Endothelial Growth Factor Family Research Articles

Upregulation of miR-33 Exacerbates Heat-Stress-Induced Apoptosis in Granulosa Cell and Follicular Atresia of Nile Tilapia (Oreochromis niloticus) by Targeting TGFβ1I1.

High temperature affects egg quality and increases follicular atresia in teleosts. The present study aimed to explore the regulated mechanism of ovary syndrome of Nile tilapia (Oreochromis niloticus) exposed to heat stress. To this end, we conducted histological and biochemical analyses and integrated miRNA-target gene analyses. The histochemical analyses confirmed that heat stress promoted the apoptosis of granulosa cell and therefore resulted in increased follicular atresia in the ovary. Heat stress led to the differential expression of multiple miRNAs (miR-27e, -27b-3p, -33, -34a -133a-5p, and -301b-5p). In a luciferase activity assay, miR-33 bound to the 3′-untranslated region (UTR) of the TGFβ1I1 (transforming growth factor-β1-induced transcript 1) gene and inhibited its expression. A TGFβ1I1 gene signal was detected in the granulosa cells of Nile tilapia by immunohistochemical analysis. Up-regulation of the miR-33 of tilapia at 6 d and 12 d exposed to heat (34.5 °C ± 0.5 °C) had significant down-regulation of the TGFβ1I1 expression of the gene and protein in tilapia ovaries. An miRNA-target gene integrated analysis revealed that miR-33 and TGFβ1I1 function in an apoptosis-related signal pathway. The signal transduction of the vascular endothelial growth factor (VEGF) family members VEGFA and its receptor (KDR) in the heat-stressed group decreased significantly compared with the control group. Transcript-levels of the Bax and Caspase-3 as apoptotic promotors were activated and Bcl-2 and Caspase-8 as apoptotic inhibitors were suppressed in the heat-stressed tilapia. These results suggest that heat stress increases the expression of miR-33, which targets TGFβ1I1 and inhibits its expression, resulting in decreased levels of follicle-stimulating hormone and 17β-estradiol and increased apoptosis by suppressing VEGF signaling, eventually inducing follicular atresia. In conclusion, our results show that the miR-33/TGFβ1I1 axis of Nile tilapia is involved in the follicular development of broodstock, and can suppress VEGF signaling to accelerate follicular atresia. Our findings demonstrate the suppressive role of miR-33 during oocyte development in Nile tilapia.

Abstract 11825: The Association Between Frailty and Vascular Endothelial Growth Factor Families in Patients With Heart Failure: The PREHOSP-CHF Study

Background: Frailty is a complex clinical syndrome associated with ageing and chronic illness, and is common in heart failure (HF). Vascular endothelial dysfunction including maladaptive angiogenesis and lymphangiogenesis is involved in the pathogenesis of HF, and is also considered to be the causes of frailty. We investigated the association of vascular endothelial growth factor (VEGF) families, central regulators of angiogenesis and lymphangiogenesis, with frailty, and prognostic role of these cytokines in frail HF patients. Methods: We performed a multicenter prospective cohort study to determine the predictive value of VEGF families for prognosis among patients with HF. A total of 1,024 patients (mean age, 75.5 years, 58.7% male) were included in the analyses. Serum levels of VEGF, VEGF-C, VEGF-D, and soluble VEGF receptor-2 (sVEGFR-2) were measured. Frailty was assessed by Canadian Study of Health and Aging Clinical Frailty Scale (CFS). Results: A total of 256 (25.1%) patients had frailty (CFS≥5) at baseline. Frail patients were older, more likely female, and had lower body mass index, higher NYHA class, and higher rates of prior hospitalization for HF, HF with preserved ejection fraction, anemia, cerebrovascular disease, and dementia. N-terminal pro brain natriuretic peptide, high sensitivity troponin I, and high sensitivity C-reactive protein levels were higher in frail patients. During the 2-year follow-up, 211 all cause death occurred. In Kaplan-Maier analysis, frail patients showed significantly higher incidence of all-cause (hazard ratio [HR], 4.12; 95% confidence interval [CI], 3.14-5.42). Regarding VEGF families, frail patients had significantly lower levels of sVEGFR-2 and VEGF-C, and higher levels of VEGF-D. Multiple regression analysis revealed that VEGF-C had inverse correlation with CFS (P, 0.03). After adjusting for clinical confounders, a low VEGF-C level was independently associated with all-cause death in frail patients (HR, 0.74; 95%CI, 0.58-0.94 for 1-SD increase), but was not in non-frail patients (HR, 0.87; 95%CI, 0.64-1.14 for 1-SD increase) (P for interaction, 0.09). Conclusions: In HF patients, a low VEGF-C value was associated with frailty and was independent risk for all-cause death in frail patients.

