Serum levels of angiogenic and anti-angiogenic factors: their prognostic relevance in locally advanced hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a prototype tumor wherein angiogenesis plays a vital role in its progression. The role of VEGF, a major angiogenic factor in HCC is known; however, the role of anti-angiogenic factors simultaneously with the angiogenic factors has not been studied before. Hence, in this study, the serum levels of major angiogenic [Vascular Endothelial Growth Factor (VEGF), angiopoietin-2 (Ang-2)] and anti-angiogenic (endostatin, angiostatin) factors were analyzed and correlated with clinico-radiological features and with outcome. A total of 150 patients (50 HCC, 50 cirrhosis and 50 chronic hepatitis) and 50 healthy controls were enrolled in this study. Serum levels of VEGF, Ang-2, endostatin, and angiostatin were estimated by enzyme-linked immunosorbent assay. HCC shows significantly elevated serum levels of angiogenic factors VEGF and Ang-2 and of anti-angiogenic factors endostatin and angiostatin. ROC curve analysis for serum VEGF yielded an optimal cut-off value of 225.14 pg/ml, with a sensitivity of 78 % and specificity of 84.7 % for a diagnosis of HCC and its distinction from other group. Using this value, the univariate and multivariate analysis revealed significantly poor outcome in patients with higher levels of serum VEGF (p = 0.009). Combinatorial analysis revealed that patients with higher levels of both angiogenic and anti-angiogenic factors showed poor outcome. Serum VEGF correlates with poor survival of HCC patients and, therefore, serves as a non-invasive biomarker of poor prognosis. Moreover, elevated levels of anti-angiogenic factors occur endogenously in HCC patients.     

Enhancement of differentiation efficiency of hESCs into vascular lineage cells in hypoxia via a paracrine mechanism

Hypoxia is one way of inducing differentiation due to the activation of the key regulatory factor, Hypoxia-inducible factor 1 alpha (HIF-1α). However, the action of HIF-1α on the differentiation of hESCs was unclear until now. To investigate the effect of hypoxia on the differentiation of hESCs, we compared the differentiation efficacy into vascular lineage cells under normoxic and hypoxic conditions. We observed HIF-1α expression and the related expression of pro-angiogenic factors VEGF, bFGF, Ang-1 and PDGF in hEBs cultured under hypoxic conditions. Along with this, differentiation efficacy into vascular lineage cells was improved under hypoxic conditions. When HIF-1α was blocked by echinomycin, both angiogenic factors and the differentiation efficacy were down-regulated, suggesting that the enhancement of differentiation efficacy was caused by intrinsic up-regulation of HIF-1α and these pro-angiogenic factors under hypoxic condition. This response might be primarily regulated by the HIF-1α signal pathway, and hypoxia might be the key to improving the differentiation of hESCs into vascular lineage cells. Therefore, this study demonstrated that microenvironmental changes (i.e., hypoxia) can improve differentiation efficacy of hESCs into a vascular lineage without exogenous factors via cell-intrinsic up-regulation of angiogenic factors. These facts will contribute to the regulation of stem cell fate.     

Altered angiogenic balance in ulcerative colitis: a key to impaired healing?

Angiogenesis is an essential component of ulcer healing since it assures delivery of oxygen and nutrients to the healing site. Previous studies demonstrated increased serum and tissue levels of vascular endothelial growth factor (VEGF, the most potent angiogenic growth factor) in patients with active ulcerative colitis (UC) and animal models of UC. However, there is no explanation why the healing of UC-related mucosal injury is impaired despite increased expression of VEGF. Expression of angiogenesis inhibitors, angiostatin and/or endostatin, in UC has not been determined before. We examined expression of VEGF, angiostatin, and endostatin in two models of experimental UC. The results revealed that in addition to increased VEGF, both endostatin and angiostatin levels were markedly (2-3-folds) increased in colonic mucosa at early stage of experimental UC. This is the first demonstration that colitis triggers increase in angiostatin and endostatin levels. The results may explain why mucosal lesions heal slowly despite increased VEGF levels, and may provide a novel and mechanistic insight into UC.     

