Diabetes Publish Ahead of Print, published online February 11, 2008 Selective Activation of PPARα and PPARγ Induces Neoangiogenesis through a VEGF-dependent Mechanism Federico Biscetti MD1, Eleonora Gaetani MD1, Andrea Flex MD1, Tamar Aprahamian PhD2, Teresa Hopkins PhD2, Giuseppe Straface MD1, Giovanni Pecorini MD1, Egidio Stigliano BS3, Roy C. Smith PhD4, Flavia Angelini BS1, John J. Castellot Jr. PhD4, Roberto Pola MD PhD1,4,5 1 Laboratory of Vascular Biology and Genetics, Department of Medicine, A. Gemelli University Hospital, Catholic University School of Medicine, Rome, Italy; 2 Molecular Cardiology, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA; 3 Department of Pathology, Catholic University School of Medicine, Rome, Italy; 4 Department of Anatomy and Cell Biology, Tufts University School of Medicine, Boston, MA; 5 IRCCS OASI, Troina, Italy Running Title: PPARs and angiogenesis Corresponding Author: Roberto Pola, MD PhD Department of Anatomy and Cell Biology Tufts University School of Medicine 136 Harrison Avenue 02111 Boston, MA [email protected] Received for publication 6 June 2007 and accepted in revised form 24 January 2008. Copyright American Diabetes Association, Inc., 2008 PPARs and angiogenesis ABSTRACT Objective: Peroxisome proliferator-activated receptors (PPARs) are therapeutic targets for fibrates and thiazolidinediones, which are commonly used to ameliorate hyperlipidemia and hyperglycemia in type 2 diabetes mellitus (T2DM). In this study, we evaluated whether activation of PPARα and PPARγ stimulates neoangiogenesis. Research design and Methods: We used selective synthetic PPARα and PPARγ agonists and investigated their angiogenic potentials in vitro and in vivo. Results: Activation of PPARα and PPARγ leads to endothelial tube formation in an endothelial/interstitial cell co-culture assay. This effect is associated with increased production of the angiogenic cytokine Vascular Endothelial Growth Factor (VEGF). Neovascularization also occurs in vivo, when PPARα and PPARγ agonists are used in the murine corneal angiogenic model. No vascular growth is detectable when PPARα and PPARγ agonists are respectively used in PPARα knock-out mice and mice treated with a specific PPARγ inhibitor, demonstrating that this angiogenic response is PPAR-mediated. PPARα- and PPARγ-induced angiogenesis is associated with local VEGF production and does not differ in extent and morphology from that induced by VEGF. In addition, PPARα- and PPARγ-induced in vitro and in vivo angiogenesis may be significantly decreased by inhibiting VEGF activity. Finally, in corneas treated with PPARα and PPARγ agonists, there is increased phosphorylation of eNOS and Akt. Conclusions: These findings demonstrate that PPARα and PPARγ activation stimulates neoangiogenesis through a VEGF-dependent mechanism. Neoangiogenesis is a crucial pathologic event in T2DM. The ability of PPARα and PPARγ agonists to induce neoangiogenesis might have important implications for the clinical and therapeutic management of T2DM. 2 PPARs and angiogenesis eroxisome proliferator-activated rupture of atherosclerotic plaques in coronary receptors (PPARs) are ligand-inducible and carotid arteries. On the other hand, P transcription factors that belong to the neoangiogenesis is important to promote nuclear hormone receptor superfamily (1). revascularization and contrast ischemia in The clinical importance of PPARs originates tissues affected by diabetic microangiopathy. with fibrates and thiazolidinediones (TZDs), Therefore, the effects of PPARα and PPARγ which respectively act on PPARα and PPARγ in angiogenesis merit careful evaluation. and are used to ameliorate hyperlipidemia and Several studies have analyzed the angiogenic hyperglycemia in subjects with type 2 properties of PPARα and PPARγ by using diabetes mellitus (T2DM). Fibrates fibrates and TZDs, respectively. However, the (gemfibrozil, clofibrate, fenofibrate, and results of these investigations have been very bezofibrate) are drugs that effectively reduce controversial (14-22). In this regard, it is triglycerides (TG) and free fatty acids (FFA) important to point out that both fibrates and and increase high density lipoproteins- TZDs do not selectively act on PPARs, but cholesterol (2-5). Fibrates also improve have pleiotropic activities that occur through glucose tolerance in T2DM patients, although PPAR-independent pathways. For instance, this activity might be attributable to the fact fibrates activate other nuclear and superficial that some of these compounds also have receptors (23,24) and also stimulate pathways potential PPARγ activity (6). TZDs (such as that do not depend on PPARα (25, 26). rosiglitazone, troglitazone, pioglitazone, and Similarly, TZDs act on intracellular