Activity Of Angiogenic Factors Research Articles

Inflammation mediated angiogenesis in chronic lymphocytic leukemia.

Chronic inflammation has been identified in leukemias as an essential regulator of angiogenesis. B-chronic lymphocytic leukemia (CLL) cells secrete high levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1 alpha (HIF1α). The aim was to assess the role of inflammation in activation of angiogenic factors: endothelial nitric oxide synthase (eNOS), HIF1α and VEGF via proliferation related signaling pathways and VEGF autocrine control. We isolated mononuclear cells (MNC) and CD19+ cells from peripheral blood of 60 patients with CLL. MNC were treated with pro-inflammatory interleukin-6 (IL-6) and VEGF, in combination with inhibitors of JAK1/2 (Ruxolitinib), mTOR (Rapamycin), NF-κB (JSH23), SMAD (LDN-193189) and PI3K/AKT (Ly294002) signaling pathways, to evaluate eNOS, VEGF and HIF1α expression by immunoblotting, immunocytochemistry and RT-qPCR. Also, we investigated IL-6 dependent neovascularization in human microvascular endothelial cells (HMEC-1) in co-culture with MNC of CLL. The angiogenic factors eNOS, VEGF and HIF1α had significantly higher frequencies in MNC of CLL in comparison to healthy controls (p < 0.001) and CD19+ cells of CLL. IL-6 increased the quantity of HIF1α (p < 0.05) and VEGF positive cells in the presence of JSH23 (p < 0.01). VEGF increased HIF1α (p < 0.05), and decreased eNOS gene expression (p < 0.01) in MNC of CLL. VEGF significantly (p < 0.001) increased the number of HIF1α positive MNC of CLL, prevented by inhibitors of JAK1/2, PI3K and mTOR signaling pathways. VEGF stimulation of SMAD (p < 0.05) and STAT5 (p < 0.01) signaling has been prevented by inhibitors of JAK1/2, mTOR, PI3K and SMAD signaling, individually (p < 0.01) or mutually (p < 0.001). Also, we showed that MNC of CLL and IL-6 individually stimulate neovascularization in co-culture with HMEC-1, without a cumulative effect. We demonstrated elevated angiogenic factors in CLL, while VEGF and IL-6 independently stimulated HIF1α. VEGF stimulation of HIF1α was mostly mTOR dependent, while IL-6 stimulation was NF-κB dependent.

The Mechanism of Action of Follistatin-like Protein-1 (FSTL-1)

The paper provides current data on some proteins of the TGF- p family which are potentially capable of exerting a protective effect in diseases of the heart, lungs, placenta, gonads, and pancreas. The study investigated the anti-inflammatory properties of follistatin-like protein-1 (FSTL-1), one of the proteins of this family, at the cellular level. It was demonstrated that FSTL-1 is responsible for heart muscle regeneration in mammals through activation of angiogenic factors. Despite the fact that this protein plays a key role in myocardial regeneration, its concentration in the epicardium decreases immediately after a heart attack, which hampers effective self-repair of the heart. The paper summarises the results of studies of the efficacy of intravenous administration of FSTL-1 in rats with myocardial infarction. However, the administration of a foreign protein can cause allergic reactions, therefore a drug that induces FSTL-1 secretion was chosen instead.The aim of the study was to provide experimental substantiation of the possibility of exogenous regulation of FSTL-1 secretion.Materials and methods: FSTL-1 concentration in rat plasma was assessed by enzyme immunoassay before and after treatment with the antioxidant drug ethyl methyl hydroxypyridine malate. The antioxidant was administered to 15 healthy male Wistar rats subcutaneously 3 times a day at a dose of 6 mg/day for 14 days. A fasting blood sample was obtained on the first day before administration of the drug and on day 15.Results: after the period of treatment with ethyl methyl hydroxypyridine malate the concentration of FSTL-1 in the plasma of the laboratory rats increased significantly (p = 0.0011) to reach 0.92 ± 0.11 ng/mL as compared to the initial concentration of 0.48 ± 0.04 ng/mL.Conclusion: the study provided experimental evidence for new properties of ethyl methyl hydroxypyridine malate, i.e. induction of FSTL-1 in healthy rats. Further studies are encouraged to assess potential use of this drug as an inductor of FSTL-1 in myocardial ischemia.