Paneling human thyroid cancer cell lines for candidate proteins for targeted anti-angiogenic therapy

Tumor angiogenesis is believed to result from an imbalance of pro- and anti-angiogenic factors, some of which are candidates for targeted therapy. Such therapy has raised hopes for patients with undifferentiated thyroid carcinomas, who are facing a grave prognosis with a survival of only months. In this study, in vivo growth of xenografted human thyroid carcinomas unexpectedly responded quite differently to neutralizing anti-vascular endothelial growth factor (VEGF) antibody. In particular, lasting inhibition as well as accelerated growth occurred after treatment. Consequently, a panel of anti-angiogenic factors was addressed in a representative sample of thyroid carcinoma lines. VEGF, fibroblast growth factor (FGF-2), and endostatin were demonstrated by Western blotting and EIA, whereas PDGF-A, PDGF-B, and IL-6 were negative. Quantification of VEGF, FGF-2, and endostatin revealed a wide range of concentrations from 500 to 4,200 pg/ml VEGF, 5 to 60 pg/ml FGF-2, and 50 to 300 pg/ml endostatin, not related to a particular histologic thyroid carcinoma background. Angiostatin (kringles 1-3) was detected in all, but one of the cell lines. Finally, aaATIII was confirmed in FTC133 cells. These data highlight the complex regulation of angiogenesis in thyroid carcinoma cell lines and suggest that the array of angiogenic factors differs markedly between individual cell lines. For the first time, angiostatin, endostatin, and possibly also aaATIII are identified as novel candidate regulators of angiogenesis in thyroid carcinoma cells.     

The effects of surgery, with or without rhGM-CSF, on the angiogenic profile of patients treated for colorectal carcinoma

Wound healing is a process with immunological and angiogenic aspects. rhGM-CSF is known to stimulate the immune system and angiogenesis via multiple pathways. In this study we investigated the combined effects of surgery, with or without rhGM-CSF, on angiogenic parameters in patients with a colorectal carcinoma. In this phase II randomized, placebo-controlled trial, 16 patients were assigned to perioperative rhGM-CSF (2.8 microg/kg body weight) treatment or saline. Patients received subcutaneous injections from three days before surgery until four days after. IL-6, VEGF, endostatin and angiostatin levels were measured perioperatively. rhGM-CSF enhanced the production of IL-6 and VEGF, but had no effect on the antiangiogenic agents endostatin and angiostatin. Surgery induced a transient decrease of endostatin. Two types of angiostatin (kringle 1-3 and kringle 1-4) became visible postoperatively. We conclude that this study demonstrated the immediate initiation of angiogenesis postoperatively, reflected by the increase of VEGF and a transient decrease of endostatin, followed by the appearance of two angiostatin bands, which confirms physiological wound healing in these cancer patients.     

Hydrogen sulfide mitigates cardiac remodeling during myocardial infarction via improvement of angiogenesis

Exogenous hydrogen sulfide (H2S) leads to down-regulation of inflammatory responses and provides myocardial protection during acute ischemia/reperfusion injury; however its role during chronic heart failure (CHF) due to myocardial infarction (MI) is yet to be unveiled. We previously reported that H2S inhibits antiangiogenic factors such, as endostatin and angiostatin, but a little is known about its effect on parstatin (a fragment of proteinase-activated receptor-1, PAR-1). We hypothesize that H2S inhibits parstatin formation and promotes VEGF activation, thus promoting angiogenesis and significantly limiting the extent of MI injury. To verify this hypothesis MI was created in 12 week-old male mice by ligation of left anterior descending artery (LAD). Sham surgery was performed except LAD ligation. After the surgery mice were treated with sodium hydrogen sulfide (30 μmol/l NaHS, a donor for H2S, in drinking water) for 4 weeks. The LV tissue was analyzed for VEGF, flk-1 and flt-1, endostatin, angiostatin and parstatin. The expression of VEGF, flk-1 and flt-1 were significantly increased in treated mice while the level of endostatin, angiostatin and parstatin were decreased compared to in untreated mice. The echocardiography in mice treated with H2S showed the improvement of heart function compared to in untreated mice. The X-ray and Doppler blood flow measurements showed enhancement of cardiac-angiogenesis in mice treated with H2S. This observed cytoprotection was associated with an inhibition of anti-angiogenic proteins and stimulation of angiogenic factors. We established that administration of H2S at the time of MI ameliorated infarct size and preserved LV function during development of MI in mice. These results suggest that H2S is cytoprotective and angioprotective during evolution of MI.     

