Stabilization of HIF-2α Induces sVEGFR-1 Production from Tumor-Associated Macrophages and Decreases Tumor Growth in a Murine Melanoma Model

Macrophage secretion of vascular endothelial growth factor (VEGF) in response to hypoxia contributes to tumor growth and angiogenesis. In addition to VEGF, hypoxic macrophages stimulated with GM-CSF secrete high levels of a soluble form of the VEGF receptor (sVEGFR-1), which neutralizes VEGF and inhibits its biological activity. Using mice with a monocyte/macrophage-selective deletion of hypoxia-inducible factor (HIF)-1α or HIF-2α, we recently demonstrated that the antitumor response to GM-CSF was dependent on HIF-2α-driven sVEGFR-1 production by tumor-associated macrophages, whereas HIF-1α specifically regulated VEGF production. We therefore hypothesized that chemical stabilization of HIF-2α using an inhibitor of prolyl hydroxylase domain 3 (an upstream inhibitor of HIF-2α activation) would increase sVEGFR-1 production from GM-CSF-stimulated macrophages. Treatment of macrophages with the prolyl hydroxylase domain 3 inhibitor AKB-6899 stabilized HIF-2α and increased sVEGFR-1 production from GM-CSF-treated macrophages, with no effect on HIF-1α accumulation or VEGF production. Treatment of B16F10 melanoma-bearing mice with GM-CSF and AKB-6899 significantly reduced tumor growth compared with either drug alone. Increased levels of sVEGFR-1 mRNA, but not VEGF mRNA, were detected within the tumors of GM-CSF- and AKB-6899-treated mice, correlating with decreased tumor vascularity. Finally, the antitumor and antiangiogenic effects of AKB-6899 were abrogated when mice were simultaneously treated with a sVEGFR-1 neutralizing Ab. These results demonstrate that AKB-6899 decreases tumor growth and angiogenesis in response to GM-CSF by increasing sVEGFR-1 production from tumor-associated macrophages. Specific activation of HIF-2α can therefore decrease tumor growth and angiogenesis.     

Adenosine infusion increases plasma levels of VEGF in humans

Background  

Many in vitro studies have shown that adenosine (Ado) can induce vascular endothelial growth factor (VEGF) mRNA and protein expression and stimulate endothelial proliferation. In the present study, we seek to determine whether Ado can increase circulating levels of VEGF protein in the intact human.     

Vascular endothelial growth factor and its soluble receptors VEGFR-1 and VEGFR-2 in the serum of patients with systemic lupus erythematosus

We investigated the serum concentration of vascular endothelial growth factor (VEGF) and its two soluble receptors, sVEGFR-1 and sVEGFR-2, in a group of 60 patients with systemic lupus erythematosus (SLE), and 20 healthy controls, using an enzyme-linked immunosorbent assay. We examined a possible association between serum levels of these proteins and certain clinical and laboratory parameters as well as SLE activity. VEGF, sVEGFR-1 and sVEGFR-2 were detectable in all patients with SLE and in all normal individuals. The VEGF level was higher in active SLE (mean, 300.8 pg/ml) than in inactive SLE (mean, 165.9 pg/ml) (p < 0.05) or in the control group (mean, 124.7 pg/ml) (p < 0.04). The highest sVEGFR-1 concentrations were also detected in active SLE patients (mean, 42.2 pg/ml) and the lowest in inactive disease (mean, 32.0 pg/ml) (p < 0.01). In contrast, the levels of sVEGFR-2 were lower in SLE (mean, 12557.6 pg/ml) than in the control group (mean, 15025.3 pg/ml) (p < 0.05). We found a positive correlation between sVEGFR-1 concentration and the SLE activity score p = 0.375 (p < 0.004) and a negative, but statistically insignificant correlation between sVEGFR-2 and SLE activity (p = -0.190, p > 0.05). Treatment with steroids and cytotoxic agents did not influence VEGF or its soluble receptors levels. In conclusion, in SLE patients the levels of VEGF and sVEGFR-1 are higher in patients with active SLE than in inactive disease or healthy persons. In contrast, the level of sVEGFR-2 is lower in active SLE than in inactive disease. The imbalance between VEGF and its soluble receptors may be important in SLE pathogenesis.     

