14-3-3 pathway

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Contents lists available at ScienceDirect

Prostaglandins and Other Lipid Mediators

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Invited review

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Prostacyclin protects vascular integrity via PPAR/14-3-3 pathway

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Ling-yun Chu a , Jun-Yang Liou a,b , Kenneth K. Wu a,b,c,∗ a

Metabolomic Medicine Research Center, China Medical University, Taichung, Taiwan Institute of Cell and System Medicine, National Health Research Institute, Chunan, Taiwan c Department of Medical Sciences, National Tsing-Hua University, Hsin-chu, Taiwan

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a r t i c l e

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i n f o

a b s t r a c t

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Article history: Received 19 January 2015 Received in revised form 25 March 2015 Accepted 13 April 2015 Available online xxx

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Keywords: Prostacyclin PPAR 14-3-3

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Contents

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1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

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Vascular integrity is protected by the lining endothelial cells (ECs) through structural and molecular protective mechanisms. In response to external stresses, ECs are dynamic in producing protective molecules such as prostacyclin (PGI2 ). PGI2 is known to inhibit platelet aggregation and controls smooth muscle cell contraction via IP receptors. Recent studies indicate that PGI2 defends endothelial survival and protects vascular smooth muscle cell from apoptosis via peroxisome-proliferator activated receptors (PPAR). PPAR activation results in 14-3-3 upregulation. Increase in cytosolic 14-3-3␧ or 14-3-3␤ enhances binding and sequestration of Akt-mediated phosphorylated Bad and reduces Bad-mediated apoptosis via the mitochondrial pathway. Experimental data indicate that administration of PGI2 analogs or augmentation of PGI2 production by gene transfer attenuates endothelial damage and organ infarction caused by ischemia–reperfusion injury. The protective effect of PGI2 is attributed in part to preserving endothelial integrity. © 2015 Published by Elsevier Inc.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endothelial cell is a major source of PGI2 production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PGI2 inhibits thromboxane A2 (TXA2 )-induced platelet aggregation and SMC contraction via IP signaling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PGI2 prevents stress-induced EC apoptosis via peroxisome-proliferator activated receptors (PPARs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PGI2 controls vascular SMC apoptosis via PPAR␣ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PPAR␦-mediated 14-3-3␧ upregulation confers resistance to apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PGI2 protects VSMCs from H2 O2 -induced apoptosis by activating PPAR␣ → 14-3-3␤ transcriptional pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PGI2 prevents vascular cell apoptosis through multiple mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prostacyclin analogs protect endothelial barrier function via cyclic AMP pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endothelial damage predisposes heart and brain to ischemia reperfusion injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PGI2 and 15d-PGJ2 protect against ischemia–reperfusion injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiological and therapeutic considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and future perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Author and contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

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Blood vessels form a closed transportation system for effective oxygen and nutrient delivery and CO2 and waste disposal. A typical

∗ Corresponding author at: Metabolomic Medicine Research Center, China Medical Q2 University, Taichung, Taiwan. Tel.: +886 713 500 6800. E-mail address: [email protected] (K.K. Wu).

medium-size artery comprises three layers of tissues and a central lumen. Facing the lumen is the intimal layer (tunica intima) which is composed of a single layer of cells, i.e. the vascular endothelial cells and subendothelial connective tissues. The medial layer (tunica media) which is separated from the intimal layer by a dense band of elastic tissue, i.e. internal elastic lamina, is composed of smooth muscle cells and supportive tissues. The adventitia layer (tunica externa) which is separated from media by external elastic lamina contains fibroblasts, nerve innovation and other cell types.

http://dx.doi.org/10.1016/j.prostaglandins.2015.04.006 1098-8823/© 2015 Published by Elsevier Inc.