Impact of hypertension on clinical outcome in patients treated with vascular endothelial growth factor inhibitors

Abstract Background Vascular endothelial growth factor (VEGF) family is overexpressed in the tumor microenvironment and induces tumor angiogenesis.1 VEGF signaling pathway (VSP) inhibitors are essential therapeutic drugs for solid cancer patients.2 However, VSP inhibitors often provoke drug-specific anti-angiogenesis in normal tissues, which was referred to as on-target toxicity.2 Hypertension (HTN) is one of the most frequent adverse events caused by VSP inhibitors. A previous report demonstrated that VSP inhibitor-induced hypertension is a favorable prognostic factor, contrarily.3However, multiple VSP inhibitors are administered in various cancer types, and then the relation between VSP inhibitor-associated hypertension and clinical outcome is still controversial. Purpose The aim of this study is to elucidate the impacts of HTN and the timing of onset on clinical outcome during cancer therapy with VSP inhibitors. Method We reviewed 2,348 patients who were treated with VSP inhibitors from the LIFE Study database, consisting of 14 municipality-level information from claims data between 2016 and 2020. According to the timing of HTN onset mode, the patients were stratified into 3 groups; (1) new-onset HTN group (n=334): de novo development after VSP inhibitor administration, (2) pre-existing HTN group (n=1,363): existing HTN at baseline, including aggravation after VSP inhibitor initiation, and (3) no HTN group (n=651) (Figure 1). The time to treatment failure (TTF) was applied as a surrogate clinical indicator of overall survival. Event-free survival analysis with the log-rank test was conducted for time to first treatment failure amongst the 3 groups. In addition, Cox proportional hazard models adjusted with clinical characteristics were performed to investigate independent factors for TTF. Results In the event-free survival analysis, both the new-onset HTN and the pre-existing HTN were associated with prolongation of TTF, compared to the non-HTN (p<0.001 and p<0.001, respectively, by Bonferroni correction) (Figure 2). The New-onset HTN was significantly associated with longer TTF than pre-existing HTN (p<0.001). In Cox proportional hazard model adjusted with age, sex, past medical history, primary cancer lesion, and type of VSP inhibitors, the new-onset HTN and the pre-existing HTN were independent favorable factors, compared to the non-HTN [new-onset HTN: Hazard ratio (HR) 0.62, 95% confidence interval (CI) 0.54 - 0.71, p<0.001; and pre-existing HTN: HR 0.85, 95% CI 0.77 - 0.94, p<0.005]. Moreover, the New-onset HTN was also a significant factor for longer TTF, compared to the pre-existing HTN [HR 0.72, 95% CI 0.64 - 0.82, p<0.001]. Conclusion In patients treated with VSP inhibitors, both new-onset and pre-existing HTN are independent factors for a favorable clinical outcome, especially new-onset HTN after VSP inhibitors administration. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) Figure 1. Patient screening and enrollmentFigure 2. HTN & TTF

Glycosylation at Asn254 Is Required for the Activation of the PDGF-C Protein.

Platelet-derived growth factor C (PDGF-C) is a member of the PDGF/VEGF (vascular endothelial growth factor) family, which includes proteins that are well known for their mitogenic effects on multiple cell types. Glycosylation is one of the most important forms of posttranslational modification that has a significant impact on secreted and membrane proteins. Glycosylation has many well-characterized roles in facilitating protein processing and contributes to appropriate folding, conformation, distribution, and stability of proteins that are synthesized intracellularly in the endoplasmic reticulum (ER) and Golgi apparatus. Although the general process and functions of glycosylation are well documented, there are most likely others yet to be discovered, as the glycosylation of many potential substrates has not been characterized. In this study, we report that the PDGF-C protein is glycosylated at three sites, including Asn25, Asn55, and Asn254. However, we found that mutations at any of these sites do not affect the protein expression or secretion. Similarly, disruption of PDGF-C glycosylation had no impact on its progression through the ER and Golgi apparatus. However, the introduction of a mutation at Asn254 (N254 A) prevents the activation of full-length PDGF-C and its capacity for signaling via the PDGF receptor. Our findings reveal that glycosylation affects PDGF-C activation rather than the protein synthesis or processing. This study characterizes a crucial modification of the PDGF-C protein, and may shed new light on the process and function of glycosylation.

P0966PLACENTAL GROWTH FACTOR DEFICIENCY AGGRAVATES DIABETIC NEPHROPATHY

Abstract Background and Aims Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family. PlGF exerts favorable angiogenetic and lymphangiogenic activity by binding to VEGF-R1 and -R3, respectively. Due to its functional synergy with VEGF-A, it is required for a correct neovascularization during pathological conditions while inactivation of PlGF contributes to decrease of pathological angiogenesis. Because reduced angiogenesis and lymphagiogenesis that contribute to defective lipid drainage are implicated in the progression diabetic kidney disease, we investigated the role of PlGF in the development of diabetic nephropathy by using PlGF-knockout mice. Method Diabetes was induced by a low-dose streptozotocin injection in 9-week-old male C57BL/6J PlGF-KO and wild-type mice and biochemical and morphological parameters were examined at 12 weeks later. Results In diabetic PlGF-KO mice, fasting blood glucose and HbA1c levels increased significantly and the development of glomerular sclerosis and mesangial area expansion were accompanied by albuminuria. Diabetic PlGF-KO mice exhibited increased expression of type IV collagen, transforming growth factor-β1 and glomerular IHC staining for nephrin, PECAM-1 and WT-1-positive cells and VEGF-R1,-R2,-R3 expression decreased, suggesting decreased endothelial cell and podocyte structure. Intrarenal expression of pLKB1, and pAMPK decreased and that of PPARα, PGC1α, ERRα, p-eNOS, and urinary Nox concentration decreased while iNOS increased, indicating disturbed lipid metabolism and endothelial dysfunction in the same group. Increased intrarenal FFA, TG, and cholesterol concentration represents presence of lipid accumulation. F4/80- and TUNEL-positive cells increased, suggesting increased inflammatory cell infiltration and apoptosis. CD68 and arginase-II increased indicating that macrophage subtype M1 polarization is involved in the inflammatory process. Expression of Bcl2/bax decreased and that of SOD1 and 2 decreased, indicating increased apoptosis and oxidative stress, respectively. Increased expression of intrarenal 8-OHdG and urinary isoprostane level indicates increased oxidative stress. Conclusion Impaired angiogenesis and lymphangiogenesis are implicated in PlGF deficiency and this promotes lipotoxicity-induced inflammation, oxidative stress and deteriorates renal functional and phenotypic parameters in the diabetic kidney.

