Targeting Vascular Endothelial Growth Factor Receptors Research Articles

Combined Anti-Angiogenic Therapy Targeting PDGF and VEGF Receptors Lowers the Interstitial Fluid Pressure in a Murine Experimental Carcinoma

Elevation of the interstitial fluid pressure (IFP) of carcinoma is an obstacle in treatment of tumors by chemotherapy and correlates with poor drug uptake. Previous studies have shown that treatment with inhibitors of platelet-derived growth factor (PDGF) or vascular endothelial growth factor (VEGF) signaling lowers the IFP of tumors and improve chemotherapy. In this study, we investigated whether the combination of PDGFR and VEGFR inhibitors could further reduce the IFP of KAT-4 human carcinoma tumors. The tumor IFP was measured using the wick-in-needle technique. The combination of STI571 and PTK/ZK gave an additive effect on the lowering of the IFP of KAT-4 tumors, but the timing of the treatment was crucial. The lowering of IFP following combination therapy was accompanied by vascular remodeling and decreased vascular leakiness. The effects of the inhibitors on the therapeutic efficiency of Taxol were investigated. Whereas the anti-PDGF and anti-VEGF treatment did not significantly inhibit tumor growth, the inhibitors enhanced the effect of chemotherapy. Despite having an additive effect in decreasing tumor IFP, the combination therapy did not further enhance the effect of chemotherapy. Simultaneous targeting of VEGFR and PDGFR kinase activity may be a useful strategy to decrease tumor IFP, but the timing of the inhibitors should be carefully determined.

Cardiovascular and renal toxicity during angiogenesis inhibition: clinical and mechanistic aspects

Inhibition of angiogenesis with humanized monoclonal antibodies to vascular endothelial growth factor (VEGF) or with tyrosine kinase inhibitors targeting VEGF receptors has become an established treatment for various tumor types. Contrary to expectations, angiogenesis inhibition by blocking VEGF-mediated signaling is associated with serious side effects including hypertension and renal and cardiac toxicity in a substantial proportion of patients. Fortunately, most of these side effects as discussed in this paper seem to be manageable, but likely become more problematic when survival increases. Although several hypotheses regarding the etiology of angiogenesis inhibition-related cardiovascular and renal side effects have been postulated, many of the underlying pathophysiological mechanisms remain to be elucidated. This may lead to the development of more specific angiogenesis inhibitors, better management of their side effects and may potentially provide new insights into the pathogenesis of cardiovascular disease in general.

Phase II Trial of Sorafenib in Metastatic Thyroid Cancer

Based on the pivotal role of Ras-Raf-MAP-ERK signaling and vascular endothelial growth factor (VEGF) in papillary thyroid cancer (PTC), we conducted a phase II clinical trial of sorafenib targeting RAF and VEGF receptor kinases in PTC. The primary end point was the objective response rate. Secondary end points included response correlation with serum thyroglobulin (Tg); functional imaging; tumor genotype; and signaling inhibition in tumor biopsies. Using a Simon minimax two-stage design, 16 or 25 chemotherapy-naïve metastatic PTC patients were to be enrolled in arm A (accessible tumor for biopsy). Arm B patients had other subtypes of thyroid carcinoma or prior chemotherapy, and did not require tumor biopsies. Patients received 400 mg orally twice per day of sorafenib. Response was assessed every 2 months using RECIST (Response Evaluation Criteria in Solid Tumors). Of 41 PTC patients, six patients had a partial response (PR; 15%; 95% CI, 6 to 29) and 23 patients (56%; 95% CI, 40 to 72) had stable disease longer than 6 months. Median duration of PR was 7.5 months (range, 6 to 14). Median progression-free survival was 15 months (95% CI, 10 to 27.5). In 14 (78%) of 18 Tg-assessable PTC patients, Tg declined more than 25%. Common grade 3 adverse events included hand-foot skin reaction, musculoskeletal pain, and fatigue. BRAF mutation was detected in 17 (77%) of 22 PTCs analyzed. Four of 10 paired tumor biopsies from PTC patients showed a reduction in levels of vascular endothelial growth factor receptor phosphorylation, ERK phosphorylation, and in VEGF expression during sorafenib therapy. No PRs were noted among non-PTC patients. Sorafenib is reasonably well-tolerated therapy with clinical and biologic antitumor activity in metastatic PTC.

