Inducers of angiogenesis promote endothelial cell (EC) survival, and inhibitors cause apoptosis. Inhibitors target molecules in the inducer-generated pathways in remodeling EC. In the original proposal we showed that inhibitors Thrombospondin-1 (TSP1) and pigment epithelial-derived factor (PEDF) increase EC CD95 ligand (CD95L), a death mediator. Inducers upregulate death receptor, CD95, which binds CD95L and triggers apoptosis, thus blocking angiogenesis. This is how EC balance apoptosis and survival by angiogenic inhibitors/stimuli. We identified another common target of the pro- and anti-angiogenic factors, the nuclear factor of activated T-cells (NFAT). NFAT also acts as molecular pivot balancing angiogenesis activation and inhibition. Promoter array analysis identified activity changes of several transcription factors due to TSP1 and PEDF including NFicB, cMyb and Egr-1. All three form common network with NFAT. We propose to elucidate signaling and transcriptional events involved in the NFAT cross-regulation by the pro- and anti-angiogenic factors. We will use two non-related inhibitors, TSP1 and PEDF, and two stimuli, vascular endothelial growth factor and basic fibroblast growth factor (VEGF and bFGF). We will determine: The upstream mediators of NFAT deactivation by TSP1 and PEDF. We will evaluate the contribution of JNKkinases, p38 and GSK in vitro by functional assays with kinase inhibitors, immunoprecipitation and western blotting. We will analyze the effect of inhibitors on the levels and activity of NFAT proximal activator, Calcineurin A (CnA) and its modulators, Ca++ mobilization and DSCR-1. Mice null for NFATc2 and CnA will be used to confirm their functional role. The regulation of NFAT targets by TSP1 and PEDF. We will screen known NFAT targets Bcl-2, cyclins A and E, c-FLIP, cyclooxygenase-2 (Cox-2), interleukins and tissue factor by Western and Northern blotting. Confirmed targets will be evaluated by EMSA and ChIP (chromatin immunoprecipitation) with NFATc2 antibodies and assessed in the in vitro angiogenesis assays with si-RNA or neutralizing antibodies. NFicB role in the TSP1 and PEDF signaling. NFicB activation will be confirmed by immunocytochemistry and EMSA. Functional importance will be demonstrated using biochemical inhibitor and/or constitutive^ active IkB (kB*). Role in the FasL regulation will be examined using inhibitor and by ChIP. Knock-out mice will be used to verify in vivo NFicB contribution. The involvement of E2F1, Egr-1 and c-Myb in NFAT action and regulation. Changes in Egr-1 and c-Myb activity due to TSP1 or PEDF will be confirmed by EMSA. Mice null for Egr and c-Myb will be used to evaluate their biological role in the anti-angiogenesis by TSP1 or PEDF. The interaction with NFAT will be studied using EMSA, IP, and ChlP. A growing body of evidence suggests that activated endothelium is poised for apoptosis induction by inhibitors. We will thus delineate molecular targets common for the inhibitors and stimuli and identify major transcription factors involved in their interaction. These studies will yield new targets for therapeutic intervention using natural inhibitors. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL068033-05A1
Application #
7091040
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Goldman, Stephen
Project Start
2001-09-30
Project End
2010-03-31
Budget Start
2006-04-01
Budget End
2007-03-31
Support Year
5
Fiscal Year
2006
Total Cost
$375,938
Indirect Cost
Name
Northwestern University at Chicago
Department
Urology
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
Veliceasa, Dorina; Biyashev, Dauren; Qin, Gangjian et al. (2015) Therapeutic manipulation of angiogenesis with miR-27b. Vasc Cell 7:6
Biyashev, Dauren; Veliceasa, Dorina; Topczewski, Jacek et al. (2012) miR-27b controls venous specification and tip cell fate. Blood 119:2679-87
Orgaz, Jose L; Benguria, Alberto; Sanchez-Martinez, Cristina et al. (2011) Changes in the gene expression profile of A375 human melanoma cells induced by overexpression of multifunctional pigment epithelium-derived factor. Melanoma Res 21:285-97
Ladhani, Omar; Sanchez-Martinez, Cristina; Orgaz, Jose L et al. (2011) Pigment epithelium-derived factor blocks tumor extravasation by suppressing amoeboid morphology and mesenchymal proteolysis. Neoplasia 13:633-42
Biyashev, Dauren; Veliceasa, Dorina; Kwiatek, Angela et al. (2010) Natural angiogenesis inhibitor signals through Erk5 activation of peroxisome proliferator-activated receptor gamma (PPARgamma). J Biol Chem 285:13517-24
Aurora, Arin B; Aurora, Aryn B; Biyashev, Dauren et al. (2010) NF-kappaB balances vascular regression and angiogenesis via chromatin remodeling and NFAT displacement. Blood 116:475-84
Mirochnik, Yelena; Aurora, Arin; Schulze-Hoepfner, Frank T et al. (2009) Short pigment epithelial-derived factor-derived peptide inhibits angiogenesis and tumor growth. Clin Cancer Res 15:1655-63
Orgaz, J L; Ladhani, O; Hoek, K S et al. (2009) 'Loss of pigment epithelium-derived factor enables migration, invasion and metastatic spread of human melanoma'. Oncogene 28:4147-61
Smith, Norm D; Schulze-Hoepfner, Frank Thilo; Veliceasa, Dorina et al. (2008) Pigment epithelium-derived factor and interleukin-6 control prostate neuroendocrine differentiation via feed-forward mechanism. J Urol 179:2427-34
Veliceasa, Dorina; Schulze-Hoepfner, Frank Thilo; Volpert, Olga V (2008) PPARgamma and Agonists against Cancer: Rational Design of Complementation Treatments. PPAR Res 2008:945275

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