Study of gene correlation between endothelial growth factor family in mouse eye

Objective To compare the function and action pathways of VEGFA, VEGFB and VEGFC in VEGF family of mouse eye. Methods Using the BXD mouse gene data in Genenetwork database as template to compare and study the similarities and differences of VEGFA, VEGFB and VEGFC molecular pathways or potential functions in the whole genome expression spectrum of BXD recombinant mouse inbred line population, with multiple analytical methods and statistical strategies were used, such as gene expression level, target genes comparison, top genes comparison associated to target genes, expression Quantitative Trait Loci (eQTL). Results Matrix comparison showed strong positive correlation between two probes of VEGFC (r=0.732, P 0.05). In the comparative analysis of the relevant Top50 genes of each VEGF gene, most of the genes in BXD mouse were not significantly correlated with VEGFA, VEGFB and VEGFC except for the weak association of individual related genes. The results of eQTL analysis showed that each probe of VEGF gene was located on different chromosomes. Conclusions The expression levels and positive and negative correlations of VEGFA, VEGFB and VEGFC were different in the VEGF family of mouse eye, suggesting that these genes may play their role through different pathways. Key words: Vascular endothelial growth factors; DNA probes; Genome components

An intron variant in the FLT1 gene increases the risk of preeclampsia in Iranian women

ABSTRACTBackground: Preeclampsia is a hypertensive disorder that affects pregnancy, mother, and fetus. Pathogenesis of preeclampsia could be associated with the angiogenesis pathways. The vascular endothelial growth factor (VEGF) family is one of the important factors for normal pregnancy and angiogenesis. Genetic variations in the gene family members may play a role in the etiology of preeclampsia. We investigated the possible association between VEGFA gene rs3025039, and VEGFR1 (FLT1) gene rs722503 polymorphisms and preeclampsia in a sample of Iranian patients. Methods: Genotyping was performed in 395 women, including, 204 pre-eclamptic pregnant women and 191 healthy normotensive pregnant women by using the PCR-RFLP method. Results: The rs722503 polymorphism was associated with preeclampsia under the dominant model (P = 0.04, OR = 1.53, 95% CI: 1.03–2.27). No significant difference was observed for the rs3025039 alleles and genotypes in the studied groups. Conclusions: Based on our study, rs722503 polymorphism in the FLT1 gene may play an important role in susceptibility to preeclampsia.

Vascular restenosis in coronary artery bypass grafting might be associated with VEGF-C/VEGFR-3 signaling pathway

The vascular endothelial growth factor (VEGF) family of peptides and caveolins (CAVs) are reported to contribute, in early graft failure in patients, a coronary artery bypass grafting (CABG). To investigate the possible association of ultimate luminal occlusion to VEGFs and CAVs expression, a functional analysis (based on the molecular biology, bioinformatics, histology, and clinical studies) was performed. Twenty-four hundred and sixty-eight CABG patients diagnosed with multivessel stable coronary artery disease (CAD) were enrolled into prospective study and assigned to two subgroups: double- and triple-vessel CAD subjects. Distal parts of all the harvested saphenous vein (SV) and internal thoracic artery (ITA) segments were used for further tests. ITA graft failure did not differ between double-vessel and triple-vessel CAD patients. The number of SV occlusions was significantly higher in triple-vessel CAD subjects. The microarray analysis performed on SV and ITA samples obtained exclusively from triple-vessel CAD patients who developed early graft occlusion revealed 383 genes with increased and 301 genes with decreased expression in ITA samples as compared to SV grafts. This was followed by functional analysis of ‘blood vessel development’ group of genes. Average VEGF-C expression in ITA grafts was higher than in corresponding SV grafts; FLT4 expression was significantly higher in SV than in ITA transplants. VEGFR-3 and CAV3 expression demonstrated immunohistochemically in SMCs of the tunica media of SV grafts predicted their early restenosis in triple-vessel CAD patients. CAV2 protein expression in SMCs of ITA grafts indicated the risk of early graft failure both in double-vessel and triple-vessel CAD subjects.

Vascular Endothelial Growth Factor-A (VEGF-A) Single Nucleotide Polymorphisms and Endometriosis: Still a Controversial Issue:

Endometriosis is a chronic, multifactorial, polygenic gynecological disease. Endometrium has a high angiogenic potential and endometriotic lesions involve large areas with a rich blood supply. Angiogenesis is controlled by numerous inducers, including the vascular endothelial growth factor (VEGF) family. Endometrium undergoes cyclical growth and regression during the menstrual cycle, which depends on ovarian steroid levels and is a rich source of angiogenic growth factors, including VEGF-A. The VEGF-A gene is located on chromosome 6p21.3 and it is highly polymorphic with more than 20 different variants. This article critically reviews the published data concerning the relationships between some of the VEGF-A single nucleotide polymorphisms and the risk, pathogenesis and stage of endometriosis. Contrasting results have been published in the literature - probably due to the different ethnic background and the number of patients enrolled in clinical trials. However, the increasing interest in the use of anti...