Axitinib Induces Paradoxical Erythropoietin Synthesis in Metastatic Renal Cell Carcinoma

TO THE EDITOR: In a recent letter, Alexandrescu et al reported thoughtful data to address the occurrence of erythrocytosis after treatment with tyrosine kinase inhibitors (TKIs) sorafenib and sunitinib for metastatic cancers. In addition, it has been previously reported that patients with von Hippel-Lindau (VHL) disease treated with semaxanib, a TKI targeting vascular endothelial growth factor receptor 2 (VEGFR-2) and cKit, may experience a supra-additive increase in hematocrit level. Experimental studies showing hepatic erythropoietin (EPO) synthesis related to VEGF blockage could explain this iatrogenic polycythemia. We report a case of polycythemia in a patient treated for metastatic renal cell carcinoma with axitinib, another TKI targeting VEGFR-2. Our findings were the opposite of those in the report by Alexandrescu et al. A 52-year-old man, with no significant past medical history, underwent a right radical nephrectomy for clear renal cell carcinoma. In October 2002, a partial response was achieved with interferon-alfa treatment for lung metastases. In March 2004, the lung metastases progressed in number and size, and the patient subsequently was enrolled onto an axitinib phase II trial. Oral axitinib was administered continuously at the fixed dose of 5 mg twice daily. Minor adverse effects were reported, including diarrhea and asthenia. During the initiation of the second cycle (week 5), we observed an increase in hematocrit, which was confirmed during the subsequent cycles. An isotopic measurement of RBCs confirmed the diagnosis of polycythemia. Treatment was continued for 36 months, with a documented complete response by the third cycle. However, repeated therapeutic phlebotomies for polycythemia remained necessary. There was no alternative cause of polycythemia, including the absence of clinical evidence of VHL disease. In addition, no somatic VHL mutation in the tumor sample was found in polymerase chain reaction analysis. Endogenous EPO blood levels and VEGF-A levels were monitored, and results are listed in Table 1. Tam et al demonstrated that erythropoiesis is regulated by endogenous VEGF. In Tam’s preclinical study, high-grade VEGF blockage induced the repression of adult hepatic EPO production. The absence of VEGFR-2 expression on hepatocytes suggests the participation of liver sinusoidal endothelial cells with homeostatic VEGFR-2– dependant paracrine signaling. In our observation, axitinib treatment was associated with a paradoxical VEGF increase, as reported both in vivo and in a cohort of patients with metastatic gastrointestinal stromal tumors treated with sunitinib, another TKI with VEGFR-2 inhibition. However, we speculate that axitinib mimics VEGF starvation in organs (such as the liver) through VEGFR-2 blockade, and leads to hepatic EPO production. Finally, we cannot exclude an axitinib interaction with the hypoxiainducible factor pathway, which induces nitric oxide release, cutaneous vascular flow modification, and increased systemic EPO expression. Further investigation in clinical trials is warranted to determine the utility of EPO blood level as a marker of VEGF/ VEGFR modulator activity.

