFR-3 domain blocked VEGF-D-induced tumor growth and lymphatic spread via reduction of angiogenesis and lymphangiogenesis. In the orthotopic approach, VEGF-D expression resulted in an increased rate of intra- and extrahepatic as well as lymph node metastasis. In conclusion, our study suggests that expression of VEGF-D is involved in growth and lymphatic spread of HCC. Therefore, VEGF-D might represent a therapeutic target in HCC.

Prognostic and predictive value of vascular endothelial growth factor and its soluble receptors, VEGFR-1 and VEGFR-2 levels in the sera of small cell lung cancer patients

Small cell lung cancer (SCLC) has a rapid growth rate and is characterized by early metastases. Tumor growth is dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis. Whether surveillance of pre- and post-treatment serum VEGF and especially its receptors VEGF-1 and VEGF-2 levels in SCLC patients have impact on clinical outcome is unknown. From February 2001 to January 2003, 39 consecutive patients with histological proven SCLC were enrolled into the study. Pre-treatment (n: 39) and post-treatment (n: 25) samples of the same patients were collected at the time of their response evaluation. The levels of VEGF and its receptors VEGFR-1 and VEGFR-2 were measured in the serum by quantitative sandwich enzyme immunoassay technique. The median pre-treatment serum VEGF, VEGFR-1, and VEGFR-2 levels which were significantly higher than the normal controls were 1,200 pg/ml (range, 1,414.3 +/- 956.2 pg/ml), 85 pg/ml (range, 97.8 +/- 70.7 pg/ml), and 11,550 pg/ml (range, 14,481 +/- 6,267 pg/ml), respectively. We detected a poor but positive correlation between VEGF and VEGFR-2 (r: 0.46, p: 0.003). Pre-treatment low serum VEGF (<728.5 pg/ml) value (p: 0.02) and good response to treatment (p: 0.008) were found as good prognostic factors by multivariate analysis. Low serum VEGF concentration is a significant and independent prognostic factor in SCLC patients. Surveillance of VEGF and its receptors to predict chemotherapy response is not useful. Whether the levels of serum VEGF and its receptors VEGFR-1 and VEGFR-2 have value in detecting treatment modalities of SCLC need further studies.

Positive Feedback between Vascular Endothelial Growth Factor-A and Autotaxin in Ovarian Cancer Cells

Tumor cell migration, invasion, and angiogenesis are important determinants of tumor aggressiveness, and these traits have been associated with the motility stimulating protein autotaxin (ATX). This protein is a member of the ectonucleotide pyrophosphatase and phosphodiesterase family of enzymes, but unlike other members of this group, ATX possesses lysophospholipase D activity. This enzymatic activity hydrolyzes lysophosphatidylcholine to generate the potent tumor growth factor and motogen lysophosphatidic acid (LPA). In the current study, we show a link between ATX expression, LPA, and vascular endothelial growth factor (VEGF) signaling in ovarian cancer cell lines. Exogenous addition of VEGF-A to cultured cells induces ATX expression and secretion, resulting in increased extracellular LPA production. This elevated LPA, acting through LPA(4), modulates VEGF responsiveness by inducing VEGF receptor (VEGFR)-2 expression. Down-regulation of ATX secretion in SKOV3 cells using antisense morpholino oligomers significantly attenuates cell motility responses to VEGF, ATX, LPA, and lysophosphatidylcholine. These effects are accompanied by decreased LPA(4) and VEGFR2 expression as well as by increased release of soluble VEGFR1. Because LPA was previously shown to increase VEGF expression in ovarian cancer, our data suggest a positive feedback loop involving VEGF, ATX, and its product LPA that could affect tumor progression in ovarian cancer cells.

An Open, Phase I Study of Cediranib in Patients with Acute Myeloid Leukemia (AML).