Stemness marker ALDH1A1 promotes tumor angiogenesis via retinoic acid/HIF-1\u03b1/VEGF signalling in MCF-7 breast cancer cells

BackgroundAldehyde dehydrogenase 1A1 (ALDH1A1), a member of aldehyde dehydrogenase family, is a marker of stemness in breast cancer. During tumor progression cancer stem cells (CSCs) have been reported to secrete angiogenic factors to orchestrate the formation of pathological angiogenesis. This vasculature can represent the source of self-renewal of CSCs and the route for further tumor spreading. The aim of the present study has been to assess whether ALDH1A1 controls the output of angiogenic factors in breast cancer cells and regulates tumor angiogenesis in a panel of in vitro and in vivo models.MethodsStemness status of breast cancer cells was evaluated by the ability to form turmorspheres in vitro. A transwell system was used to assess the angiogenic features of human umbilical vein endothelial cells (HUVEC) when co-cultured with breast cancer cells MCF-7 harboring different levels of ALDH1A1. Under these conditions, we survey endothelial proliferation, migration, tube formation and permeability. Moreover, in vivo, MCF-7 xenografts in immunodeficient mice allow to evaluate blood flow, expression of angiogenic factors and microvascular density (MVD).ResultsIn MCF-7 we observed that ALDH1A1 activity conferred stemness property and its expression correlated with an activation of angiogenic factors. In particular we observed a significant upregulation of hypoxia inducible factor-1α (HIF-1α) and proangiogenic factors, such as vascular endothelial growth factor (VEGF). High levels of ALDH1A1, through the retinoic acid pathway, were significantly associated with VEGF-mediated angiogenesis in vitro. Co-culture of HUVEC with ALDH1A1 expressing tumor cells promoted endothelial proliferation, migration, tube formation and permeability. Conversely, downregulation of ALDH1A1 in MCF-7 resulted in reduction of proangiogenic factor release/expression and impaired HUVEC angiogenic functions. In vivo, when subcutaneously implanted in immunodeficient mice, ALDH1A1 overexpressing breast tumor cells displayed a higher expression of VEGF and MVD.ConclusionIn breast tumors, ALDH1A1 expression primes a permissive microenvironment by promoting tumor angiogenesis via retinoic acid dependent mechanism. In conclusion, ALDH1A1 might be associated to progression and diffusion of breast cancer.

Cyclic strain alters the expression and release of angiogenic factors by human tendon cells.

Angiogenesis is associated with the tissue changes underlying chronic overuse tendinopathy. We hypothesized that repetitive, cyclic loading of human tendon cells would lead to increased expression and activity of angiogenic factors. We subjected isolated human tendon cells to overuse tensile loading using an in vitro model (1 Hz, 10% equibiaxial strain). We found that mechanically stimulated human tendon cells released factors that promoted in vitro proliferation and tube formation by human umbilical vein endothelial cells (HUVEC). In response to cyclic strain, there was a transient increase in the expression of several angiogenic genes including ANGPTL4, FGF-2, COX-2, SPHK1, TGF-alpha, VEGF-A and VEGF-C, with no change in anti-angiogenic genes (BAI1, SERPINF1, THBS1 and 2, TIMP1-3). Cyclic strain also resulted in the extracellular release of ANGPTL4 protein by tendon cells. Our study is the first report demonstrating the induction of ANGPTL4 mRNA and release of ANGPTL4 protein in response to cyclic strain. Tenocytes may contribute to the upregulation of angiogenesis during the development of overuse tendinopathy.

