Proangiogenic Environment Research Articles

Diabetic retinopathy is associated with a switch in splicing from anti- to pro-angiogenic isoforms of vascular endothelial growth factor

Proliferative diabetic retinopathy results from excess blood vessel growth into the vitreous fluid of the eye. Retinal angiogenesis is regulated by expression of vascular endothelial growth factor (VEGF), and many studies have shown that VEGF is critically involved in proliferative diabetic retinopathy. VEGF is alternatively spliced to form the angiogenic (VEGF(xxx)) and potentially anti-angiogenic (VEGF(xxx)b) family of isoforms. The VEGF(xxx)b family is found in normal tissues, but down-regulated in renal and prostate cancer. Previous studies on endogenous expression of VEGF in the eye have not distinguished between the two families of isoforms. We measured VEGF(xxx)b isoform expression in normal human eye tissue (lens, sclera, retina and iris) and vitreous fluid using enzyme-linked immunosorbent assay and Western blotting with a VEGF(xxx)b-specific antibody. VEGF(xxx)b protein was expressed in lens, sclera, retina, iris and vitreous fluid. Multiple isoforms were seen, including VEGF(165)b, VEGF(121)b, VEGF(145)b, VEGF(183)b and VEGF(189)b. In non-diabetic patients, 64+/-7% of the VEGF in the vitreous was VEGF(xxx)b (n=18), whereas in diabetic patients only 12.5+/-3.6% of total VEGF was VEGF(xxx)b. Since VEGF(xxx)b inhibits VEGF(xxx)-induced angiogenesis in a one-to-one stoichiometric manner, these results show that in the eye of diabetic patients VEGF splicing was switched from an anti-angiogenic to a pro-angiogenic environment. This occurred through changes to the ratio of VEGF(xxx):VEGF(xxx)b. Alterations to splicing, and through that to the balance of VEGF isoforms, could therefore be a potential therapeutic strategy for diabetic retinopathy.

Ultraviolet B-Induced Skin Angiogenesis Is Associated with a Switch in the Balance of Vascular Endothelial Growth Factor and Thrombospondin-1 Expression

We have previously shown that targeted overexpression of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in the epidermis prevented chronic ultraviolet B (UVB)-induced angiogenesis and cutaneous photodamage in mice, suggesting that angiogenesis plays a critical role in the mediation of UVB effects on the skin. Nevertheless, the molecular regulation of angiogenesis factors and inhibitors by acute UVB irradiation still remains to be elucidated. We performed quantitative analyses of cutaneous vascularity and of vascular endothelial growth factor (VEGF) and TSP-1 expression after acute UVB irradiation of mouse skin. Skin vascularity in the upper dermis was greatly increased until day 8 after a single dose of UVB irradiation (200 mJ per cm2) and associated with upregulation of VEGF mRNA expression, with downregulation of TSP-1 mRNA, and with protein expression in the hyperplastic epidermis. After 13 days, skin vascularity was normalized with downregulation of VEGF mRNA expression and upregulation of TSP-1 mRNA expression to the levels observed in non-UVB-irradiated skin. In contrast, the angiogenic UVB response was prolonged in TSP-1-deficient mice. We found a pronounced induction of the TSP-1 receptor CD36 in CD31-positive vessels on day 8 after UVB irradiation, associated with vascular endothelial cell apoptosis. These results demonstrate that acute UVB irradiation leads to a shift toward a proangiogenic environment and they suggest that the balance between VEGF and TSP-1 plays a critical role in the control of angiogenesis and vascular regression induced by acute UVB irradiation.

Implications for oxidative and nitrative stress in the pathogenesis of AIDS-related Kaposi's sarcoma.

