Vascular Endothelial Growth Factor, a Key Modulator of the Anti-Tumor Immune Response.

During tumor growth, angiogenesis is required to ensure oxygen and nutrient transport to the tumor. Vascular endothelial growth factor (VEGF) is the major inducer of angiogenesis and appears to be a key modulator of the anti-tumor immune response. Indeed, VEGF modulates innate and adaptive immune responses through direct interactions and indirectly by modulating protein expressions on endothelial cells or vascular permeability. The inhibition of the VEGF signaling pathway is clinically approved for the treatment of several cancers. Therapies targeting VEGF can modulate the tumor vasculature and the immune response. In this review, we discuss the roles of VEGF in the anti-tumor immune response. In addition, we summarize therapeutic strategies based on its inhibition, and their clinical approval.

Bioenergetic effects of hydrogen sulfide suppress soluble Flt-1 and soluble endoglin in cystathionine gamma-lyase compromised endothelial cells.

Endothelial dysfunction is a hallmark of preeclampsia, a life-threatening complication of pregnancy characterised by hypertension and elevated soluble Fms-Like Tyrosine Kinase-1 (sFlt-1). Dysregulation of hydrogen sulfide (H2S) by inhibition of cystathionine γ-lyase (CSE) increases sFlt-1 and soluble endoglin (sEng) release. We explored whether compromise in CSE/H2S pathway is linked to dysregulation of the mitochondrial bioenergetics and oxidative status. We investigated whether these effects were linked to CSE-induced sFlt-1 and sEng production in endothelial cells. Here, we demonstrate that CSE/H2S pathway sustain endothelial mitochondrial bioenergetics and loss of CSE increases the production of mitochondrial-specific superoxide. As a compensatory effect, low CSE environment enhances the reliance on glycolysis. The mitochondrial-targeted H2S donor, AP39, suppressed the antiangiogenic response and restored the mitochondrial bioenergetics in endothelial cells. AP39 revealed that upregulation of sFlt-1, but not sEng, is independent of the mitochondrial H2S metabolising enzyme, SQR. These data provide new insights into the molecular mechanisms for antiangiogenic upregulation in a mitochondrial-driven environment. Targeting H2S to the mitochondria may be of therapeutic benefit in the prevention of endothelial dysfunction associated with preeclampsia.

Role of Chemotherapy, VEGFR Inhibitors, and mTOR Inhibitors in Advanced Perivascular Epithelioid Cell Tumors (PEComas).

PURPOSE: Perivascular epitheliod cell tumors (PEComas) are rare mesenchymal neoplasms for which the role of systemic treatments is not established as there are no published prospective clinical trials or sufficiently large retrospective case series. The aim of this study is to clarify the activity of conventional chemotherapy and biological agents in advanced/metastatic PEComas. EXPERIMENTAL DESIGN: This was an observational, retrospective, international study that included patients with advanced/metastatic PEComa treated with systemic therapy at 5 European sarcoma reference centers and within the Italian Rare Cancer Network. Survival analyses were performed using the Kaplan-Meier method and the Cox hazards regression models. RESULTS: A total of 53 patients were included. Cytotoxic chemotherapy regimens were active only in a small proportion of PEComas. Gemcitabine-based regimens [objective response rate (ORR): 20%, median progression-free survival (PFS): 3.4 months] seemed to have the same activity of anthracycline-based regimens (ORR: 13%, median PFS: 3.2 months). Antiangiogenic agents resulted in disease stabilization in some patients, with a number having density changes/tissue response on imaging, with an ORR of 8.3% and a median PFS of 5.4 months. mTOR inhibitors were the most active agents, with an ORR of 41% and a median PFS of 9 months. CONCLUSIONS: Our study provides data for the selection of systemic therapy in patients with advanced/metastatic PEComa: mTOR inhibitors are the most active agents. Antiangiogenics and chemotherapy with gemcitabine-based regimens or anthracycline-based regimens are options in further line, but with a lower response rate and PFS.

Hit discovery of 4-amino-N-(4-(3-(trifluoromethyl)phenoxy)pyrimidin-5-yl)benzamide: A novel EGFR inhibitor from a designed small library.

Searching for hit compounds within the huge chemical space resembles the attempt to find a needle in a haystack. Cheminformatics-guided selection of few representative molecules of a rationally designed virtual combinatorial library is a powerful tool to confront this challenge, speed up hit identification and cut off costs. Herein, this approach has been applied to identify hit compounds with novel scaffolds able to inhibit EGFR kinase. From a generated virtual library, six 4-aryloxy-5-aminopyrimidine scaffold-derived compounds were selected, synthesized and evaluated as hit EGFR inhibitors. 4-Aryloxy-5-benzamidopyrimidines inhibited EGFR with IC50 1.05-5.37 µM. Cell-based assay of the most potent EGFR inhibitor hit (10ac) confirmed its cytotoxicity against different cancerous cells. In spite of no EGFR, HER2 or VEGFR1 inhibition was elicited by 4-aryloxy-5-(thio)ureidopyrimidine derivatives, cell-based evaluation suggested them as antiproliferative hits acting by other mechanism(s). Molecular docking study provided a plausible explanation of incapability of 4-aryloxy-5-(thio)ureidopyrimidines to inhibit EGFR and suggested a reasonable binding mode of 4-aryloxy-5-benzamidopyrimidines which provides a basis to develop more optimized ligands.

