Original Article
Comparative analysis of the in vivo angiogenic properties of stable prostacyclin analogs: a possible role for peroxisome proliferator-activated receptors

https://doi.org/10.1016/j.yjmcc.2003.10.016Get rights and content

Abstract

Objective. – Until recently, prostacyclin (PGI2) biological activities were thought to be exclusively mediated by cell surface receptors named IP. Recent studies have instead identified a novel pathway of PGI2 signaling, occurring through activation of peroxisome proliferator-activated receptors (PPARs) located in the nucleus. The availability of stable PGI2 analogs with different affinity for IP receptors and PPARs provides the possibility to test the importance and function of this dual pathway in vitro and in vivo. In this study, the in vivo angiogenic properties of different PGI2 analogs and the potential relationship between PPAR-mediated pathways, vascular endothelial growth factor (VEGF), and angiogenesis were investigated.

Methods and results. – By using the murine corneal model of angiogenesis, we found that PGI2 analogs able to act on nuclear PPARs, such as iloprost and carbaprostacyclin (cPGI), induce angiogenesis in vivo. In contrast, cicaprost, a PGI2 analog that only acts on IP receptors, has no in vivo angiogenic activity. Interestingly, angiogenesis induced by iloprost and cPGI does not differ in extent and morphology from that induced by VEGF and is associated with local increment of VEGF mRNA expression and protein levels. Finally, iloprost-induced angiogenesis is significantly decreased by systemic inhibition of VEGF activity, obtained by gene transfer of a soluble form of the VEGF receptor Flt-1.

Conclusions. – These data demonstrate that stable PGI2 analogs may have angiogenic properties in vivo, depending on their ability to act on PPARs. The resulting angiogenic process appears to be mediated by VEGF. These findings indicate that important physiological activities in the cardiovascular system, such as angiogenesis and VEGF induction, may be modulated by PGI2 through specific activation of the PPAR signaling pathway in vivo, with potentially important fundamental and clinical implications.

Introduction

Prostacyclin (PGI2) is the most abundant product of arachidonic acid in vascular tissues and has well-characterized antiplatelet and vasodilating effects [1]. Endogenous PGI2 is a very labile molecule that undergoes spontaneous inactivation within minutes in vivo [2]. Owing to its valuable clinical implications in vascular biology, several synthetic analogs with more stable chemical structures, such as iloprost, carbaprostacyclin (cPGI), and cicaprost, have been developed [3], [4], [5].

The multiple biological activities of the PGI2 system are mediated by dual signaling pathways, one using exported prostaglandins (PGs) from the endoplasmic reticulum and the other using nuclear PGs. The classical PGI2 signaling pathway uses a G protein-coupled cell surface receptor named IP [6], whose activation leads to increased production of intracellular cAMP and consequent activation of the protein kinase A cascade, or calcium mobilization via phospholipase C activation [6], [7]. In addition to this classical signaling pathway, PGI2 also utilizes a class of nuclear receptors known as peroxisome proliferator-activated receptors (PPARs), that belong to a family of ligand-activated transcription factors with activities relevant to broad aspects of cardiovascular biology [8], [9], [10], [11], [12], [13], [14], [15], [16]. Recent findings indicate that PPARs may be activated by several members of the PG system [8]. In particular, certain eicosanoids are among the ligands for PPARα, while ligands for PPARγ include the prostaglandin 15d-PGJ2 [8]. On the other hand, PGI2 agonists, such as iloprost and cPGI-can effectively induce DNA binding and transcriptional activation by PPARα and PPARδ [8], [11]. Interestingly, other PGI2 analogs, such as cicaprost, only act on IP receptors and are incapable to induce signaling activation through the PPAR system, indicating that a different affinity for this class of receptors exists among synthetic analogs of PGI2.

Recently, it has been proposed that PPARs may play a role in angiogenesis. Some reports indicate that, in endothelial cells (ECs) and macrophages, oxidized LDL, troglitizone, and 15d-PGJ2 increase the expression of vascular endothelial growth factor (VEGF), a prototypical angiogenic agent, via PPARγ activation [12], [13]. VEGF expression and secretion may be upregulated also in smooth muscle cells by 15d-PGJ2 [14]. In addition, a gene related to angiopoietins, termed PGAR (for PPARγ angiopoietin-related gene), is a physiologic target for both PPARα and PPARγ and its expression is upregulated by hypoxia and hypoxia-inducible factor-1α (HIF-1α) [15]. Finally, PPARδ has recently been identified as a new biological target for PGI2 during blastocyst implantation, a process that requires angiogenesis [16]. Interestingly, it has also been reported that, in COX-2-deficient mice, the reduced expression of the VEGF receptor KDR in the uterus may be upregulated by PGI2 administration [16], thus suggesting that PPARδ might play a role in inducing factors that are involved in angiogenesis.

In this study, we investigated the in vivo angiogenic properties of stable PGI2 analogs with different affinity for IP receptors and PPARs. We found that angiogenesis may be induced in vivo only by iloprost and cPGI, that have the ability to act on PPARs, while cicaprost, that only acts on IP receptors, has not in vivo angiogenic properties. We also found that iloprost- and cPGI-induced angiogenesis in vivo occurs in association with local VEGF upregulation and that iloprost-induced neovascularization may be inhibited by blocking systemic VEGF activity, thus suggesting a crucial role for VEGF in mediating the angiogenic properties of PPAR-targeted PGI2 analogs.

Section snippets

Animals

Male 8–12-week-old C57BL/6J mice were used for all the experiments. Mice were anesthetized with an intramuscular injection of ketamine and sacrificed with an overdose of ketamine. The investigation was in accordance with the A. Gemelli University Hospital Institutional Animal Care and Use Committee.

Mouse cornea neovascularization assay

This in vivo model of angiogenesis has been previously described [17]. Briefly, a single corneal pocket was created in each eye using a modified von Graefe cataract knife. Pellets containing 0.3 μg

Results

Six days after pellet implantation, corneas were evaluated for the presence of angiogenesis. No blood vessels were found in corneas where control pellets were implanted (Fig. 1a ). In contrast, VEGF-treated corneas exhibited a robust angiogenic response (Fig. 1b). Similarly, pellets containing iloprost and cPGI were able to induce a strong corneal angiogenic process (Fig. 1c,d). Interestingly, no angiogenesis was detected in corneas where pellets containing cicaprost were implanted (Fig. 1e).

Discussion

In recent years, there has been increasing appreciation of the interdependent relationship existing between the PGI2 system and angiogenesis. It is well known that PGI2 mediates several activities of VEGF and is considered one key molecule in linking post-receptor signaling networks to downstream biological effects of VEGF [19], [20], [21], [22], [23], [24]. Not only VEGF uses PGI2 as modulator of its angiogenic properties. Indeed, it has been documented that PGI2 is also an essential mediator

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