Vascular Changes in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most vascular solid tumors, in which angiogenesis plays an important role. The status of angiogenesis in HCC correlates with the disease progression and prognosis, and thus provides a potential therapeutic target. This review summarizes the vascular changes and molecular and cellular basis of angiogenesis in HCC. Development of HCC is characterized by arterialization of its blood supply and sinusoidal capillarization. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that plays a critical role in mediating angiogenesis in HCC. The VEGF can function on various types of cells, such as endothelial cells, hepatic stellate cells, endothelial progenitor cells and hemangiocytes, to induce vascular changes in HCC. Therefore, blockade of VEGF-mediated pathways, either by anti-VEGF neutralizing antibody or tyrosine kinase inhibitors that target VEGF receptors, suppresses carcinogenesis and angiogenesis in HCC. In addition to VEGF, several other angiogenic factors in HCC have recently been identified. These factors can also regulate angiogenic processes through interaction with VEGF or VEGF-independent pathways. Despite the fact that treatment of HCC remains a tough task due to lack of effective systemic therapy, antiangiogenic therapy has already entered clinical trials in HCC patients and sheds light on a promising novel treatment for this disease.

VEGF receptor antagonist Cyclo-VEGI reduces inflammatory reactivity and vascular leakiness and is neuroprotective against acute excitotoxic striatal insult.

BackgroundExcitotoxic brain insult is associated with extensive neuronal damage but could also cause inflammatory reactivity and vascular remodeling. The effects of the vascular endothelial growth factor (VEGF) inhibitor, Cyclo-VEGI on expression of VEGF, microgliosis and astrogliosis, blood-brain barrier (BBB) integrity and neuronal viability have been studied following intra-striatal injection of the excitotoxin, quinolinic acid (QUIN). The purpose of this study was to examine VEGF-dependent inflammatory responses in excitotoxin-injected brain and their dependence on pharmacological antagonism of VEGF receptors.MethodsSingle and double immunofluorescence staining of cellular (microglia, astrocyte, neuron) responses and dye and protein infiltration of blood-brain barrier have been applied in the absence, and presence, of pharmacological modulation using a VEGF receptor antagonist, Cyclo-VEGI. Dunn-Bonferroni statistical analysis was used to measure for significance between animal groups.ResultsDetailed analysis, at a single time point of 1 d post-QUIN injection, showed excitotoxin-injected striatum to exhibit marked increases in microgliosis (ED1 marker), astrogliosis (GFAP marker) and VEGF expression, compared with PBS injection. Single and double immunostaining demonstrated significant effects of Cyclo-VEGI treatment of QUIN-injected striatum to inhibit microgliosis (by 38%), ED1/VEGF (by 42%) and VEGF striatal immunoreactivity (by 43%); astrogliosis and GFAP/VEGF were not significantly altered with Cyclo-VEGI treatment. Leakiness of BBB was indicated by infiltration of Evans blue dye and plasma protein fibrinogen into QUIN-injected striatum with barrier permeability restored by 62% (Evans blue permeability) and 49% (fibrinogen permeability) with Cyclo-VEGI application. QUIN-induced toxicity was demonstrated with loss of striatal neurons (NeuN marker) and increased neuronal damage (Fluoro-Jade marker) with significant neuroprotection conferred by Cyclo-VEGI treatment (33% increase in NeuN and 38% decrease in Fluoro-Jade).ConclusionAn antagonist for VEGF receptor-mediated signaling, Cyclo-VEGI, has shown efficacy in a broad spectrum of activity against striatal excitotoxic insult including inhibition of microgliosis, reduction in leakiness of BBB and parenchymal infiltration of plasma fibrinogen and in conferring significant protection for striatal neurons. Antagonism of VEGF-mediated activity, possibly targeting VEGF receptors on reactive microglia, is suggested as a neuroprotective mechanism against inflammatory reactivity and a novel strategy to attenuate acute excitotoxic damage.