Vitamin D Supplementation Prevents Placental Ischemia Induced Endothelial Dysfunction by Downregulating Placental Soluble FMS-Like Tyrosine Kinase-1.

Maternal vitamin D deficiency in pregnancy has been associated with an increased risk of preeclampsia. Vascular endothelial dysfunction is a major phenotype of pregnancies with preeclampsia, contributing to increased maternal hypertension and proteinuria. We sought to determine whether vitamin D supplementation would alleviate preeclampsia associated endothelial dysfunction and explore the underlying mechanism using the reduced uterine perfusion pressure (RUPP) rat model. RUPP operated rats were supplemented with 1,25(OH)2D (RUPP+VD) on day 1, 7, and 14 of pregnancy by subcutaneous injection. On day 19 of pregnancy, after the measurement of blood pressure and urine collection, maternal blood serum and placenta samples were collected. 1,25(OH)2D treatment significantly improved endothelial dysfunction by reducing apoptosis and increasing nitric oxide (NO) production in blood vessels of RUPP operated rats compared to untreated RUPP rats. 1,25(OH)2D significantly down-regulated the expression of placental soluble FMS-like tyrosine kinase-1 (sFlt-1) in RUPP rats. Furthermore, the circulating sFlt-1 levels in maternal serum were positively correlated with the expression of placental sFlt-1 and were restored to a normal pregnant level by 1,25(OH)2D treatment in RUPP rats. Incubation of endothelial cell line with rat serum from RUPP+VD group significantly increased NO production and decreased caspase-3 activity compared with serum from untreated RUPP rats. Moreover, neutralization of sFlt-1 using the specific antibody mimicked the effect of 1,25(OH)2D, which abolished the deleterious effect of RUPP rat's serum on NO production and apoptosis. These results suggest that vitamin D supplementation is protective against RUPP induced endothelial dysfunction by downregulating placental sFlt-1, which can possibly alleviate preeclampsia associated symptoms.

Phase I study of dovitinib (TKI258), an oral FGFR, VEGFR, and PDGFR inhibitor, in advanced or metastatic renal cell carcinoma.

PURPOSE: Signaling through the fibroblast growth factor (FGF) pathway may account for tumor resistance to antiangiogenic therapies targeting the VEGF pathway. Here, dovitinib (TKI258), a potent oral inhibitor of FGF receptor, VEGF receptor (VEGFR), and platelet-derived growth factor receptor tyrosine kinases, is studied in a dose escalation trial. EXPERIMENTAL DESIGN: Patients with advanced or metastatic renal cell carcinoma (RCC) with predominant clear cell histology were treated with oral dovitinib 500 or 600 mg/day (5-days-on/2-days-off schedule). RESULTS: Twenty heavily pretreated patients (median 3 prior regimens) were enrolled, with 16, 11, and 12 patients having previously received at least 1: VEGFR inhibitor, mTOR inhibitor, and immunotherapy, respectively. Fifteen and 5 patients were treated in 500- and 600-mg cohorts, respectively. Three patients experienced dose-limiting toxicities: grade 2 bradycardia (500 mg), grade 4 hypertensive crisis (600 mg), and grade 3 asthenia with grade 2 nausea and vomiting (600 mg). The most common adverse events related to dovitinib were nausea (75%), diarrhea (70%), vomiting (70%), and asthenia (50%), the majority of which were mild (grade 1 or 2), with grade 3 events 5% or less (except asthenia, 15%) and only one grade 4 event (hypertensive crisis). Two patients achieved a partial response (500 mg), and 12 patients had stable disease, including 2 patients with long lasting disease stabilizations (>1 year) in the 500-mg cohort. CONCLUSIONS: Dovitinib was tolerable and showed antitumor activity at a maximum tolerated dose of 500 mg on a 5-days-on/2-days-off schedule in heavily pretreated RCC patients.

Synthesis and biological profile of the pan-vascular endothelial growth factor receptor/tyrosine kinase with immunoglobulin and epidermal growth factor-like homology domains 2 (VEGF-R/TIE-2) inhibitor 11-(2-methylpropyl)-12,13-dihydro-2-methyl-8-(pyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (CEP-11981): a novel oncology therapeutic agent.