Circulating VEGF and its soluble receptors sVEGFR-1 and sVEGFR-2 in patients with acute leukemia

Angiogenesis plays an important role in the pathogenesis of acute leukemia, and vascular endothelial growth factor (VEGF) is a crucial, positive regulator of this process. The biological activity of VEGF is mediated by two different receptor tyrosine kinases: VEGFR-2 and VEGFR-1. The soluble form of VEGFR-1 is likely to be a negative regulator of VEGF availability, but the physiological role of sVEGFR-2 is still unclear. The plasma levels of sVEGFR-1 and sVEGFR-2 in patients with acute leukemia have not been investigated. We measured the plasma concentrations of VEGF and its two soluble receptors in 39 AML and 15 ALL patients as well as in the control group, using the ELISA assay. We also correlated the plasma levels of these proteins with disease status and known prognostic factors. The sVEGFR-1 level was significantly higher in patients with AML and ALL than in the healthy subjects (p < 0.002 and p < 0.03 respectively). The sVEGFR-2 level was significantly higher in AML patients compared with the control group (p < 0.03). The VEGF levels in AML and ALL patients and in healthy subjects did not differ significantly. The sVEGFR-1 level was higher in AML patients with > 50% of blasts in the bone marrow (BM), WBC > 20 G/L and elevated LDH level, than in the group with BM blasts < 50% (p < 0.01), WBC < 20 G/L (p < 0.02) and a normal LDH level (p < 0.05). Positive correlations between sVEGFR-1 level and WBC (p < 0.02),% of BM blasts (p < 0.05), the absolute blast count in peripheral blood (ABC) (p < 0.009) and LDH (p < 0.000001) were found. The sVEGFR-1/VEGF ratio (R1) was calculated, and a positive correlation between R1 and ABC in AML (p < 0.03) was determined. A higher (above median) sVEGFR-1/VEGF ratio correlated with a lower CR rate and a shorter survival (p < 0.03 and p = 0.0007 respectively). In conclusion, the plasma concentration of sVEGFR-1 is higher in leukemia patients than in healthy subjects and correlates with tumour burden and poor prognosis. The sVEGFR-1/VEGF ratio may be of greater prognostic value than VEGF alone. Further investigation is recommended to better determine their function.     

Hypoxia Inducible Factors-Mediated Inhibition of Cancer by GM-CSF: A Mathematical Model

Under hypoxia, tumor cells, and tumor-associated macrophages produce VEGF (vascular endothelial growth factor), a signaling molecule that induces angiogenesis. The same macrophages, when treated with GM-CSF (granulocyte/macrophage colony-stimulating factor), produce sVEGFR-1 (soluble VEGF receptor-1), a soluble protein that binds with VEGF and inactivates its function. The production of VEGF by macrophages is regulated by HIF-1α (hypoxia inducible factor-1α), and the production of sVEGFR-1 is mediated by HIF-2α. Recent experiments measured the effect of inhibiting tumor growth by GM-CSF treatment in mice with HIF-1α-deficient or HIF-2α-deficient macrophages. In the present paper, we represent these experiments by a mathematical model based on a system of partial differential equations. We show that the model simulations agree with the above experiments. The model can then be used to suggest strategies for inhibiting tumor growth. For example, the model qualitatively predicts the extent to which GM-CSF treatment in combination with a small molecule inhibitor that stabilizes HIF-2α will reduce tumor volume and angiogenesis.     