Inhibition of Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Phosphorylation on Tumor-Associated Endothelial Cells Leads to Treatment of Orthotopic Human Colon Cancer in Nude Mice

The purpose of our study was to determine whether the dual inhibition of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) signaling pathways in tumor-associated endothelial cells can inhibit the progressive growth of human colon carcinoma in the cecum of nude mice. SW620CE2 human colon cancer cells growing in culture and orthotopically in the cecum of nude mice expressed a high level of transforming growth factor alpha (TGF-α) and vascular endothelial growth factor (VEGF) but were negative for EGFR, human epidermal growth factor receptor 2 (HER2), VEGFR. Double immunofluorescence staining revealed that tumorassociated endothelial cells expressed EGFR, VEGFR2, phosphorylated EGFR (pEGFR), phosphorylated VEGFR (pVEGFR). Treatment of mice with either 7H-pyrrolo [2,3-d]-pyrimidine lead scaffold (AEE788; an inhibitor of EGFR and VEGFR tyrosine kinase) or CPT-11 as single agents significantly inhibited the growth of cecal tumors (P < .01); this decrease was even more pronounced with AEE788 combined with CPT-11 (P < .001). AEE788 alone or combined with CPT-11 also inhibited the expression of pEGFR and pVEGFR on tumor-associated endothelial cells, significantly decreased vascularization and tumor cell proliferation, increased the level of apoptosis in both tumorassociated endothelial cells and tumor cells. These data demonstrate that targeting EGFR and VEGFR signaling on tumor-associated endothelial cells provides a viable approach for the treatment of colon cancer.

Serum level of vascular endothelial growth factor (VEGF) as an independent prognostic factor in metastatic renal cell carcinoma (MRCC)

5044 Background: The French Immunotherapy Group previously showed that circulating interleukin-6 (IL-6) and VEGF correlate with survival in MRCC patients (pts) (Négrier JCO 2004). We aimed to confirm the prognostic value of IL-6 and VEGF, and to test that of macrophage colony-stimulating factor (M-CSF) and macrophage inflammatory protein 3-alpha (MIP-3a) for survival in MRCC pts of good and intermediate prognoses. Patients and Methods: Pre-treatment samples were obtained from 302 MRCC pts enrolled in 2 parallel multicenter randomized cytokine trials. Baseline serum levels of IL-6, M-CSF, VEGF, and MIP3a were determined by immunoassays; their prognostic value for progression-free survival (PFS) and overall survival (OS) was tested. Characteristics of pts with a 0.15 significance level in univariate analysis (ECOG PS, prior therapy, metastatic sites and biological parameters) were entered in a multivariate Cox model. A backward stepwise procedure discriminated non-influential variables at 0.05 level. Results: Median IL6, VEGF, M-CSF and MIP3a levels were respectively 7.3 (range 0–392 pg/mL), 0.5 (0–2.3 ng/mL), 406 (0–4,785 pg/mL) and 0 (0–1,705 ng/mL). IL6 or M-CSF and VEGF (p&lt;0.0001), or IL6 and M-CSF (p=0.0005) were significantly correlated. Death occurred in 192/302 pts, with 20.9 month median OS. OS was highly correlated with IL6, VEGF and M-CSF (p &lt;0.001) but not with MIP3a (p=0.06) in univariate analysis. Only VEGF remained independently prognostic for OS in multivariate analysis (39% increased probability of death if VEGF level doubled) after adjustment on PS, time from diagnosis to metastasis, number of metastatic sites, pleural metastasis, abnormal sedimentation rate, haemoglobin and LDH levels. Similar results were found for PFS, with a hazard ratio of 1.19 for a 2-fold VEGF increase. Conclusions: The independent prognostic value of serum IL6 level is not confirmed in our cohort that excluded pts with poor outcome, but serum VEGF level appears as an independent prognostic factor, thus supporting the therapeutic interest for these pts of newly available drugs targeting VEGF receptors. No significant financial relationships to disclose.