A substantial body of evidence supports the utility of antiangiogenesis inhibitors as a strategy to block or attenuate tumor-induced angiogenesis and inhibition of primary and metastatic tumor growth in a variety of solid and hematopoietic tumors. Given the requirement of tumors for different cytokine and growth factors at distinct stages of their growth and dissemination, optimal antiangiogenic therapy necessitates inhibition of multiple, complementary, and nonredundant angiogenic targets. 11-(2-Methylpropyl)-12,13-dihydro-2-methyl-8-(pyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (11b, CEP-11981) is a potent orally active inhibitor of multiple targets (TIE-2, VEGF-R1, 2, and 3, and FGF-R1) having essential and nonredundant roles in tumor angiogenesis and vascular maintenance. Outlined in this article are the design strategy, synthesis, and biochemical and pharmacological profile for 11b, which completed Phase I clinical assessing safety and pharmacokinetics allowing for the initiation of proof of concept studies.

Isolation and structural characterization of glycosides from an anti-angiogenic extract of Monnina obtusifolia H.B.K.

Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is of physiological and pathological importance. Substantial data over the last decade has implicated uncontrolled angiogenesis with various pathological states. Vascular endothelial growth factors (VEGFs) play a critical role in its regulation, and have become one of the most interesting anti-angiogenesis targets. We have investigated the anti-angiogenic potential of plant extracts in a preliminary ELISA screening. The n-BuOH extract obtained from the leaves of Monnina obtusifolia (Polygalaceae) demonstrated an inhibition of VEGF-A or Placental Growth Factor interaction with Flt-1 (VEGF receptor 1), with an inhibition over 50% in particular for VEGF-A/Flt-1 interaction at a concentration of 500 µg/mL. Successively fractionation of the bioactive n-BuOH extracts of M. obtusifolia aerial parts led to the isolation of six new compounds, 1-O-(4-hydroxy-2-methylene-butanoic acid)-6-O-ß-D-(4-hydroxy-2-methylene-butanoyl)-glucopyranose (1), 1-O-(isopentenyl)-6-O-ß-D-(4-hydroxy-2-methylene-butanoyl)-glucopyranose (2), 1-O-(4-hydroxy-2-methylene-butanoic acid)-6-O-ß-D-(isovaleroyl)-glucopyranose (3), 1-O-(3-methylbut-3enyl)-6-O-ß-D-(isovaleroyl)-glucopyranose (4), two new sucrose esters, 3,4-O-ß-D-di-feruloyl-fructofuranosyl-6-O-α-D-(p-coumaroyl)-glucopyranoside (5), and 3,4-O-ß-D-di-feruloyl-fructofuranosyl-6-O-α-D-(caffeoyl)-glucopyranoside (6), together with known flavonoids. Their structures were established on the basis of detailed spectral analysis. Since none of the isolated compounds showed a relevant inhibition of VEGFs, the biological activity observed for the butanolic extract might be due to the presence of a combination of compounds acting synergistically.

Competitive ELISA-based screening of plant derivatives for the inhibition of VEGF family members interaction with vascular endothelial growth factor receptor 1.

The activation of vascular endothelial growth factor receptor-1 (VEGFR-1, also known as Flt-1) is crucial in many physiological and pathological conditions, like angiogenesis, cancer, inflammation, hematopoiesis, bone marrow precursors/stem cells recruitment in tumor angiogenesis, and metastasis formation. Many recent reports indicate that molecules able to antagonize Flt-1 activity have gained a strong interest in the view of therapeutic approaches. In order to identify new compounds able to interfere in the Flt-1 recognition by VEGFs family members, we have developed a highly sensitive competitive ELISA-based screening to study plant extracts and derivatives. Several fractions of the N-butanol extract of Pteleopsis suberosa leaves and of the chloroform extract of Parinari campestris leaves demonstrated by a bioassay-guided fractionation an evident inhibition of VEGF-A or placental growth factor (PlGF) interaction with Flt-1, with an inhibition over 50 % in particular for the VEGF-A/Flt-1 interaction at a concentration of 100 microg/mL. This activity seems be due to the presence of a combination of compounds acting synergistically.

Role of anti-angiogenesis agents in treating NSCLC: focus on bevacizumab and VEGFR tyrosine kinase inhibitors.