ciglitazone) are insulin-sensitizing drugs and mechanisms regulated by pERK, p38 MAP- have constituted a major advance in the recent kinase, PAI-1, MMP-2, Cyclin D1, Bcl- therapeutic management of T2DM (7-9). In xL/Bcl-2, and TNFα, in a PPARγ- addition to improve insulin sensitivity, TZDs independent way (27-33). In addition, TZDs have also effects on TG, FFA, and ketone are not PPARγ-specific, since high body level in several animal models of concentrations of these compounds may also T2DM. Recently, PPARα/γ dual agonists activate PPARα and PPARδ (34). A clear have also been produced, hypothesizing that demonstration of the PPARγ-independent the simultaneous activation of these nuclear properties of TZDs is provided by the fact receptors might provide better glucose and that these molecules are able to inhibit the lipid control than single subtype selective release of pro-inflammatory mediators in cells agents (10-13). that lack the PPARγ gene (35). In recent years, there has been increasing In the present study, we used selective appreciation of the fact that PPARα and PPARα and PPARγ synthetic agonists and PPARγ might be involved in the molecular tested their potential ability to stimulate mechanisms that regulate neoangiogenesis, neoangiogenesis in well-established in vitro defined as the growth of new blood vessels and in vivo assays. We found that specific and from pre-existing vascular networks, through selective activation of PPARα and PPARγ the action of growth factors and cytokines that leads to increased production of vascular stimulate migration, proliferation, and endothelial growth factor (VEGF), a survival of endothelial cells (ECs). prototypical angiogenic agent, and formation Neoangiogenesis plays a dual role in T2DM. of endothelial tubules when ECs are co- On one hand, it is involved in the cultured with interstitial cells. In vivo, PPARα pathogenesis of diabetic retinopathy and, and PPARγ synthetic agonists stimulate according to some studies, contributes to angiogenesis in the mouse corneal 3 PPARs and angiogenesis neovascularization assay, while fibrates and compared by an MTT assay performed TZDs are unable to induce angiogenesis in the according to manufacturer’s instructions same experimental setting. PPARα- and (ATCC). We also evaluated whether PPARγ-angiogenic process is associated with WY14643 and GW1929 were able to increase increased expression of VEGF and increased VEGF levels in ECs, by using a commercially phosphorylation of eNOS and Akt. Finally, it available ELISA kit (R&D Systems). All the may be inhibited by blocking VEGF activity. experiments were performed in triplicate. EC/interstitial cell co-culture assay. We used MATERIALS AND METHODS a commercially available assay (TCS PPAR selective agonists. The WY14643 and CellWorks), in which proliferating early the GW1929 compounds were used to passage normal human ECs are co-cultured with early passage normal human interstitial specifically activate PPARα and PPARγ, cells in 24-well plates, in a specially respectively. GW9662 was used to inhibit formulated culture medium. In this assay, PPARγ. VEGF was used as positive control. cells spontaneously proliferate and then enter All molecules were from Sigma. a migratory phase, during which they move EC migration and proliferation assays. through the matrix to form, after 11 days in Human umbilical vein ECs (HUVECs) and culture, a network of capillary-like tubules human dermal microvascular ECs (36). This degree of spontaneous angiogenic (HDMVECs) (Cambrex) were cultured in activity was evaluated and used as control to EGM2. Migration of ECs was determined quantify the angiogenic effects induced by the using a monolayer denudation assay. 80-90% addition of PPAR agonists. The following confluent ECs were scraped with a 1000 µl concentrations of WY14643 and GW1929 pipette tip. Cultures were washed three times were used: 1 nmol/L, 10 nmol/L, 100 nmol/L, with HBSS and incubated with EBM2/0.5% FBS. Control cultures received media alone, and 1 µmol/L. VEGF and suramin were while experimental groups received PPAR respectively used as positive and negative agonists at concentrations of 1 nmol/L, 10 controls, at the concentration suggested my nmol/L, 100 nmol/L, 1 µmol/L, 5 µmol/L, 10, the manufacturer (TCS CellWorks). On day and 20 µmol/L. VEGF was used as positive 11, cells were fixed with ice cold 70% ethanol control at a concentration of 10 nmol/L. The and tubule formation was visualized by rate of wound closure was determined by immunostaining for the EC marker CD31 and photographing cells at 3 pre-marked areas at quantified by image analysis (TCS AngioSys time 0 and at 6 and 12 hours. Using a digital software), as previously described (37,38). imaging system, the rate