AIDS-related Kaposi's sarcoma (AIDS-KS), which is the most prevalent AIDS related cancer, arises in a unique environment characterized by profound immunosuppression in conjunction with sustained immunostimulation. Persistent inflammation and the accompanying increased production of reactive species can promote carcinogenesis by numerous routes including sustained cell proliferation, initiation of nuclear and mitochondrial DNA mutations and induction of a proangiogenic environment. Furthermore, during conditions of continuous inflammation, protein nitration can result in irreversible inactivation of enzymes including the cytoprotective and reactive species degrading enzyme, mitochondrial superoxide dismutase (MnSOD). Because MnSOD serves as a putative tumor suppressor gene in addition to its reactive species inactivating capacities, the loss of MnSOD's cytoprotective functions could markedly facilitate malignant transformation. The purpose of this study was to investigate biochemical and molecular pathways by which reactive species facilitate AIDS-KS pathogenesis. Immunohistochemical studies of AIDS-KS tumors showed intense AIDS-KS lesional cell staining for MnSOD, inducible nitric oxide synthase (NOS 2) and the presence of a cellular 'fingerprint' of nitrative stress, 3-nitrotyrosine. Collectively, these results that imply reactive species stress occurs in situ. Similarly, cultured AIDS-KS cells derived from the AIDS-KS tumors contained both MnSOD protein and the 'high output' isoform, NOS 2. Co-localization studies established that the mitochondria are a primary site for 3-nitrotyrosine localization and immunoprecipitation/immunoblotting experiments confirmed that MnSOD tyrosine nitration occurs in AIDS-KS cells. Functional SOD assays showed that AIDS-KS cells possess significantly lower MnSOD activity relative to matched control cells; findings which correspond with ongoing MnSOD tyrosine nitration and subsequent inactivation within AIDS-KS cells. These results, which show in situ evidence of reactive species stress within AIDS-KS tumors and functional deficits attributable to nitrative stress in tumor-derived AIDS-KS lesional cells, imply that reactive species are intimately associated with AIDS-KS pathogenesis and provide insights for development of novel strategies for AIDS-KS clinical treatments.

Cyclooxygenase-2 overexpression inhibits cathepsin D-mediated cleavage of plasminogen to the potent antiangiogenic factor angiostatin.

Overexpression of cyclooxygenase (COX)-2 and enhanced synthesis of prostaglandin E2 (PGE2) have been implicated in human endometrial pathologies. To investigate the molecular role of COX-2, the Ishikawa human endometrial epithelial cell line was stably transfected with the pIRES2 vector containing COX-2 cDNA in either the sense or antisense directions. PGE2 concentrations were significantly elevated in the cells transfected with the COX-2 sense compared with wild-type cells or cells transfected with the antisense cDNA (P < 0.01). Elevated PGE2 synthesis was associated with enhanced expression and signaling of PGE2 receptors (EP). cDNA array analysis revealed differential expression of cathepsin D between the COX-2 sense and antisense cells. Cathepsin D RNA and protein expression was 6.7- and 2.1-fold lower in the COX-2 sense compared with COX-2 antisense cells respectively. Cathepsin D is known to cleave plasminogen to the potent antiangiogenic factor angiostatin. To investigate differential angiostatin generation, conditioned media from COX-2 sense, COX-2 antisense and wild-type cells were incubated with plasminogen and subsequently subjected to Western blot analysis. In comparison to wild-type cells, the cleavage of plasminogen to angiostatin was abolished when incubated in COX-2 sense cells conditioned media and elevated when incubated in COX-2 antisense cells conditioned media. Coincubation of plasminogen with the cathepsin D inhibitor pepstatin A inhibited the cleavage of plasminogen to angiostatin in the COX-2 antisense conditioned media. These data demonstrate that COX-2 exerts a negative feedback on the expression of cathepsin D. This in turn reduces the generation of the antiangiogenic factor angiostatin, hence promoting a proangiogenic environment.

Thrombospondin-1 Expression in Epithelial Ovarian Carcinoma: Association with p53 Status, Tumor Angiogenesis, and Survival in Platinum-Treated Patients