Prognosis is poor for the majority of AML patients, particularly the elderly and those with relapsed or refractory AML, and more effective treatment options are needed. Vascular endothelial growth factor-A (VEGF) and its receptors (VEGFRs) may contribute to the pathophysiology of AML and are considered as potential therapeutic targets. Cediranib (RECENTIN™; AZD2171) is an oral, highly potent and selective VEGF signaling inhibitor with activity versus VEGFR-1, -2 and -3, and additional activity against c-Kit. This two-part study evaluated the safety and tolerability of multiple once-daily oral doses of cediranib in patients with relapsed or refractory AML and elderly patients with de novo or secondary AML, with secondary assessments of pharmacokinetics (PK), pharmacodynamics (PD) and efficacy. Part A was a dose-escalation phase to determine the maximum tolerated dose (MTD); Part B was an expansion phase to explore the MTD and a lower, biologically active dose. A total of 35 patients (mean age, 68 years [range 50–81]; 15 male, 20 female) entered the study and received treatment with cediranib (Part A, n=23; Part B, n=12). Part A comprised 10mg (n=4), 20mg (n=6) and 30mg (n=13) dose cohorts, and intra-patient dose escalation was permitted. In Part B, patients received cediranib 20mg (n=3) or 30mg (n=9). Cediranib ≤30mg was generally well tolerated; diarrhea (n=19), hypertension (n=14) and fatigue (n=13) were the most common adverse events (AEs). Cediranib 30mg was the highest dose studied, and was declared the MTD based on dose-limiting toxicities of hypertension and diarrhea experienced by 3 patients receiving 30mg in Part A. Maximal plasma concentrations at steady-state were attained 0.83–4.1 h post-dosing. Steady-state plasma concentrations were attained after approximately 7 days of repeated once-daily dosing. Following multiple oral doses of cediranib 30mg in Parts A and B, the unbound geometric mean Css,min was 5x the IC50 value required to inhibit proliferation of human umbilical vein endothelial cells in preclinical assays, supporting the once-daily oral dosing regimen. The relationship between PK and two PD parameters (VEGF levels and blood pressure) were investigated and there appeared to be a positive correlation between exposure (Css,max and AUCss) and plasma VEGF levels, but not blood pressure. Time-dependent changes in soluble VEGFR-2 levels were observed. A best investigator assessment of AML objective response according to predefined criteria (Cheson et al, J Clin Oncol 2003; 21:4642–9) was observed for 6 patients (1 morphological complete remission with incomplete blood recovery [Cri], 4 partial responses and 1 minor response). However, none of the responses resulted in improvement in normal hemopoiesis. No responses were observed in 6 patients with Flt-3 ITD mutations. One patient remained on treatment for 455 days. Once-daily oral cediranib ≤30mg was generally well tolerated in patients with AML, with encouraging preliminary evidence of activity as a monotherapy. The AE and PK profiles of cediranib in AML are consistent with those seen in solid tumors.

Vascular endothelial growth factor and its soluble receptor in benign and malignant ovarian tumors

An imbalance between pro-angiogenic and anti-angiogenic factors is hypothesized in the pathogenesis of ovarian cystic disease. The aim of the following study was to explore the possible role of free vascular endothelial growth factor receptor 1 (sVEGFR-1), a soluble regulator of vascular endothelial growth factor (VEGF) action, in ovarian cystoadenoma, endometriomata and cystoadenocarcinoma. Forty-eight women, of whom fourteen had ovarian serous cysts, twenty-eight had stage III–IV ovarian endometriomata, and six had stage IIIB–IIIC ovarian carcinoma, were included. Sampling of serum, peritoneal and ovarian cystic fluids and of tumor tissue was performed before, during and following surgery, respectively. Levels of VEGF and sVEGFR-1 were measured in serum, peritoneal and cystic fluid. VEGF and sVEGFR-1 expression was evaluated in tumor tissue. There were no differences in serum VEGF and sVEGFR-1 levels nor in VEGF/VEGFR-1 ratio between study groups. Peritoneal fluid VEGF levels were higher in cystoadenocarcinoma patients than in endometriosis and in cystoadenoma patients, while sVEGFR-1 peritoneal fluid concentrations were significantly higher only in endometriosis-affected women. VEGF/VEGFR-1 ratio was highest in the peritoneal fluid of cancer patients with respect to the other two groups of women. Cystic fluid VEGF and VEGFR-1 concentrations were higher in endometriomata and in cystoadenocarcinomas than in cystadenomas but the VEGF/VEGFR-1 ratio was highest in cancer patients. Western blot evidenced a marked expression of VEGF and soluble VEGFR-1 in endometriosis tissue with respect to benign cyst tissue but a lower expression of both molecules, contrary to that expected, in cancer tissue. In conclusion, all in all, our data indicate that an excess of local VEGF with respect to its soluble receptor VEGFR-1 may be a key factor in the onset and maintenance of pathological neo-angiogenesis in ovarian cyst formation.