Midgestation Maternal Serum 25-Hydroxyvitamin D Level and Soluble Fms-Like Tyrosine Kinase 1/Placental Growth Factor Ratio as Predictors of Severe Preeclampsia

Recent studies have shown that low serum 25-hydroxyvitamin D (25[OH]D) level is a risk factor for preeclampsia. The clinical significance of in vitro findings that vitamin D regulates vascular endothelial growth factor production is unclear. We sought to determine whether there is an association between midgestation serum 25(OH)D levels and angiogenic factor activity and to compare their predictive value for the development of severe preeclampsia. We conducted a nested case-control study of women with severe preeclampsia (n=41) versus women with uncomplicated term birth (n=123) who had second trimester genetic screening (15-20 weeks). Using banked frozen serum, we measured levels of 25(OH)D, vascular endothelial growth factor, soluble fms-like tyrosine kinase 1, and placental growth factor and compared their correlations and predictive values. We found no correlation between serum 25(OH)D and angiogenic factors levels. 25(OH)D alone was comparable to vascular endothelial growth factor and soluble fms-like tyrosine kinase 1/placental growth factor ratio as a predictive marker for severe preeclampsia. A composite of both 25(OH)D level and soluble fms-like tyrosine kinase 1/placental growth factor ratio was more predictive than either alone (area under curve: 0.83 versus 0.74 and 0.67, respectively). In conclusion, combining midpregnancy 25(OH)D level with soluble fms-like tyrosine kinase 1/placental growth factor ratio provides a better prediction for the development of severe preeclampsia.

Oxidative stress is closely associated with tumor angiogenesis of hepatocellular carcinoma

Oxidative stress (OS) plays an important role in the progression of chronic liver disease and hepatocarcinogenesis. However, the role of OS in the progression of hepatocellular carcinoma (HCC) is unclear. The aim of this study was to assess whether OS promotes angiogenesis in HCC. The expressions of vascular endothelial growth factor (VEGF), VEGF receptor2 (VEGFR2), and phosphorylated Akt were assessed, and microvessel density (MVD) and the cancer-associated fibroblast (CAF) population were examined by immunohistological staining in 55 HCC samples. The OS level in these tissues was assessed using 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal (4-HNE) immunostaining, and an 8-OHdG enzyme-linked immunosorbent assay (ELISA). The expression and activation of angiogenic factors and the effect of growth stimulation of human umbilical vein endothelial cells (HUVECs) were also assessed in vitro, using HLE hepatoma-derived cells and conditioned medium with or without treatment with hydrogen peroxide (H₂O₂); a phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin; and an anti-oxidative agent, N-acetyl-L-cysteine (NAC). A higher OS grade was significantly associated with higher MVD, VEGF expression, Akt activity, and OS grade of CAFs, but not with the percentage of the CAF population in HCC tissues. Additionally, cancer cells constituted a major population of OS marker-positive cells in HCC tissues. In vitro, H₂O₂ treatment induced up-regulation of VEGF at both the mRNA and protein levels, activated Akt, and resulted in the proliferation of HUVECs; the addition of wortmannin and NAC counteracted the effects of OS. OS enhances the malignant potential of HCC through the stimulation of angiogenesis by activation of the Akt-VEGF pathway.

Abstract 5369: AN019 in combination with ionizing radiation retards intracranial tumor growth and inhibits angiogenic induction in vitro and in vivo