Abstract 5735: Comprehensive analysis of serum levels of VEGF and its receptors in patients with uterine cervical cancer

Abstract Objective: Angiogenesis is one of the processes that is critical for the growth, invasion and metastasis of solid tumors, including uterine cervical cancer (CC). The vascular endothelial growth factor (VEGF) family is one of the major pathways involved in tumor angiogenesis. The aim of this study was to determine whether serum levels of these angiogenic factors could be used as biomarkers in patients with CC.Methods: A total of 107 patients with International Federation of Gynecology and Obstetrics (FIGO) stage IB to IIB CC who were treated at Tottori University Hospital between 2006 and 2015 were enrolled in this study. The study was approved by the Institutional Review Board of the School of Medicine of Tottori University. All patients gave written informed consent before the collection of specimens according to institutional guidelines. Serum samples were collected before initial surgery and levels of VEGF-A, -C, VEGFR-1, and VEGFR-2 were analyzed by enzyme-linked immunosorbent assay (ELISA). We evaluated the association between the levels of these angiogenic factors and clinicopathologic variables. With a median follow-up duration of 1743 days, survival analysis of 93 patients treated between 2006 and 2013 was performed. We also determined the mRNA expression of VEGF-A by real-time RT-PCR in fresh frozen tumors and the protein expression by immunohistochemical staining in paraffin-embedded tumors from CC patients. The mRNA levels of VEGF-A relative to GAPDH were used for the analysis.Results: Median levels of serum VEGF-A, -C, VEGFR-1, and VEGFR-2 were 313, 8122, 68, and 6210 pg/ml, respectively. We found a significant positive correlation between VEGF-A levels and the maximum tumor diameter (P=0.010). Patients with pelvic lymph node involvement (PLNI) showed significantly higher levels of VEGF-A and VEGFR-2 than those without PLNI. Patients with bulky tumor or parametrial infiltration (PI) also showed significantly higher levels of VEGF-A. In contrast, patients with a bulky tumor or PLNI showed significantly lower levels of VEGFR-1. Both histological types and FIGO stage were not related to levels of these angiogenic factors. We set the cutoff values of these factors at the median levels of the angiogenic factors. The 5-year overall survival rate (OS) for patients with high VEGF-A levels was significantly lower than those with low levels (92.9% vs. 72.8%, P = 0.014). The 5-year OS for patients with high VEGFR-2 levels was significantly lower than those with low levels (94.7% vs. 73.8%, P = 0.012). PLNI and PI were prognostic factors for overall survival. Multivariate analysis revealed that PLNI, VEGF-A, and VEGFR-2 levels were independent prognostic factors. The mRNA and protein expressions of VEGF-A were strongly observed in the cancer cells. There was no correlation between mRNA levels and serum levels of VEGF-A.Conclusion: These results suggest that serum VEGF-A may be a promising prognostic biomarker for CC. Citation Format: Daiken Osaku, Tetsuro Oishi, Mayumi Sawada, Hiroaki Komatsu, Jun Chikumi, Akiko Kudoh, Michiko Nonaka, Shinya Sato, Muneaki Shimada, Hiroaki Itamochi, Tasuku Harada. Comprehensive analysis of serum levels of VEGF and its receptors in patients with uterine cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5735. doi:10.1158/1538-7445.AM2017-5735

Abstract 4583: Potential role of neuropilin-2 in the regulation of c-MET-induced PD-L1 expression in renal cancer cells

Abstract Neuropilin-1 and -2 (NRP1 and NRP2) are the transmembrane glycoproteins that act as co-receptors for class III semaphorins and several members of the vascular endothelial growth factor (VEGF) family. In conjunction with VEGF receptors and plexins, NRPs and semaphorins modulate various biological responses. Recent studies suggest that NRPs are up-regulated in many types of cancer cells, and may play a major role in tumor growth. In addition to semaphorins and VEGF, NRP1 and NRP2 can also bind to hepatocyte growth factor (HGF), which is the ligand for the tyrosine kinase receptor c-MET. c-MET is highly over-expressed in renal cancer cells; however, the impact of NRPs on c-MET-mediated tumorigenic pathways have not been studied. Recently, we have reported that HGF-/c-MET-induced signaling increases the expression of immunosuppressive and negative-co-stimulatory molecule programmed death-1 ligand (PD-L1) on renal cell carcinoma (RCC) cells, and it protects tumor cells from immune-mediated killing. In this study, we investigated whether NRPs and its ligands can modulate the expression of HGF-/c-MET-induced PD-L1. We have observed that NRP2, and to a lesser extent NRP1, is over-expressed in human RCC cell lines (786-O, ACHN and Caki-1) compared with normal renal epithelial cells at both mRNA and protein level. siRNA-mediated silencing of NRP1 and NRP2 promoted apoptosis of renal cancer cells, suggesting possible role(s) of NRPs in tumor growth. The knock-down of NRP2 down-regulated the cell surface expression of PD-L1 possibly through the reduced interaction of growth-promoting ligands (like, HGF) with NRP2. Interestingly, SEMA3F, a class III semaphorin ligand for both NRP1 and NRP2, has been reported to have anti-tumorigenic effects. We observed that SEMA3F was significantly down-regulated in RCC cells compared with normal renal epithelial cells; and when we added SEMA3F to the cultures of renal cancer cells, it down-regulated both basal and HGF-/c-MET-induced PD-L1 expression. Together, our findings suggest that NRPs (primarily NRP2) can play a critical role in regulating HGF-/c-MET-induced PD-L1 expression in renal cancer cells to mediate immune escape of tumor cells. Therefore targeting NRP2 and/or its activation through SEMA3F/synthetic ligands may restrict c-MET-PD-L1-induced tumor promoting pathways in renal cancer cells. Citation Format: Murugabaskar Balan, Soumitro Pal. Potential role of neuropilin-2 in the regulation of c-MET-induced PD-L1 expression in renal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4583.