Objective. The regulation of the metastatic process in epithelial ovarian cancer has not been well defined. Similar to other tumor types, the angiogenic phenotype in ovarian cancer strongly influences clinical outcome, suggesting that the acquisition of a pro-angiogenic environment is essential to the process of ovarian cancer proliferation and metastasis. Thrombospondin-1 (TSP-1) is a potent peptide shown in other tumor systems to be associated with angiogenesis and possibly regulated by p53, a gene which is mutated in as high as 50% of advanced ovarian cancers. The purpose of this study was to investigate TSP-1 expression in invasive epithelial ovarian cancer and to examine the relationship between TSP-1 expression and the degree of angiogenesis. In addition, we examined whether TSP-1 expression was associated with overexpression of p53.Methods. Frozen sections obtained from 85 patients with invasive epithelial ovarian cancer were examined immunohistochemically for expression of TSP-1 and p53. The sections were examined microscopically by two investigators, who were blinded to the clinicopathologic variables. Outcome variables included the correlation among TSP-1, angiogenesis, and p53, as well as the association between TSP-1 expression and survival.Results. The majority (62%) of cases demonstrated high levels (3+) of TSP-1 expression; 7% demonstrated no TSP-1 expression. p53 was overexpressed in 55% of cases, and expression was inversely correlated with TSP-1 staining. Thirteen cancers had 0 or 1+ TSP-1 staining; 12 (92%) of these overexpressed the p53 protein. In contrast, only 49% of tumors with high expression of TSP-1 have overexpression of p53 (P = 0.02). TSP-1 was suggestive for improved survival in patients with advanced disease; high TSP-1 expression was associated with a median survival of 2.4 years compared to 1.5 years for patients with tumors having a lower degree of TSP-1 expression (P = 0.06).Conclusion. These data suggest that TSP-1 may possess a tumor inhibitory function in patients with advanced epithelial ovarian carcinoma. The reduction of TSP-1 expression associated with overexpression of p53 may be coupled with the development of a pro-angiogenic environment and malignant phenotype.

Macrophage infiltration in human non-small-cell lung cancer: the role of CC chemokines.

Bronchogenic carcinoma is the leading cause of malignancy-related mortality in the United States, with an overall 5-year survival rate of less than 15%. This aggressive behavior reflects, among other traits, the capacity of the tumor to evade normal host immune defenses, and to induce a pro-angiogenic environment. A central feature of any immune response toward tumors is the recruitment of specific immune cell populations. In the present study we investigated the infiltration of monocytes in human specimens of non-small-cell lung cancer (NSCLC). The presence of macrophages in NSCLC tumors was documented by immunohistochemistry. In vitro chemotaxis assays demonstrated higher monocyte chemotactic activity in NSCLC tumor homogenates than in normal lung tissue. We next investigated the expression of CC chemokines within specimens of NSCLC tumors. Levels of the CC chemokines were higher in NSCLC tumor tissue than in normal lung tissue. Immunolocalization showed that the cells associated with antigenic CC chemokines were the malignant tumor cells, as well as occasional stromal cells. Maximal inhibition of monocyte chemotaxis induced by NSCLC in vitro occurred in the presence of neutralizing antibodies to MCP-1 and MIP-1beta. On follow-up of 15 patients in whom we quantified macrophage infiltration, we found that those with recurrence of disease had higher levels of macrophage infiltration in their initial tumors. However, the functional significance of CC-chemokine-mediated macrophage infiltration into NSCLC remains to be determined.

Ultrasonic couplings: Joseph Lucas (Industries) Ltd British Patent 1, 213, 471 (1970) Filed 26 Jan 1968

Pregnant women with sickle cell disease (SCD) are at high risk for sickle cell-related complications, obstetrical complications, and perinatal morbidity. Chronic inflammation and the proangiogenic environment associated with SCD have been associated with endothelial damage.It is unknown whether SCD complications could be associated with placental dysfunction or abnormal placental morphology. Moreover, circulating angiogenic factors in pregnant women with SCD are unexplored.Clinical records, placental and blood samples were collected at term delivery for 21 pregnant patients with SCD and 19 HbAA pregnant controls with adapted to gestational age birth weight newborns. Histological and stereological analyses and scanning electron microscopy (SEM) of the placenta, and PlGF and sFlt1 measurements in blood were performed.In the SCD group, the parenchyma-forming villi of placentas were thinner than in controls, and increased fibrinoid necrosis and an overabundance of syncytial knots were seen. SEM revealed elongated intermediate villous endings with a reduction in the number of terminal villi compared to controls, indicating a significant branching defect in SCD placentas. Finally, SCD patients had an imbalance in the angiogenic ratio of sFlt1/PlGF (p = 0.008) with a drop of PlGF concentrations.We evidence for the first time both abnormal placenta morphology and altered sFlt1/PlGF ratio in SCD patients, uncorrelated with maintained placental efficiency and fetal growth.