Anti-Vascular Endothelial Growth Factor (VEGF) Therapy: A New Cause of Hypertension

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that has multiple effects on the vascular endothelium and many other cells. Many cancers utilize VEGF for proliferation, progression and metastasis, making it a logical target of anti-tumor therapy. Numerous clinical oncology trials have employed this therapeutic approach to effectively treat various malignancies. Hypertension has emerged as one of the most important adverse effects of anti-VEGF therapy. This is supported by multiple clinical trials, using either anti-VEGF antibody, or small molecules targeting VEGF receptor tyrosine kinases. The incidence of hypertension associated with anti-VEGF antibody therapy ranges from 11 to 32% in most phase III clinical trials, while the incidence is about 16 to 43% with therapy targeted at VEGF receptors. The mechanisms underlying anti-VEGF associated hypertension are not understood. Several potential mechanisms have been proposed including vascular rarefaction, endothelial dysfunction, altered nitric oxide metabolism and aberrant neurohormones. More importantly, anti-VEGF related hypertension may have significant clinical implications. Poorly controlled hypertension may be associated with worsening cardiac and renal function, and reversible posterior encephalopathy syndrome. Further studies should focus on uncovering the mechanisms of anti-VEGF induced hypertension, as well as guiding therapy for this adverse effect.

Cross-species Vascular Endothelial Growth Factor (VEGF)-blocking Antibodies Completely Inhibit the Growth of Human Tumor Xenografts and Measure the Contribution of Stromal VEGF

To fully assess the role of VEGF-A in tumor angiogenesis, antibodies that can block all sources of vascular endothelial growth factor (VEGF) are desired. Selectively targeting tumor-derived VEGF overlooks the contribution of host stromal VEGF. Other strategies, such as targeting VEGF receptors directly or using receptor decoys, result in inhibiting not only VEGF-A but also VEGF homologues (e.g. placental growth factor, VEGF-B, and VEGF-C), which may play a role in angiogenesis. Here we report the identification of novel anti-VEGF antibodies, B20 and G6, from synthetic antibody phage libraries, which block both human and murine VEGF action in vitro. Their affinity-improved variants completely inhibit three human tumor xenografts in mice of skeletal muscle, colorectal, and pancreatic origins (A673, HM-7, and HPAC). Avastin, which only inhibits the tumor-derived human VEGF, is approximately 90% effective at inhibiting HM-7 and A673 growth but is <50% effective at inhibiting HPAC growth. Indeed, HPAC tumors contain more host stroma invasion and stroma-derived VEGF than other tumors. Thus, the functional contribution of stromal VEGF varies greatly among tumors, and systemic blockade of both tumor and stroma-derived VEGF is sufficient for inhibiting the growth of tumor xenografts.

Antivascular Therapy of Oral Tongue Squamous Cell Carcinoma with PTK787

Vascular endothelial growth factor (VEGF) is an important mediator in tumor vascularization, growth, and metastasis. We investigated whether blockade of the VEGF receptor (VEGF-R) signaling pathway by the tyrosine kinase inhibitor PTK787 combined with CPT-11, a semisynthetic camptothecin analogue, can inhibit the tumor growth and angiogenesis of squamous cell carcinoma of the oral tongue in an orthotopic nude mouse model. JMAR human oral squamous cell carcinoma cells were injected into the tongues of nude mice. Seven days later, the mice were randomized to receive a placebo, daily oral PTK787, weekly CPT-11 injection, or PTK787 plus CPT-11. After 4 weeks of treatment, the mice underwent necropsy, and the tongue tumors, cervical lymph nodes, and lungs were removed for immunohistochemical analyses. CPT-11, PTK787, and PTK787 plus CPT-11 significantly decreased tumor volumes and prolonged survival. The combination treatment group had the most significant decrease in volume and increase in survival. PTK787 alone or in combination with CPT-11 reduced the phosphorylation of VEGF-R in tumor cells and tumor-associated endothelial cells, was associated with decreased microvessel density, a decreased proliferative index, and an increased apoptotic index. PTK787 alone or the combination therapy resulted in apoptosis of both tumor cells and tumor-associated endothelial cells. These results suggest that targeting VEGF-R tyrosine kinase activity can be an effective therapeutic approach in squamous cell carcinoma of the oral tongue.