Abstract 3387: Serum VEGF-A may predict prognosis in patients with epithelial ovarian cancer

Abstract Objective: Angiogenesis is one of the processes that is critical for the growth, invasion and metastasis of solid tumors, including epithelial ovarian cancer (EOC). The vascular endothelial growth factor (VEGF) family is one of the major pathways involved in tumor angiogenesis. The aim of this study was to determine whether serum levels of these angiogenic factors could be used as biomarkers in patients with EOC. Methods: A total of 133 patients with EOC who were treated at Tottori University Hospital between 2006 and 2014 were enrolled in this study. The study was approved by the Institutional Review Board of the School of Medicine of Tottori University. All patients gave written informed consent before the collection of specimens according to institutional guidelines. Serum samples were collected before initial surgery and levels of VEGF-A, -C and VEGFR1 were analyzed by enzyme-linked immunosorbent assay (ELISA). We evaluated the association between the levels of VEGF-A, -C, and VEGFR1 and clinicopathologic variables. We also determined the mRNA expression VEGF-A, -C, and VEGFR1 by real-time RT-PCR in fresh frozen tumors and the protein expression by immunohistochemical staining in paraffin-embedded tumors from EOC patients. Additionally, serum samples were collected before and after bevacizumab treatment, and levels of angiogenic factors were examined. Results: The mRNA and protein expressions of VEGF-A, -C, and VEGFR1 were strongly observed in the cancer cells. Median levels of serum VEGF-A, -C and VEGFR1 were 284, 4861 and 417 pg/ml, respectively. The levels of VEGF-A ans VEGFR1 in patients at stage III-IV were significantly higher than those in patients at stage I-II. Both histologic subtypes and lymph node involvement were not related to levels of these angiogenic factors. We found a significant positive correlation between VEGF-A levels and the ascitic volume. The overall survival rate was significantly lower in FIGO stage III or IV patients with EOC. We set the cutoff value of these factors at the median levels of each angiogenic factors. The 5-year survival rate for patients with high VEGF-A levels was significantly lower than those with low levels (60.7% vs. 45.5%, P = 0.029). Both VEGFR1 and VEGF-C levels were not related to outcome of patients. Multivariate analysis revealed that serum VEGF-A level and FIGO stage were independent prognostic factors. VEGF-A levels dramatically decreased after treatment with bevacizumab. Conclusion: These results suggest that serum VEGF-A may be a promising prognostic biomarker for EOC. Further study is needed to determine if could be predictive biomarker. Citation Format: Hiroaki Komatsu, Tetsuro Oishi, Hiroaki Itamochi, Mayumi Sawada, Michiko Nonaka, Seiya Sato, Jun Chikumi, Shinya Sato, Muneaki Shimada, Tasuku Harada. Serum VEGF-A may predict prognosis in patients with epithelial ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3387.

Abstract 3386: Serum VEGF-A may predict prognosis in patients with uterine cervical cancer