A naturally occurring soluble form of vascular endothelial growth factor receptor 2 detected in mouse and human plasma

Angiogenesis and vasculogenesis are regulated in large part by several different growth factors and their associated receptor tyrosine kinases (RTKs). Foremost among these is the vascular endothelial growth factor (VEGF) family including VEGF receptor (VEGFR)-2 and -1. VEGFR ligand binding and biological activity are regulated at many levels, one of which is by a soluble, circulating form of VEGFR-1 (sVEGFR-1). This sVEGFR-1 can act as a competitive inhibitor of its ligand, serve as a possible biomarker, and play important roles in cancer and other diseases such as preeclampsia. Recombinant forms of sVEGFR-2 have been shown to have antiangiogenic activity, but a naturally occurring sVEGFR-2 has not been described previously. Here, we report such an entity. Having a molecular weight of approximately 160 kDa, sVEGFR-2 can be detected in mouse and human plasma with several different monoclonal and polyclonal anti-VEGFR-2 antibodies using both ELISA and immunoprecipitation techniques. In vitro studies have determined that the sVEGFR-2 fragment can be found in the conditioned media of mouse and human endothelial cells, thus suggesting that it may be secreted, similar to sVEGFR-1, or proteolytically cleaved from the cell. Potential biological activity of this protein was inferred from experiments in which mouse sVEGFR-2 could bind to VEGF-coated plates. Similar to sVEGFR-1 and other soluble circulating RTKs, sVEGFR-2 may have regulatory consequences with respect to VEGF-mediated angiogenesis as well as potential to serve as a quantitative biomarker of angiogenesis and antiangiogenic drug activity, particularly for drugs that target VEGF or VEGFR-2.     

Assessment of pro- and antiangiogenic factors blood serum concentrations in patients with hormonal inactive adrenal tumors

INTRODUCTION: The growth and persistence of solid tumors and their metastases is connected with angiogenesis. This process is determined by activity of pro- and antyangiogenic factors. VEGF is the one of the most important factors having a stimulant effect on angiogenesis. Soluble forms of VEGF receptors are inhibitors of angiogenesis. The soluble forms of VEGF receptors containing extra cellular part of receptor, which binds ligand, seem to be real inhibitors of VEGF. THE AIM OF THE STUDY: Evaluation the value of serum VEGF and soluble forms of VEGF receptors concentration as a marker of malignancy in patients with hormonal inactive adrenal tumors. MATERIAL AND METHODS: Twenty seven patients (18 female, 9 male; mean age 48+/-4.3 years) with adrenocortical carcinoma (N=8), adrenal metastases (N=4) and adrenocortical adenoma (N=15) were included in this study. Age- and gender-matched control samples were acquired from healthy volunteers (N=10). Serum VEGF and sVEGFR levels were determinated by means of ELISA assay. Statistical analysis was performed using the Student-t test, the Pearson's test and the series test. RESULTS: In healthy controls mean VEGF level was 197.2 pg/ml, sVEGFR-1 43.5 pg/ml and sVEGFR-2 8976.3 pg/ml. Patients with adrenocortical carcinoma had the levels of VEGF (1263.8 pg/ml) significantly higher and of sVEGFR-2 (5893.7 pg/ml) significantly lower in comparison to control group (p<0.05). On the other hand the mean VEGF (334.2 pg/ml) concentration in patients with benign adrenocortical adenoma wasn't significant different than in control group (p>0.05) but mean sVEGFR-1 (21.7 pg/ml) and sVEGFR-2 (7106.4 pg/ml) concentrations were significantly lower than in the control (p<0.05). In metastases group mean VEGF (485.9 pg/ml) level was higher and sVEGFR-2 (5455.2 pg/ml) was lower than in control group (p<0.05). CONCLUSION: These data suggest that determination of VEGF and sVEGFR concentration in the serum of patients with hormonal inactive adrenal tumors may be applied as an additional marker of malignancy.     

Regulation of soluble vascular endothelial growth factor receptor (sFlt-1/sVEGFR-1) expression and release in endothelial cells by human follicular fluid and granulosa cells

Background  

During the female reproductive cycle, follicular development and corpus luteum formation crucially depend on the fast generation of new blood vessels. The importance of granulosa cells and follicular fluid in controlling this angiogenesis is still not completely understood. Vascular endothelial growth factor (VEGF) produced by granulosa cells and secreted into the follicular fluid plays an essential role in this process. On the other hand, soluble VEGF receptor-1 (sFlt-1) produced by endothelial cells acts as a negative modulator for the bioavailability of VEGF. However, the regulation of sFlt-1 production remains to be determined.     