Concerted inhibition of HIF-1α and -2α expression markedly suppresses angiogenesis in cultured RPE cells

HIF-1α is known to play an important role in the induction of VEGF by hypoxia in retinal pigment epithelial (RPE) cells. However, the involvement of the other isoform, HIF-2α, in RPE cells remains unclear. Thus, the purpose of present study was to clarify the role of HIF-2α during induction of angiogenic genes in hypoxic RPE cells. When human RPE cells (ARPE-19) were cultured under hypoxic conditions, HIF-1α and HIF-2α proteins increased. This induced an increase in mRNA for VEGF, causing secretion of VEGF protein into the medium. This conditioned medium induced tube formation in human vascular endothelial cells (HUVEC). The increased expression of mRNA for VEGF in hypoxic RPE cells was partially inhibited by HIF-1α siRNA, but not by HIF-2α siRNA. However, co-transfection of HIF-1α siRNA and HIF-2α siRNA augmented downregulation of VEGF mRNA and protein in hypoxic RPE cells and inhibited formation of tube-like structures in HUVEC. GeneChip and PCR array analyses revealed that not only VEGF, but also expression of other angiogenic genes were synergistically downregulated by co-transfection of hypoxic RPE cells with HIF-1α and HIF-2α siRNAs. These findings suggest an important compensatory role for the HIF-2α isoform in the regulation of angiogenic gene expression. Thus, suppression of angiogenic genes for HIF-1α and HIF-2α may be a possible therapeutic strategy against retinal angiogenesis in Age-related macular degeneration (ARMD).     

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 PD-L1 and HIF-1α Coexpression with Poor Prognosis in Hepatocellular Carcinoma

OBJECTIVE: To investigate the correlation between the expression of PD-L1 and HIF-1α in hepatocellular carcinoma (HCC) tissue and further analyze the association with clinical parameters and the prognostic value of coexpression in HCC patients. METHODS: We assessed the expression of PD-L1 and HIF-1α by immunohistochemistry in tumor tissue from 90 HCC patients who underwent curative hepatectomy. The results were validated in an independent cohort of additional 90 HCC patients. RESULTS: PD-L1 and HIF-1α exhibited in tumor tissue high expression rates of 41.11% (37/90) and 43.33% (43/90), respectively, and their expressions were positively correlated (r = 0.563, P < .01). High expression of PD-L1 was significantly associated with low albumin levels (P < .05); high expression of HIF-1α was significantly correlated with high alpha-fetoprotein (AFP) levels and low albumin levels (P < .05); high expression of both PD-L1 and HIF-1α was also significantly associated with high AFP levels and low albumin levels (P < .05). High expression of PD-L1, HIF-1α, as well as both PD-L1 and HIF-1 α was respectively significantly associated with worse overall survival (OS) and disease-free survival (DFS) (P < .05). Patients with co-overexpression of PD-L1 and HIF-1α had the worst prognosis compared with other groups. Additionally, multivariate Cox regression models suggested that high expression of PD-L1, HIF-1α, as well as both PD-L1 and HIF-1α was an independent prognostic factor for OS and DFS (P < .05). Furthermore, the positive correlation and prognostic values of PD-L1 and HIF-1α were validated in an independent data set. CONCLUSION: We demonstrated that HCC patients with co-overexpression of PD-L1 and HIF-1α in tumor tissue had a significantly higher risk of recurrence or metastasis and death compared with others. Therefore, more frequent follow-up is needed for patients with co-overexpression of PD-L1 and HIF-1α. At the same time, a combinational therapy with HIF-1α inhibitors in conjunction with PD-L1 blockade may be beneficial for HCC patients with co-overexpression in the future.     