Abstract Objective: Angiogenesis is one of the processes that is critical for the growth, invasion and metastasis of solid tumors, including uterine cervical cancer (CC). The vascular endothelial growth factor (VEGF) family is one of the major pathways involved in tumor angiogenesis. The aim of this study was to determine whether serum levels of these angiogenic factors could be used as biomarkers in patients with CC. Methods: A total of 115 patients with International Federation of Gynecology and Obstetrics (FIGO) stage IB to IIB CC who were treated at Tottori University Hospital between 2006 and 2015 were enrolled in this study. The study was approved by the Institutional Review Board of the School of Medicine of Tottori University. All patients gave written informed consent before the collection of specimens according to institutional guidelines. Serum samples were collected before initial surgery and levels of VEGF-A, -C and VEGFR1 were analyzed by enzyme-linked immunosorbent assay (ELISA). We evaluated the association between the levels of VEGF-A, -C, and VEGFR1 and clinicopathologic variables. Survival analysis was performed with 86 patients treated between 2006 and 2013. We also determined the mRNA expression VEGF-A, -C, and VEGFR1 by real-time RT-PCR in fresh frozen tumors and the protein expression by immunohistochemical staining in paraffin-embedded tumors from CC patients. Results: The mRNA and protein expressions of VEGF-A, -C, and VEGFR1 were strongly observed in the cancer cells. Median levels of serum VEGF-A, -C and VEGFR1 were 313, 8404 and 67.2 pg/ml, respectively. The levels of VEGF-A in patients with lymph node involvement were significantly higher than those in patients without it. On the contrary, the levels of VEGFR1 in patients with lymph node involvement were significantly lower than those in patients without it. Both histological types and FIGO stage were not related to levels of these angiogenic factors. We found a significant positive correlation between VEGF-A levels and the maximum tumor diameter. There was a significant negative correlation between VEGFR1 levels and the maximum tumor diameter. The overall survival rate was significantly lower in FIGO stage IIB than stage IB- IIA patients. We set the cutoff value of these factors at the median levels of each angiogenic factors. The 5-year survival rate for patients with high VEGF-A levels was significantly lower than those with low levels (92.9% vs. 72.8%, P = 0.014). Both VEGFR1 and VEGF-C levels were not related to outcome of patients. Multivariate analysis revealed that serum VEGF-A level and FIGO stage were independent prognostic factors. Conclusion: These results suggest that serum VEGF-A may be a promising prognostic biomarker for CC. Citation Format: Mayumi Sawada, Tetsuro Oishi, Hiroaki Komatsu, Hiroaki Itamochi, Michiko Nonaka, Seitya Sato, Jun Chikumi, Sinya Sato, Sinya Sato, Muneaki Shimada, Tasuku Harada. Serum VEGF-A may predict prognosis in patients with uterine cervical cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3386.

Inhibitory effects of IBI302 on experimental choroidal neovascularization

Objective To investigate the inhibitory effects of IBI302 on experimental choroidal neovascularization (CNV). Methods Affinity of IBI302 to vascular endothelial growth factor (VEGF) family cytokines (including VEGF-A165, VEGF-A121 and placental growth factor PlGF) and complements (C3b, C4b) was determined by enzyme-linked immunosorbent assay (ELISA). The antagonist effect of IBI302 on VEGF was measured by proliferation, migration and tube formation tests of human umbilical vein endothelial cells (HUVEC). The anti-complement activity of IBI302 was measured by hemolysis test mediated by complement classical pathway and alternative pathway. Rhesus laser-induced CNV model was divided into 5 groups including model control group, bevacizumab group, IBI302 0.25 mg group, IBI302 0.50 mg group and IBI302 1.25 mg group. Fluorescein angiography and optical coherence tomography were performed on these monkeys at 14 and 28 days after drug delivery to observe the fluorescein leakage area and retinal thickness. The aqueous VEGF concentration was measured at 29 days after drug delivery. Results IBI302 showed good affinity to VEGF-A165, VEGF-A121 and PlGF, as well as C3b and C4b. IBI302 significantly inhibited the proliferation, migration and tube formation of HUVEC induced by VEGF-A165. IBI302 inhibited the hemolysis induced by complements obviously. At 14 and 28 days after drug delivery, the area of fluorescein leakage and retinal thickness in IBI302 0.25 mg group, IBI302 0.50 mg group, IBI302 1.25 mg group were reduced. The differences of the area of fluorescein leakage and retinal thickness in three IBI302 groups were not significant (P>0.05). At 29 days after drug delivery, the VEGF concentration in the aqueous of rhesus monkey in bevacizumab group [(38.644±6.521) pg/ml] was decreased than that in model control group [(94.203±17.360) pg/ml], the difference was significant (P<0.05). The VEGF concentration in the aqueous of rhesus monkey in three IBI302 groups were less than 31.300 pg/ml. Conclusion IBI302 inhibited experimental CNV through blocking the activity of VEGF and complement. Key words: Choroidal neovascularization/prevention & control; Recombinant fusion proteins/antagonists & inhibitors; Animal experimentation

Functional roles of angiogenic factors and receptors on non-endothelial cells in the oropharyngeal cavity of the chukar partridge (Alectoris chukar)

The vascular endothelial growth factor (VEGF) family belong to the platelet-derived growth factor supergene family and is involved in angiogenesis and mitogenesis. The VEGF–VEGFR system regulates endothelial cell proliferation, migration, vascular permeability, secretion and other non-endothelial cells functions. To clarify the possible role of endothelial and non-endothelial cells, VEGF and its receptors, vascular endothelial cell growth inhibitor (VEGI) were immunohistochemically examined in oropharyngeal organs. Ten adult partridges were used in this study and the pharynx and larynx were dissected together with the palate and tongue. VEGI, VEGF and its receptor were highly expressed in luminal epithelial and stromal cells, when compared to glandular epithelial and muscle cells (P < 0.05). Moreover, VEGF, its receptors and VEGI were expressed rather strongly in the endothelial cells of the blood capillaries and in both the endothelial and smooth muscle cells of the large and small blood vessels. In conclusion, VEGF and its receptors (flt1/fms, flk1/KDR and flt4) and VEGI were expressed by various cell groups at varying intensity in the oropharyngeal organs. This demonstrates that they play a critical role in the regulation and maintenance of the functions in cells different from endothelial ones as well as in cell proliferation, differentiation, apoptosis and angiogenesis.