Effects of nerve growth factor (NGF) on blood vessels area and expression of the angiogenic factors VEGF and TGFbeta1 in the rat ovary

Background  

Angiogenesis is a crucial process in follicular development and luteogenesis. The nerve growth factor (NGF) promotes angiogenesis in various tissues. An impaired production of this neurotrophin has been associated with delayed wound healing. A variety of ovarian functions are regulated by NGF, but its effects on ovarian angiogenesis remain unknown. The aim of this study was to elucidate if NGF modulates 1) the amount of follicular blood vessels and 2) ovarian expression of two angiogenic factors: vascular endothelial growth factor (VEGF) and transforming growth factor beta 1 (TGFbeta1), in the rat ovary.     

Dysregulated angiogenesis in B-chronic lymphocytic leukemia: Morphologic, immunohistochemical, and flow cytometric evidence

Background

The extent of enhanced bone marrow angiogenesis in chronic lymphocytic leukemia (CLL) and relationship to proangiogenic factors and prognostic indicators is largely unexplored.

Methods

To further investigate the role of angiogenesis in CLL by evaluating the topography and extent of angiogenesis in a group of CLL bone marrow biopsies, to study the expression of pro and antiangiogenic vascular factors in CLL cells to more precisely document the cell types producing these factors, and to evaluate the role, if any, of localized hypoxia in upregulation of angiogenesis in CLL We used immunohistochemistry (IHC) (n = 21 pts) with antibodies to CD3 and CD20, proangiogenic (VEGF, HIF-1a) and antiangiogenic (TSP-1) factors, and VEGF receptors -1 and -2 to examine pattern/extent of CLL marrow involvement, microvessel density (MVD), and angiogenic characteristics; flow cytometry (FC) was performed on 21 additional cases for VEGF and TSP-1.

Results

CLL patients had higher MVD (23.8 vs 14.6, p~0.0002) compared to controls (n = 10). MVD was highest at the periphery of focal infiltrates, was not enhanced in proliferation centers, and was increased irrespective of the presence or absence of cytogenetic/immunophenotypic markers of aggressivity. By IHC, CLL cells were VEGF(+), HIF-1a (+), TSP-1(-), VEGFR-1(+), and VEGFR-2(+). By FC, CLL cells were 1.4–2.0-fold brighter for VEGF than T cells and were TSP-1(-).

Conclusion

CLL demonstrates enhanced angiogenesis, with increased MVD, upregulated VEGF and downregulated TSP-1. Upregulation of HIF-1a in all CLL cases suggests localized tissue hypoxia as an important stimulant of microvessel proliferation. The presence of VEGF receptors on CLL cells implies an autocrine effect for VEGF. Differences in MVD did not correlate with traditional genetic/immunophenotypic markers of aggressiveness.     

Association of mast cell-derived VEGF and proteases in Dengue shock syndrome

Background

Recent in-vitro studies have suggested that mast cells are involved in Dengue virus infection. To clarify the role of mast cells in the development of clinical Dengue fever, we compared the plasma levels of several mast cell-derived mediators (vascular endothelial cell growth factor [VEGF], soluble VEGF receptors [sVEGFRs], tryptase, and chymase) and -related cytokines (IL-4, -9, and -17) between patients with differing severity of Dengue fever and healthy controls.