The role of hypoxia related angiogenesis in uterine smooth muscle tumors

Abstract

Mechanisms of hypoxia-related angiogenesis are important for uterine smooth muscle tumors. Factors that are related to angiogenesis during hypoxia include vascular endothelial growth factor (VEGF), hypoxia inducible factor 1α (HIF1α), T-cell intracellular antigen1 (TIA1), eukaryotic translation initiation factor 2α (eIF2α) and thrombospondin 1 (TSP1). We investigated immunoreactivities of VEGF, HIF1α, TIA1, eIF2α and TSP1 using an indirect immunoperoxidase method for formalin fixed, paraffin embedded tumors that had been diagnosed as leiomyoma (LMY), cellular leiomyoma (CLM) or leiomyosarcoma (LMS). TSP1 immunoreactivity was scored as moderate, mild or minimal, while VEGF, eIF2α and TIA1 immunoreactivities were scored as mild, moderate and strong in LMY, CLM and LMS samples, respectively. HIF1α immunoreactivity was scored as mild to minimal in LMY, CLM and LMS samples, but showed no statistically significant differences among samples. Although angiogenic factors showed strong immunohistochemical staining intensity in LMS, anti-angiogenic factors showed minimal immunohistochemical intensity. There was no difference in HIF-1α immunoreactivity compared to LMY, CLM and LMS samples. We suggest that HIF1α protein synthesis could be suppressed by eIF2α and TIA1. Furthermore, VEGF could be activated by pathways such as COX2, Ras, NF-?B or c-myc instead of HIF1α. Angiogenesis could trigger and accelerate tumor development; therefore, anti-angiogenic therapy could be useful for treatment of tumors.     

GABARBP down-regulates HIF-1α expression through the VEGFR-2 and PI3K/mTOR/4E-BP1 pathways

Human γ-aminobutyrate type A (GABA_A) receptor-binding protein (GABARBP), a tumor suppressor protein with apoptotic function, can be inhibited in response to angiogenesis through the PI3K/Akt signaling cascades. Here, we investigated whether GABARBP over-expression could regulate vascular endothelial growth factor (VEGF)/hypoxia-inducible factor-1α (HIF-1α) expression and angiogenic activity in a carcinoma model system. GABARBP dramatically inhibited VEGF-induced endothelial cell proliferation, migration, and tube formation, as well as VEGFR-2 phosphorylation in vitro. At the same time, GABARBP exposed potent anti-angiogenic activity and remarkably down-regulated the levels of VEGF and HIF-1α protein expression, key components for angiogenesis. In addressing its biological molecular mechanism, GABARBP was found to effectively inhibit the phosphorylation of down-stream PI3K components, such as PDK1, Akt, mTOR, TSC-2, p70S6K, and 4E-BP1 by directly binding with VEGFR-2. In contrast, p38/JNK phosphorylation was not suppressed by GABARBP. These findings disclose a novel function of GABARBP in suppressing VEGF and HIF-1α protein expression, which is important for tumor angiogenesis and tumor growth. Thus, our data strongly provides novel biological mechanistic insights into the regulatory function of GABARBP in ovarian tumor progression, and the important of pre-clinical certification of GABARBP as a potential angiogenesis agent targeting ovarian tumorigenesis.     