Abstract 5298: Serum angiogenic factors may predict prognosis in patients with epithelial ovarian cancer

Abstract Objective: Angiogenesis is one of the processes that is critical for the growth, invasion and metastasis of solid tumors, including epithelial ovarian cancer (EOC). The vascular endothelial growth factor (VEGF) family is one of the major pathways involved in tumor angiogenesis, and VEGF expression is activated by hypoxia-inducible factor (HIF)-1α. The aim of this study was to determine whether serum levels of these angiogenic factors could be used as prognostic biomarkers in patients with EOC. Methods: A total of 133 patients with EOC who were treated at Tottori University Hospital between 2006 and 2012 were enrolled in this study. The study was approved by the Institutional Review Board of the School of Medicine of Tottori University. All patients gave written informed consent before the collection of specimens according to institutional guidelines. Serum samples were collected before initial surgery and levels of VEGF-A, -C and HIF-1α were analyzed by enzyme-linked immunosorbent assay (ELISA). We evaluated the association between the levels of VEGF-A, -C, and HIF-1α and clinicopathologic variables. We also determined the protein expression of VEGF-A, -C, and HIF-1α by immunohistochemical staining in paraffin-embedded tumors from EOC patients Results: The protein expressions of VEGF-A, -C, and HIF-1α were strongly observed in the cancer cells. Median levels of serum VEGF-A, -C and HIF-1α were 280, 4866 and 125 pg/ml, respectively. The levels of VEGF-A in patients at stage IV were significantly higher than those in patients at stage I. Both histologic subtypes and lymph node involvement were not related to levels of these angiogenic factors. We found a significant positive correlation between VEGF-A levels and the ascitic volume. The overall survival rate was significantly lower in FIGO stage III or IV patients with EOC. We set the cutoff value of these factors at the median levels of each angiogenic factors. The 5-year survival rate for patients with high VEGF-A levels was significantly lower than those with low levels (61.7% vs. 41.8%, P = 0.0147). Both HIF-1α and VEGF-C levels were not related to outcome of patients. Multivariate analysis revealed that serum VEGF-A level and FIGO stage were independent prognostic factors. Conclusion: These results suggest that serum VEGF-A may be a promising prognostic biomarker for EOC. Citation Format: HIROAKI KOMATSU, Tetsuro Oishi, Hiroaki Itamochi, Akiko Kudoh, Michiko Nonaka, Seiya Sato, Jun Chikumi, Shinya Sato, Muneaki Shimada, Junzo Kigawa, Tasuku Harada. Serum angiogenic factors may predict prognosis in patients with epithelial ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5298. doi:10.1158/1538-7445.AM2015-5298

Is there a Role for Bevacizumab in Non-Glial Tumors?

Angiogenesis is one of the hallmarks of cancer, including brain tumors. The vascular endothelial growth factor (VEGF) family and their receptors are of utmost importance in the complex interaction between pro- and anti-angiogenic factors, and have a crucial role in tumor angiogenesis. Up to date, targeting the VEGF pathway with specific drugs has yielded interesting results in oncology. In particular bevacizumab (Bev), a humanized monoclonal antibody against VEGF-A, has been approved by the Food and Drug Administration (FDA) for use in recurrent glioblastomas failing standard radiochemotherapy. Bevacizumab is now being extensively investigated in several non-glial brain tumors, such as vestibular schwannomas, meningiomas, ependymomas, medulloblastomas and miscellaneous histotypes. The aim of this review is to reevaluate the literature on the use of Bev in non-glial brain tumors.

Immune regulatory mechanisms of neuropilins and its receptors

Neuropilins (NRP) are co-receptors for class 3 semaphorins (Sema) and members of the vascular endothelial growth factor (VEGF) family of angiogenic cytokines. Genetic analysis of the role of NRP in mice shows that NRP1 is essential for embryonic neuronal pathfinding and cardiovascular development, which is mediated via Sema and VEGF, respectively. NRP are thought to mediate functional responses, especially cell migration, as a result of complex formation with other receptors, such as plexins in the case of Sema and the VEGF receptor (VEGFR)2, resulting in enhanced signaling via some intracellular pathways. Recent findings indicate that NRP may play important biological roles in other physiological and disease-related processes. In particular, NRP are highly expressed in diverse tumour cell lines and human neoplasms. NRP also implicate in several biological processes regulating tumour growth in vivo, suggesting that NRP1 may be a future therapeutic target in cancer. In the immune system, NRP1 acts as a negative regulator of the immune system via Sema3A and it may be a therapeutic target in autoimmune diseases. In this article, we will review the immune regulatory mechanisms of NRP and its receptors. Key words: Neuropilins; Semaphorins; Vascular endothelial growth factor; Immune regulation

Abstract IA17: Targeting placental growth factor/ neuropilin-1 pathway inhibits growth and spread of medulloblastoma