Methodology/Principal Findings

The study was performed at Children''s Hospital No. 2, Ho Chi Minh City, and Vinh Long Province Hospital, Vietnam from 2002 to 2005. Study patients included 103 with Dengue fever (DF), Dengue hemorrhagic fever (DHF), and Dengue shock syndrome (DSS), as diagnosed by the World Health Organization criteria. There were 189 healthy subjects, and 19 febrile illness patients of the same Kinh ethnicity. The levels of mast cell-derived mediators and -related cytokines in plasma were measured by ELISA. VEGF and sVEGFR-1 levels were significantly increased in DHF and DSS compared with those of DF and controls, whereas sVEGFR-2 levels were significantly decreased in DHF and DSS. Significant increases in tryptase and chymase levels, which were accompanied by high IL-9 and -17 concentrations, were detected in DHF and DSS patients. By day 4 of admission, VEGF, sVEGFRs, and proteases levels had returned to similar levels as DF and controls. In-vitro VEGF production by mast cells was examined in KU812 and HMC-1 cells, and was found to be highest when the cells were inoculated with Dengue virus and human Dengue virus-immune serum in the presence of IL-9.

Conclusions

As mast cells are an important source of VEGF, tryptase, and chymase, our findings suggest that mast cell activation and mast cell-derived mediators participate in the development of DHF. The two proteases, particularly chymase, might serve as good predictive markers of Dengue disease severity.     

Nifedipine decreases sVCAM-1 concentrations and oxidative stress in systemic sclerosis but does not affect the concentrations of vascular endothelial growth factor or its soluble receptor 1

Microvascular injury, oxidative stress, and impaired angiogenesis are prominent features of systemic sclerosis (SSc). We compared serum markers of these phenomena at baseline and after treatment with nifedipine in SSc patients. Forty successive SSc patients were compared with 20 matched healthy subjects. All SSc patients stopped taking calcium-channel blockers 72 hours before measurements. Twenty SSc patients were also examined after 14 days of treatment with nifedipine (60 mg/day). Quantitative ELISA was used to measure the serum concentrations of vascular endothelial growth factor (VEGF), soluble VEGF receptor 1 (sVEGFR-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), carbonyl residues, and advanced oxidation protein products (AOPP). The median concentrations of VEGF, sVEGFR-1, sVCAM-1, carbonyl residues, and AOPP were significantly higher in SSc patients than in healthy subjects at baseline. A correlation was found between VEGF concentration and carbonyl residue concentration (r = 0.43; P = 0.007). Nifedipine treatment led to a significant decrease in concentrations of sVCAM-1, carbonyl residues, and AOPP but did not affect concentrations of VEGF and sVEGFR-1. Nifedipine treatment ameliorated endothelium injury in patients with SSc, as shown by the concentrations of adhesion molecules and oxidative damage markers. The fact that VEGF and sVEGFR-1 concentrations were not changed whereas oxidative stress was ameliorated by nifedipine is consistent with the hypothesis that VEGF signalling is impaired in SSc. However, more experimental evidence is needed to determine whether the VEGF pathway is intrinsically defective in SSc.     

Angiotensin II and angiotensin-(1-7) decrease sFlt1 release in normal but not preeclamptic chorionic villi: an <Emphasis Type="Italic">in vitro</Emphasis> study

Background  

During preeclampsia, placental angiogenesis is impaired. Factors released from the placenta including vascular endothelial growth factor (VEGF), placental growth factor (PLGF), soluble VEGF receptor 1 (sFlt1), and soluble endoglin (sEng) are regulatory molecules of placental development and function. While the renin angiotensin system has been shown to regulate angiogenic factors in other research fields, these mechanisms have not been extensively studied during pregnancy.     

Acupuncture Promotes Angiogenesis after Myocardial Ischemia through H3K9 Acetylation Regulation at VEGF Gene

Background

Acupuncture exerts cardioprotective effects on several types of cardiac injuries, especially myocardial ischemia (MI), but the mechanisms have not yet been well elucidated. Angiogenesis mediated by VEGF gene expression and its modification through histone acetylation has been considered a target in treating myocardial ischemia. This study aims to exam whether modulation of angiogenesis through H3K9 acetylation regulation at VEGF gene is one possible cardioprotective mechanism of acupuncture.