Impaired Angiogenesis After Hindlimb Ischemia in Type 2 Diabetes Mellitus

Deficient angiogenesis after ischemia may contribute to worse outcomes of peripheral arterial disease in patients with diabetes mellitus (DM). Vascular endothelial growth factor (VEGF) and its receptors promote angiogenesis. We hypothesized that in peripheral arterial disease, maladaptive changes in VEGF ligand/receptor expression could account for impaired angiogenesis in DM. Skeletal muscle from diet-induced, type 2 diabetic (DM) and age-matched normal chow (NC)-fed mice was collected at baseline and 3 and 10 days after hindlimb ischemia and analyzed for expression of VEGF (n=10 per group), full-length VEGF receptor (VEGFR)-1, soluble VEGFR-1, and markers of downstream VEGF signaling (n=20 per group) using ELISA, reverse transcriptase-polymerase chain reaction, and Western blots. In the absence of ischemia, DM mice had increased VEGF (NC versus DM: 26.6±2.6 versus 53.5±8.8 pg/mg protein; P &lt;0.05), decreased soluble and membrane-bound VEGFR-1 (NC versus DM: 1.44±0.30 versus 0.85±0.08 and 1.03±0.10 versus 0.72±0.10, respectively; P &lt;0.05), decreased phospho-AKT/AKT and phospho–endothelial NO synthase/endothelial NO synthase (NC versus DM: 0.76±0.2 versus 0.38±0.1 and 0.36±0.06 versus 0.25±0.04, respectively; P &lt;0.05), and no change in VEGFR-2. After ischemia, both DM and NC had comparable increases in VEGF-A. VEGFR-1 and soluble VEGFR-1 expression increased in both groups, but the fold increase was significantly greater in DM. These data demonstrate that soluble VEGFR-1, an angiogenesis inhibitor, is regulated in skeletal muscle by type 2 DM and ischemia. In the absence of ischemia, despite reductions in both soluble VEGFR-1 and VEGFR-1, VEGF ligand signaling is lower in DM compared with controls. After ischemia, maladaptive upregulation of these receptors further reduces the capacity of VEGF to induce an angiogenic response, which may provide a novel target for therapy.

Abstract 333: Vascular Endothelial Growth Factor-B (VEGF-B) Induces Myocardium-specific Angiogenesis via VEGFR-1 and Neuropilin-1

Growth factor gene therapy is a new, promising tool for treatment of myocardial ischemia. Development of safe and effective clinical applications requires optimal selection of growth factors and knowledge of the mechanisms involved. We have previously discovered that adenovirally over-expressed (Ad) VEGF-B 186 induces tissue-specific angiogenesis in the myocardium. Another VEGFR-1 specific ligand, placental growth factor (PlGF) induced angiogenesis both in the heart and skeletal muscle. We hypothesized that these two VEGFR-1 ligands utilize different angiogenic mechanisms. Unlike PlGF, the effect of VEGF-B was not dependent on the up-regulation of endogenous VEGF-A since soluble decoy VEGFR-2 did not block the effect. In contrast to PlGF or VEGF-A, the therapeutic effect of VEGF-B was not blocked by inhibition of nitric oxide synthesis by feeding the animals L-NAME, indicating that a novel angiogenic pathway was involved. Western blot (WB) analysis of the transduced tissues showed that in the myocardium AdVEGF-B 186 was proteolytically processed, while majority of the protein in the skeletal muscle was unprocessed. Processed form of VEGF-B binds to another angiogenic receptor neuropilin-1 (Nrp-1). Immunohistochemical staining and WB showed strong Nrp-1 expression in the myocardium while Nrp-1 expression was below detection limit in the skeletal muscle. In addition, down-stream molecules GIPC and GAIP were co-localized with Nrp-1 suggesting Nrp-1 receptor activation. Co-transduction with soluble decoy AdsNrp-1 or AdsVEGFR-1 blocked the angiogenic effects of VEGF-B 186 in the myocardium indicating that both receptors are involved in the angiogenic pathway. VEGF-B induces angiogenesis in the myocardium via VEGFR-1 and Nrp-1. This novel myocardium-specific angiogenic pathway offers a safe tool for growth factor therapies and provides new information about repair mechanisms of the heart.

Vasoactive and Permeability Effects of Vascular Endothelial Growth Factor-165 in the Term in Vitro Dually Perfused Human Placental Lobule

Vascular endothelial growth factor (VEGF) is an important vasodilator and effector of permeability in systemic blood vessels. Molecular and tissue culture techniques have provided evidence for its placental synthesis and release. Using an in vitro dual-perfusion model of the term placental lobule from normal pregnancy, we report here the relative secretion of total VEGF, soluble VEGF receptor (VEGFR)-1, and free VEGF into the maternal and fetoplacental circulations of the placenta. We tested the hypothesis that VEGF has vasomotor and permeability effects in the fetoplacental circulation of the human placenta, and we examined the broad intracellular pathways involved in the vasodilatory effect that we found. We show that total VEGF is released into the fetal and maternal circulations in a bipolar fashion, with a bias toward maternal side output. Soluble VEGFR-1 was also secreted into both circulations with bias toward the maternal side. Consequently, free VEGF (12.8 +/- 2.4 pg/ml, mean +/- se) was found only in the fetoplacental circulation. VEGF-165 was found to be a potent vasodilator of the fetoplacental circulation (maximum response: 77% of previous steady-state fetal-side inflow hydrostatic pressure after preconstriction with U46619; EC(50) = 71 pm). This vasodilatory effect was mediated by the VEGFR-2 receptor and nitric oxide in a manner-independent of the involvement of prostacyclin and the src-family tyrosine kinases. However, nitric oxide could explain only 50% of the vasodilatory effect. Finally, we measured the permeability of the perfused placenta to inert hydrophilic tracers and found no difference in the presence and absence of VEGF.