OPINION STATEMENT: Successful inhibition of angiogenesis with the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab has improved the efficacy seen with standard cytotoxic therapy in NSCLC. The addition of bevacizumab to first-line chemotherapy improved response rate and progression free survival and added 2 months to median overall survival for those patients with advanced stage NSCLC on the treatment arm of E4599. Bevacizumab is now a standard agent to add to frontline carboplatin and paclitaxel for patients with newly diagnosed NSCLC who meet the eligibility criteria from the landmark E4599 trial. Unfortunately about half of all patients are not eligible either because they have squamous histology, brain metastases, or are on anti-coagulation. Ongoing trials are further exploring the safety of bevacizumab in these patient populations, as well as in combination with other cytotoxic regimens. Exploration of other applications of bevacizumab in the second-line and adjuvant setting are ongoing as well. The largest class of drugs that block angiogenesis are the multi-targeted tyrosine kinase inhibitors (TKIs) that target the VEGF receptor (VEGFR). These drugs are still in development, and though two are now on the market for treating other malignancies, their role in NSCLC is under investigation. These agents have the advantages of hitting multiple targets, convenient oral administration, and potential for lower cost. Their lack of target specificity leads to unexpected toxicity, but also promising efficacy. For example, the overall objective response rate of 9.5% with single agent sunitinib compares similarly to that of pemetrexed or docetaxel in previously treated NSCLC patients, but toxicity, notably fatigue, lead to discontinuation in 38% of patients. Hypertension, hemorrhage and cavitation are common toxicities amongst this class of agents. Rash, fatigue, myalgia, and hand-foot syndrome are more specifically seen with TKIs. These compounds may also be synergistic or additive with traditional cytotoxic chemotherapy drugs and other novel compounds. In early trials sorafenib as a single agent has shown no clinical response in previously treated NSCLC patients, whereas clinical benefit in combination with erlotinib or chemotherapy has been seen in early studies. Vandetanib has demonstrated objective responses as a single agent and in combination with chemotherapy in previously treated NSCLC patients. A phase I trial of AZD2171 with carboplatin and paclitaxel in newly diagnosed advanced stage NSCLC also demonstrated promising results with 6 of 15 patients achieving partial responses. NSCLC specific trials are also underway, or in development for pazopanib, axitinib, AMG 706, XL647, enzastaurin, and other TKIs. Other anti-angiogenesis agents with different mechanisms of action include thalidomide and its derivatives, monoclonal antibodies to the VEGFRs, and VEGF Trap, a chimeric molecule which combines extracellular portions of VEGFR1 and VEGFR2 with the Fc portion of immunoglobulin G1 to form a molecule that binds and "traps" VEGF. Despite modest improvements, prognosis continues to be poor for patients with advanced NSCLC. Bevacizumab is a first step into the world of angiogenesis inhibitors for NSCLC and though it only offers a modest survival benefit in a limited patient population, it paves the way for the development of the next generation of anti-angiogenesis inhibitors. We can hope that further improvements in survival will follow.

Green tea extract inhibits angiogenesis of human umbilical vein endothelial cells through reduction of expression of VEGF receptors.

Epidemiological and animal studies have indicated that consumption of green tea is associated with a reduced risk of developing certain forms of cancer. However, the inhibitory mechanism of green tea in angiogenesis, an important process in tumor growth, has not been well established. In the present study, green tea extract (GTE) was tested for its ability to inhibit cell viability, cell proliferation, cell cycle dynamics, vascular endothelial growth factor (VEGF) and expression of VEGF receptors fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/Kinase insert domain containing receptor (Flk-1/KDR) in vitro using human umbilical vein endothelial cells (HUVECs). GTE in culture media did not affect cell viability but significantly reduced cell proliferation dose-dependently and caused a dose-dependent accumulation of cells in the G1 phase. The decrease of the expression of Flt-1 and KDR/Flk-1 in HUVEC by GTE was detected with immunohistochemical and Western blotting methods. These results suggest that GTE may have preventive effects on tumor angiogenesis and metastasis through reduction of expression of VEGF receptors.

A new model for random screening inhibitors of vascular endothelial growth factor receptor 1 kinase.

AIM: To establish a 96-well plate based kinase assay using a recombinant vascular endothelial growth factor (VEGF) receptor 1 kinase domain protein. METHODS: A human VEGF receptor 1 kinase domain protein was expressed in E coli, and its activity was monitored by its ability of phosphorylating the polyE4Y substrate coated on the walls of 96-well plates with antibody recognition and a colorimetric readout. A random screening of a sample organic compound library was carried out, and the hits were characterized with a transformed cell line stably expressing VEGF receptor 1 protein. RESULTS: An efficient E coli expression system for human VEGF receptor 1 kinase domain protein was constructed, and the purified recombinant protein was used to establish a practical screening assay for kinase inhibitors in vitro. Two thousand eight hundred organic compounds were screened, and two disubstituted furans (A1 and A5) with new structure showed inhibition of VEGF receptor 1 kinase. Compound A1 inhibited only phosphorylation of substrate, while compound A5 inhibited both autophosphorylation and substrate phosphorylation. Both inhibitors affected phosphorylation in the transformed cells. CONCLUSION: The recombinant receptor kinase based assay is simple and effective in identifying kinase inhibitors.