Abstract Background: Medulloblastoma (MB) is the most common pediatric brain tumor and a heterogeneous disease. Several studies have characterized tumor genetic heterogeneity with the hope of identifying targets for personalized therapy. In fact, inhibitors of Sonic Hedgehog (Shh) - a developmental pathway aberrantly activated in a subset of MBs - are currently being evaluated. Unfortunately, mutations and potential developmental adverse effects in the pediatric population can limit these therapies. We propose targeting the tumor stroma as an efficacious alternative treatment option. In particular, we aim to identify and target crucial cancer cell-stromal cell crosstalk signaling that sustains tumor growth and spread. In this context, we have evaluated the effects of blocking signaling of Placental Growth Factor (PIGF) - a member of the vascular endothelial growth factor (VEGF) family - that is dispensable in development but relevant during disease, in the growth and progression of MB. Here, we show the expression of PIGF and its receptor Neuropilin-1 (NRP-1) across primary tumors; dissect the mechanisms that regulate PIGF secretion and downstream signaling; and demonstrate the dramatic efficacy of specific anti-PIGF and anti-NRP-1 blocking antibodies in three MB models in vivo. Methods: Expression of PIGF and NRP-1 was analyzed in a cohort of 32 surgical samples of pediatric MBs by immunohistochemistry, array-comparative genomic hybridization, and deep-gene sequencing. The correlation between NRP-1 expression and 5-year overall survival was evaluated retrospectively in an independent, clinically annotated cohort of 42 MBs. Human orthotopic xenograft (D283-Med; D341-Med) and spontaneous (Smo/Smo) established tumors were treated with anti-PIGF (Thrombogenics or Genentech) or anti-NRP-1 (Genentech) antibodies. Tumor growth, progression and response to therapy was followed by 1) clinical evaluation of posterior fossa symptoms and body weight; 2) whole-body luminescence and blood GLuc levels; 3) small animal MRI; or 4) Optical Frequency Domain Imaging (OFDI). Results: We report that: 1) PIGF is expressed by more than 90% of primary pediatric MBs, regardless of their genetic subtype or histological classification; and high expression of NRP-1 correlates with poor overall survival of patients; 2) MB cells release Shh ligands that stimulate paracrine production of stromal PIGF by cerebellar granule neurons; 3) PIGF signals through NRP-1 - and independent of vascular endothelial growth factor receptor 1 (VEGFR1) - to activate the Erk/MEK pathway and sustain tumor survival; 4) Blockade of PIGF or NRP-1 with specific antibodies causes dramatic regression of MB, decreases spinal metastatic burden, and prolongs survival in in vivo orthotopic and spontaneous mouse models. Conclusion: This work identifies PIGF as the first common target across MB subgroups and provides insight into the critical role of tumor-stroma interactions. The study demonstrates that targeting PIGF/NRP-1 may provide a safe and efficient treatment option for pediatric MBs and lays the ground for evaluation of anti-PIGF therapy in clinical trials. Reference: M. Snuderl et al, Cell, 152(2):1065-76 (2013). Citation Format: Matija Snurdel, Ana Batista, Nathaniel Kirkpatrick, Carmen Almodovar, Lars Riedemann, Teresa Peterson, Napoleone Ferrara, Michael Klagsbrun, Dan G. Duda, Dai Fukumura, Lei Xu, Peter Carmeliet, Rakesh Jain. Targeting placental growth factor/ neuropilin-1 pathway inhibits growth and spread of medulloblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr IA17.

Abstract 86: Heterozygous expression of an oncogenic Pik3ca mutation during murine development results in fatal embryonic and extra-embryonic defects

Abstract The PI3K/Akt signalling pathway regulates many important cellular functions including proliferation, migration, survival and angiogenesis. The PIK3CA gene, encoding the p110α catalytic subunit of the PI3K enzyme, is somatically mutated in a range of human cancers and overgrowth syndromes. We have previously described a novel mouse model with a Cre-recombinase (Cre)-mediated knock-in of the common cancer-associated Pik3ca(H1047R) mutation that can be targeted to selected tissues using various Cre-expressing mouse strains. As the mutation is knocked in to the endogenous gene, the mutant protein is expressed at normal physiological levels. We have used the Cre-deleter mouse to induce expression of the Pik3ca(H1047R) mutation ubiquitously from the 2-cell stage of development. This results in embryonic lethality by E10.5 with embryos at E9.5 demonstrating stunted growth, failed ‘turning’ and abnormal cardiovascular development in both the embryo proper and extraembryonic yolk sac. These tissues also showed increased expression of vascular endothelial growth factor (VEGF) family proteins. We then selectively targeted the mutation to the endothelial cells of the developing embryo using the Tie2-Cre mouse strain. No Tie2-Cre:Pik3ca(H1047R) mutant embryos survived to E11.5 and at E10.5, while successfully turned, embryos were severely underdeveloped and no longer viable. At E9.5, vascular remodelling was disorganised and truncated in the embryo proper and yolk sacs. Development of the heart was also affected with clear disruption to anterior cardinal vein and dorsal aorta development. Our results demonstrate the importance of PI3K/Akt signalling in embryonic development and cardiovascular formation in the mouse. The observed cell autonomous role in vessel development was further confirmed upon targeted expression to endothelial cells while the increased expression of various VEGF family proteins suggests probable crosstalk between the PI3K/Akt and VEGF signalling pathways during cardiovascular development. The lethality associated with expression of Pik3ca(H1047R) mutation during development likely explains the lack of inherited germline PIK3CA mutations in humans. Citation Format: Lauren M. Hare, Quenten Schwarz, Rajendra Gurung, Karen G. Montgomery, Christina Mitchell, Wayne A. Phillips. Heterozygous expression of an oncogenic Pik3ca mutation during murine development results in fatal embryonic and extra-embryonic defects. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 86. doi:10.1158/1538-7445.AM2014-86