Please cite this article in press as: Chu L-y, et al. Prostacyclin protects vascular integrity via PPAR/14-3-3 pathway. Prostaglandins Other Lipid Mediat (2015), http://dx.doi.org/10.1016/j.prostaglandins.2015.04.006

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Each layer possesses special properties which contribute to vascular integrity and function. The intimal layer provides structural barrier to control vascular permeability and produce vasoprotective molecules to defend against invading toxins and insulting agents. The chief function of the medial layer is to provide regulated vascular contractility. Adventitia is a gateway for blood vessels to communicate with the outside world. Circulating in the lumen of blood vessel are normal blood constituents including blood cells, proteins, small-molecule nutrients and wastes, electrolytes and metals. Under physiological conditions, blood constituents do not interact with the lining endothelium and their passage through the vascular wall is tightly controlled. However, foreign microorganisms and toxins as well as endogenously produced cytokines and immune mediators often place endothelium under stresses which threaten to disrupt barrier, damage endothelial cells and perturb vascular integrity. Blood vessels are resilient and able to withstand the stresses thanks to dynamic innate protective mechanisms. Vascular endothelial cells (EC) are a key player of the innate protection. They are endowed with cellular and molecular ammunitions to defend against the insulting factors. ECs possess intercellular tight junctions to serve as a barrier and constitutively express thrombomodulin and protein C receptors at the luminal surface to prevent coagulation and thrombosis. Furthermore, they contain dynamic metabolic pathways which respond to the environmental insults by producing biologically active molecules to protect the vascular wall. Two pathways have been well characterized: (1) l-arginine catabolism to generate nitric oxide (NO) and (2) arachidonic acid (AA) metabolism to generate protective eicosanoids notably prostacyclin (PGI2 ) and epoxyeicosatrienoic acids (EETs). In response to vascular injury, NO and PGI2 are concurrently generated and act synergistically to block platelet activation and aggregation and relax vascular smooth muscle. Earlier investigations have centered on the actions of PGI2 and NO on blood platelets and vascular smooth muscle cell (SMC) contraction. Recent studies indicate that they protect ECs and SMCs from apoptosis and defend against inflammation and tissue injury. This review will focus on the anti-apoptotic actions of PGI2 and the underlying mechanisms. 2. Endothelial cell is a major source of PGI2 production In response to the environmental stresses, AA metabolism is initiated with activation of phospholipase A2 (PLA2 ). PLA2 is translocated from plasma membrane to the outer surface of endoplasmic reticulum (ER) and nuclear envelope (NE) where it catalyzes the liberation of AA from membrane phospholipids notably phosphatidylcholine. Free AA is converted to diverse metabolites by three major pathways: (1) cyclooxygenase (COX) pathway; (2) lipoxygenase (LOX) pathway and (3) cytochrome p450 (CYP) oxygenase pathway. Within each pathway, multiple metabolites are synthesized by their specific enzymes. The COX pathway generates several structure-related prostaglandins, thromboxane and prostacyclin (PGI2 ), while the LOX pathway produces leukotrienes, 5-, 12- and 15-HETEs (hydroxyeicosatetraenoic acid). The CYP pathway produces EETs and 20-HETE. All the AA metabolites share a 20-carbon unsaturated fatty acid backbone and hence are collectively called eicosanoids. Eicosanoids have diverse biological activities and play a broad spectrum of physiological roles. They are involved in myriad pathophysiological processes. Each cell type expresses a specific set of enzymes for synthesis of a selective list of eicosanoids. Vascular ECs produce eicosanoids from all three metabolic pathways among which prostacyclin (PGI2 ) is a predominant COX metabolite with vasoprotective actions. Prostacyclin was discovered as a metabolite of prostaglandin endoperoxide produced by vascular wall [1,2]. Its synthetic

PGI2 PGIS PLA2

ER or NE membrane

COX AA

PGI2 PGH2

Fig. 1. Schematic illustration of functional coupling of PGI2 synthetic enzymes. Abbreviations: PLA2 , phospholipase A2 ; AA, arachidonic acid; COX, cyclooxygenase; PGIS, PGI2 (prostacyclin) synthase; ER, endoplasmic reticulum; NE, nuclear envelope.