Abstract Successful treatment of malignant glioma is hampered by the development of resistance to alkylating agents such as Temozolomide (TMZ). The addition of radiation to a TMZ treatment regime has shown promising results with reported survivability of up to 5 years. One of the side effects of Temozolomide is the increased activation of angiogenic factors which may work contrary to the desired result. In this report we demonstrate the development of a novel compound AN019, showing specific VEGF suppression accompanied with anti-angiogenic and anti-proliferative properties in gliomas. We used U87, a glioma cell line, and EGFRvIII over-expressing glioma xenograft 4910 cells for this study. Deduced IC50 values indicated that AN019 had greater anti-proliferative effect than Temozolomide (U87: AN019=103µM, TMZ=768µM; 4910: AN019=169µM, TMZ=582µM). Matrigel invasion assay indicated that AN019 retarded the invasive potential of both U87 and 4910 glioma cells over TMZ treated cells which was enhanced after radiation treatment. In-vitro angiogenic and migration assays showed an almost complete inhibition of angiogenic induction and migration in U87 and 4910 cells after AN019 treatment when compared to TMZ. Cell cycle analysis revealed that 100µg/ml concentration of AN019 was sufficient to achieve &amp;gt;70% apoptosis in both U87 and 4910 cells, whereas 150µg/ml of TMZ was required to achieve the same response. Western blot and ELISA for the expression of VEGF levels indicated that lower concentrations of AN019 (U87=86±3µg/ml and 12±1µg/ml with radiation; 4910=100±1µg/ml and 6µg/ml with radiation) were required for 50% inhibition of VEGF expression. TMZ treatment required a higher concentration to achieve similar 50% inhibition of VEGF expression (U87=80±10µg/ml and 12±3µg/ml with radiation; 4910=100±3µg/ml and 27±3µg/ml with radiation). Animal studies showed that oral doses of AN019 (20mg/kg) regressed tumors in all animals and 70% of the animals showed complete absence of intracranial tumors, whereas TMZ (100mg/kg) treated animals also showed regression of tumors with a 30% cure rate. Overall, our results demonstrate that AN019 has anti-angiogenic, anti-proliferative and anti-tumorogenic properties which may have clinical implications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5369.

Anti-angiogenic potential of CoenzymeQ 10, riboflavin and niacin in breast cancer patients undergoing tamoxifen therapy

Tumour angiogenesis is a complex mechanism consisting of multi-step events including secretion or activation of angiogenic factors by tumour cells, activation of proteolytic enzymes, proliferation, migration and differentiation of endothelial cells. Both primary and metastatic tumours in the breast are dependent on angiogenesis. In the present study, 84 breast cancer patients were randomized to receive a daily supplement of CoQ 10 100 mg, riboflavin 10 mg and niacin 50 mg (CoRN), one dosage per day along with tamoxifen (TAM) 10 mg twice a day. Serum pro-angiogenic levels were elevated in untreated breast cancer patients (Group II) and their levels were found to be reduced in breast cancer patients undergoing TAM therapy for more than 1 year (Group III). When these group III breast cancer patients were supplemented with CoRN for 45 days (Group IV) and 90 days (Group V) along with TAM, a further significant reduction in pro-angiogenic marker levels were observed. Supplementing CoRN to breast cancer patients has found to decrease the levels of pro-angiogenic factors and increase the levels of anti-angiogenic factors. A reduction in pro-angiogenic marker levels attributes to reduction in tumour burden and may suggest good prognosis and efficacy of the treatment, and might even offer protection from cancer metastases and recurrence.

Tumor Angiogenesis: Initiation and Targeting - Therapeutic Targeting of an FGF-Binding Protein, an Angiogenic Switch Molecule, and Indicator of Early Stages of Gastrointestinal Adenocarcinomas -

Tumor angiogenesis has been related to the initiation as well as progression toward more aggressive behavior of human tumors. In particular, the activity of angiogenic factors is crucial for tumor progression. We previously characterized a secreted fibroblast growth factor-binding protein (FGF-BP) as a chaperone molecule, which binds to various FGFs, enhances FGF-mediated biochemical and biologic events and importantly is a crucial rate-limiting factor for tumor-dependent angiogenesis. We generated monoclonal antibodies that target FGF-BP protein and used them as a tool to evaluate frequency and pattern of FGF-BP expression during the malignant progression of pancreas and colorectal carcinoma in archival tissue samples. We found that FGF-BP is dramatically upregulated during the initiation of colorectal and pancreatic adenocarcinoma. Crucial genetic events underlying the initiation and progression of colorectal and pancreatic adenocarcinoma with a particular focus on the modulation of angiogenesis and antiangiogenic therapies are discussed. We propose that the upregulation of the secreted FGF-BP protein during early phases of pancreas and colon cancer could make this protein a possible serum marker indicating the presence of high-risk premalignant lesions. Furthermore, the biological activity of FGF-BP is neutralized by monoclonal antibodies suggesting the potential for antibody-based therapeutic targeting.