Results

We generated rat MI models by ligating the left anterior descending coronary artery and applied electroacupuncture (EA) treatment at the Neiguan (PC6) acupoint. Our results showed that acupuncture reversed the S-T segment change, reduced Q-wave area, decreased CK, CK-MB, LDH levels, mitigated myocardial remodeling, and promoted microvessel formation in the MI heart. RNA-seq analysis showed that VEGF-induced angiogenesis signaling was involved in the modulation of EA. Western blot results verified that the protein expressions of VEGF, Ras, phospho-p44/42 MAPK, phospho-p38 MAPK, phospho-SAPK/JNK and Akt, were all elevated significantly by EA treatment in the MI heart. Furthermore, increased H3K9 acetylation was also observed according with the VEGF. ChIP assay confirmed that EA treatment could notably stimulate the recruitment of H3K9ace at the VEGF promoter.

Conclusions

Our study demonstrates for the first time that acupuncture can effectively up-regulate VEGF expression through H3K9 acetylation modification directly at the VEGF promoter and hence activate VEGF-induced angiogenesis in rat MI models. We employed high throughput sequencing in this study and, for the first time, generated genome-wide gene expression profiles both in the rat MI model and in acupuncture treatment.     

Rat brain VEGF expression in alveolar hypoxia: possible role in high-altitude cerebral edema

The mechanism by which hypoxia causes high-altitudecerebral edema (HACE) is unknown. Tissue hypoxia triggers angiogenesis, initially by expressing vascular endothelial growth factor (VEGF), which has been shown to increase extracerebral capillary permeability. This study investigated brain VEGF expression in 32 rats exposed toprogressively severe normobaric hypoxia (9-6%O₂) for 0 (control), 3, 6, or 12 h or 1, 2, 3, or 6 days. O₂concentration was adjusted intermittently to the limit of tolerance byactivity and intake, but no attempt was made to detect HACE. Northernblot analysis demonstrated that two molecular bands of transcribed VEGFmRNA (~3.9 and 4.7 kb) were upregulated in cortex and cerebellumafter as little as 3 h of hypoxia, with a threefold increase peaking at12-24 h. Western blot revealed that VEGF protein was increased after 12 h of hypoxia, reaching a maximum in ~2 days. The expression of flt-1 mRNA was enhanced after 3 days of hypoxia. We conclude that VEGF production in hypoxia isconsistent with the hypothesis that angiogenesis may be involved inHACE.

     

Gelatinases, endonuclease and Vascular Endothelial Growth Factor during development and regression of swine luteal tissue

Background  

The development and regression of corpus luteum (CL) is characterized by an intense angiogenesis and angioregression accompanied by luteal tissue and extracellular matrix (ECM) remodelling. Vascular Endothelial Growth Factor (VEGF) is the main regulator of angiogenesis, promoting endothelial cell mitosis and differentiation. After the formation of neovascular tubes, the remodelling of ECM is essential for the correct development of CL, particularly by the action of specific class of proteolytic enzymes known as matrix metalloproteinases (MMPs). During luteal regression, characterized by an apoptotic process and successively by an intense ECM and luteal degradation, the activation of Ca⁺⁺/Mg⁺⁺-dependent endonucleases and MMPs activity are required. The levels of expression and activity of VEGF, MMP-2 and -9, and Ca⁺⁺/Mg⁺⁺-dependent endonucleases throughout the oestrous cycle and at pregnancy were analyzed.     

Differential expression of Vegfr-2 and its soluble form in preeclampsia

Background

Several studies have suggested that the main features of preeclampsia (PE) are consequences of endothelial dysfunction related to excess circulating anti-angiogenic factors, most notably, soluble sVEGFR-1 (also known as sFlt-1) and soluble endoglin (sEng), as well as to decreased PlGF. Recently, soluble VEGF type 2 receptor (sVEGFR-2) has emerged as a crucial regulator of lymphangiogenesis. To date, however, there is a paucity of information on the changes of VEGFR-2 that occur during the clinical onset of PE. Therefore, the aim of our study was to characterize the plasma levels of VEGFR-2 in PE patients and to perform VEGFR-2 immunolocalization in placenta.