Tamoxifen and flaxseed alter angiogenesis regulators in normal human breast tissue in vivo

The incidence of breast cancer is increasing in the Western world and there is an urgent need for studies of the mechanisms of sex steroids in order to develop novel preventive strategies. Diet modifications may be among the means for breast cancer prevention. Angiogenesis, key in tumor progression, is regulated by the balance between pro- and anti-angiogenic factors, which are controlled in the extracellular space. Sampling of these molecules at their bioactive compartment is therefore needed. The aims of this study were to explore if tamoxifen, one of the most used anti-estrogen treatments for breast cancer affected some of the most important endogenous angiogenesis regulators, vascular endothelial growth factor (VEGF), angiogenin, and endostatin in normal breast tissue in vivo and if a diet supplementation with flaxseed had similar effects as tamoxifen in the breast. Microdialysis was used for in situ sampling of extracellular proteins in normal breast tissue of women before and after six weeks of tamoxifen treatment or before and after addition of 25 g/day of ground flaxseed to the diet or in control women. We show significant correlations between estradiol and levels of VEGF, angiogenin, and endostatin in vivo, which was verified in ex vivo breast tissue culture. Moreover, tamoxifen decreased the levels of VEGF and angiogenin in the breast whereas endostatin increased significantly. Flaxseed did not alter VEGF or angiogenin levels but similar to tamoxifen the levels of endostatin increased significantly. We conclude that one of the mechanisms of tamoxifen in normal breast tissue include tipping of the angiogenic balance into an anti-angiogenic state and that flaxseed has limited effects on the pro-angiogenic factors whereas the anti-angiogenic endostatin may be modified by diet. Further studies of diet modifications for breast cancer prevention are warranted.     

Hepatitis C virus stabilizes hypoxia-inducible factor 1alpha and stimulates the synthesis of vascular endothelial growth factor

Hepatitis C virus (HCV) infection is one of the major causes of chronic hepatitis, liver cirrhosis, which subsequently leads to hepatocellular carcinoma (HCC). The overexpression of the angiogenic factors has been demonstrated in HCC. In this study, we investigated the potential of HCV gene expression in inducing angiogenesis. Our results show that HCV infection leads to the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha). We further show that this stabilization was mediated via oxidative stress induced by HCV gene expression. The activation of NF-kappaB, STAT-3, PI3-K/AkT, and p42/44 mitogen-activated protein kinase was necessary for HIF-1alpha stabilization. HIF-1alpha induction in turn led to the stimulation of vascular endothelial growth factor. By using the chick chorioallantoic membrane assay, we show that HCV-infected cells released angiogenic cytokines, leading to neovascularization in vivo. These results indicate the potential of HCV gene expression in angiogenesis.     

Betulinic acid inhibits the expression of hypoxia-inducible factor 1α and vascular endothelial growth factor in human endometrial adenocarcinoma cells

Although betulinic acid (BA) is known to induce apoptosis and antiangiogenic response in tumor cells, the underlying mechanism of its action is unknown. Deregulation of tissue collagen metabolism is one of the consequences of neoplastic transformation. The final step of collagen degradation is mediated by prolidase [E.C.3.4.13.9] which may play a role in angiogenesis. The formation of new blood vessels is regulated by the hypoxia-inducible factor 1 (HIF-1). The expression of HIF-1 correlates with hypoxia-induced angiogenesis as a result of the induction of vascular endothelial cell growth factor (VEGF). Since BA evokes anticancer activity, its effect on collagen biosynthesis, HIF-1α and VEGF expressions, as well as prolidase activity and expression was studied in cultured endometrial adenocarcinoma (EA) cells. It was found that BA inhibits collagen biosynthesis in EA cells (5[³H] proline incorporation assay). It was accompanied by a parallel decrease in prolidase activity and expression and decrease in expressions of α₁ and α₂ integrins, HIF-1α, and VEGF (western immunoblot analysis) in cultured human EA cells. The data suggest that BA may have anti-angiogenic potential by inhibition of prolidase, HIF-1α and VEGF expressions, and inhibition of collagen biosynthesis.