The second international congress on myeloproliferative and myelodysplastic syndromes

This meeting was convened by Richard T. Silver, M.D. and co-chaired by Jerry L. Spivak, M.D. It was held from 16 to 18 October 2003 in New York City, New York, USA. Thirty-nine invited speakers from nine different countries participated in the conference. There were more than 350 attendees. There were formal presentations and discussions on biology, clinical aspects, and management of patients with these diverse bone marrow stem cell disorders linked by a variable progression to acute myeloid leukemia. Of considerable interest, a clinical symposium exclusively for patients was held the day preceding the meeting at which John Bennett, Tiziano Barbui, Richard Silver, Jerry Spivak, and Ayalew Tefferi spoke on various topics pertaining to these diseases. This proved to be highly informative to the patients who reported that they enjoyed the program immensely. This was sponsored by the Cancer Research & Treatment Fund, Inc. Representatives of the Myelodysplasia Foundation were also present. This meeting report provides a summary of five different sections prepared by one or more of the session chairs. The keynote address was given by Shahin Rafii (Cornell Medical Center). Most appropriately, this talk focused on the expression and activation of angiogenic factors which play a crucial role in the progression of both myeloproliferative disorders and myelodysplastic syndromes (MDS). Among the known factors, vascular endothelial growth tyrosine kinase receptors (VEGF-R1, R2, and R3) support proliferation, survival, and mobility. Rafii’s team has demonstrated that these receptors are expressed on subsets of primary hematopoietic cells as well as leukemic cells. Some leukemic cells express both VEGF-A and VEGF-R2, resulting in the generation of an autocrine loop that supports survival and within the osteoblastic zone translocating these cells to the vascular enriched niche for receipt of molecular instructions required for proliferation and differentiation. A pathologic correlation can be seen in some patients with the identification of abnormal localization of immature precursors (ALIP) in the central portions of the medullary cavity. Misplaced megakaryocytes can release pro-fibrotic factors, including platelet derived growth factors and transforming growth factor-beta. Collectively, these data suggest that chronic disregulation of angiogenic factors alter the microenvironment dislocating marrow stem cells that force both proliferation and differentiation in varying degrees, contributing to these hematological disorders.

Elevated endothelial-cell-stimulating angiogenic factor activity in rodent glycolytic skeletal muscles.

1. Capillary density is greater in skeletal muscles comprised of predominantly oxidative (type I) fibres than in those comprised of mainly glycolytic (type II) fibres. In order to investigate further the angiogenic mechanisms involved in muscle capillarization, endothelial-cell-stimulating angiogenic factor activities in various rodent skeletal muscles were compared. 2. Eleven untrained adult male Wistar rats were killed and the predominantly oxidative (type I) muscle,s soleus and heart, the predominantly glycolytic (type II) muscle, extensor digitorum longus, and the mixed-fibre muscle, gastrocnemius, were removed. Each sample was separately homogenized and centrifuged and the supernatants were diafiltered to isolate the low-molecular-mass fraction containing endothelial-cell-stimulating angiogenic activity. This was assayed for its ability to activate latent collagenase and was expressed as units, where 1 unit represents the percentage activation of the enzyme h-1 (mg of protein in the supernatant)-1. 3. The results (medians and ranges) demonstrated significantly greater endothelial-cell-stimulating angiogenic factor activity in extensor digitorum longus muscle (2.14 units, 0.62-2.87 units, n = 13) than in soleus (0.82 units, 0.59-1.79 units, n = 15), gastrocnemius (0.34 units, 0.28-0.40 units, n = 4) or heart (0.43 units, 0.16-0.52 units, n = 11) (P less than 0.01 for each) muscle. 4. These findings suggest that endothelial-cell-stimulating angiogenic activity in muscle is either inversely or not related to the local capillary density, which may be at or near a maximum in physiologically contracting, predominantly oxidative muscles.