Methodology/Principal findings

By ELISA, we observed that the VEGFR-2 plasma levels were reduced during PE compared with normal gestational age matched pregnancies, whereas the VEGFR-1 and Eng plasma levels were increased. The dramatic drop in the VEGFR-1 levels shortly after delivery confirmed its placental origin. In contrast, the plasma levels of Eng and VEGFR-2 decreased only moderately during the early postpartum period. An RT-PCR analysis showed that the relative levels of VEGFR-1, sVEGFR-1 and Eng mRNA were increased in the placentas of women with severe PE. The relative levels of VEGFR-2 mRNA as well as expressing cells, were similar in both groups. We also made the novel finding that a recently described alternatively spliced VEGFR-2 mRNA variant was present at lower relative levels in the preeclamptic placentas.

Conclusions/Significance

Our results indicate that the plasma levels of anti-angiogenic factors, particularly VEGFR-1 and VEGFR-2, behave in different ways after delivery. The rapid decrease in plasma VEGFR-1 levels appears to be a consequence of the delivery of the placenta. The persistent circulating levels of VEGFR-2 suggest a maternal endothelial origin of this peptide. The decreased VEGFR-2 plasma levels in preeclamptic women may serve as a marker of endothelial dysfunction.     

<Emphasis Type="Italic">In vivo</Emphasis> monitoring of fetoplacental Vegfr2 gene activity in a murine pregnancy model using a Vegfr2-luc reporter gene and bioluminescent imaging

Background  

Vascular endothelial growth factor receptor-2 (VEGFR2) plays a pivotal role in angiogenesis by eliciting vascular endothelial cell growth when bound to VEGF, a powerful pro-angiogenic ligand. While Vegf and Vegfr2 are expressed throughout gestation, the latter third of gestation in mice is characterized by a marked increase in fetoplacental angiogenesis. Thus, the objective of this study was to determine the feasibility of monitoring fetoplacental Vegfr2 gene activity non-invasively using a Vegfr2-luc reporter transgenic mouse and bioluminescent imaging.     

Retinoblastoma Binding Protein 2 (RBP2) Promotes HIF-1α–VEGF-Induced Angiogenesis of Non-Small Cell Lung Cancer via the Akt Pathway

Background

Pathological angiogenesis plays an essential role in tumor aggressiveness and leads to unfavorable prognosis. The aim of this study is to detect the potential role of Retinoblastoma binding protein 2 (RBP2) in the tumor angiogenesis of non-small cell lung cancer (NSCLC).

Methods

Immunohistochemical staining was used to detect the expression of RBP2, hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and CD34. Two pairs of siRNA sequences and pcDNA3-HA-RBP2 were used to down-regulate and up-regulate RBP2 expression in H1975 and SK-MES-1 cells. An endothelial cell tube formation assay, VEGF enzyme-linked immunosorbent assay, real-time PCR and western blotting were performed to detect the potential mechanisms mediated by RBP2 in tumor angiogenesis.

Results

Of the 102 stage I NSCLC specimens analyzed, high RBP2 protein expression is closely associated with tumor size (P = 0.030), high HIF-1α expression (P = 0.028), high VEGF expression (P = 0.048), increased tumor angiogenesis (P = 0.033) and poor prognosis (P = 0.037); high MVD was associated with high HIF-1α expression (P = 0.034), high VEGF expression (P = 0.001) and poor prognosis (P = 0.040). Multivariate analysis indicated that RBP2 had an independent influence on the survival of patients with stage I NSCLC (P = 0.044). By modulating the expression of RBP2, our findings suggested that RBP2 protein depletion decreased HUVECs tube formation by down-regulating VEGF in a conditioned medium. RBP2 stimulated the up-regulation of VEGF, which was dependent on HIF-1α, and activated the HIF-1α via phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Moreover, VEGF increased the activation of Akt regulated by RBP2.

Conclusions

The RBP2 protein may stimulate HIF-1α expression via the activation of the PI3K/Akt signaling pathway under normoxia and then stimulate VEGF expression. These findings indicate that RBP2 may play a critical role in tumor angiogenesis and serve as an attractive therapeutic target against tumor aggressiveness for early-stage NSCLC patients.