enzyme, prostacyclin synthase (PGIS) was isolated from arteries [3] which was subsequently cloned [4,5]. Biochemical characterization of PGI synthase reveals that it belongs to cytochrome p450 (CYP450) superfamily. However, it is an atypical CYP450 as it does not possess oxygenase activity but acts as an isomerase [6,7]. It anchors to the outer membrane of ER and NE by a single transmembranous domain and its substrate channel is attached to the outer membrane of ER and NE by hydrophobic interactions [8]. As illustrated in Fig. 1, PGI synthase is thought to be functionally coupled to the upstream enzymes at ER and NE membranes to facilitate PGI2 synthesis. A majority of the produced PGI2 is released into the extracellular milieu. A fraction of PGI2 is thought to enter nucleus. However, this presumption has not been proved by experimental data. Blood vessels are the principal source of PGI2 production. Early studies have provided quantitative data about PGI2 production by vascular cells [9]. Endothelial cells (EC) have a robust synthesis of PGI2 . Vascular smooth muscle cells (SMC) produce a smaller amount, about 1/7–1/10 of that produced by EC. Vascular adventitial fibroblasts produce only a trace amount of PGI2 . PGI2 production by ECs is regulated at the COX step. There are two COX isoforms in ECs. COX-1 is a house keeping enzyme which catalyzes the production of a basal level of PGI2 . By contrast, COX2 is highly responsive to exogenous stimuli. Shear stress as well as chemical stimuli such as cytokines, endotoxins, environmental toxins, immune and pro-inflammatory mediators induces COX-2 expression at the transcriptional level resulting in production of abundant PGI2 [10–13]. Stress-coupled PGI2 production is considered to play an important protective role [14]. 3. PGI2 inhibits thromboxane A2 (TXA2 )-induced platelet aggregation and SMC contraction via IP signaling pathway Effects of PGI2 on platelet reactivity and SMC contractility have been extensively investigated. PGI2 inhibits platelet aggregation induced by various physiological agonists. Of particularly importance is its inhibition of platelet aggregation induced by thromboxane A2 (TXA2 ). TXA2 was identified as a metabolite of prostaglandin endoperoxide [15] which is produced in platelets by a specific enzyme, thromboxane synthase [16,17]. It was initially identified as an autacoid to induce platelet aggregation and subsequently reported to constrict arteries. Interestingly, PGI2 has actions opposite to TXA2 : it inhibits TXA2 -induced platelet aggregation and vaso-constriction. The yin-yang relationship of PGI2 vs. TXA2 proves to be physiologically relevant [18] and pharmacologically important. It is suggested that the cardiovascular complications of selective COX-2 inhibitors are attributable to loss of the vaso-protective PGI2 , leaving the actions of TXA2 un-opposed with consequent increase in the risk of vascular thrombosis and excessive vasoconstriction [19]. PGI2 inhibits platelet aggregation and vascular SMC contractility by binding to a specific membrane G-protein coupled receptor, i.e. the I-type prostaglandin (IP) receptor, which activates adenylyl

Please cite this article in press as: Chu L-y, et al. Prostacyclin protects vascular integrity via PPAR/14-3-3 pathway. Prostaglandins Other Lipid Mediat (2015), http://dx.doi.org/10.1016/j.prostaglandins.2015.04.006

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cyclase and generates cyclic AMP [20,21]. Cyclic AMP serves as a second messenger to activate protein kinase A (PKA) which mediates relaxation of vascular SMCs and inhibition of platelet reactivity. The IP signaling pathway is essential for defending against thrombosis and vascular diseases as genetic deletion of IP receptors in mice increases risk of thrombi formation, intimal hyperplasia and atherogenesis [22–24]. 4. PGI2 prevents stress-induced EC apoptosis via peroxisome-proliferator activated receptors (PPARs) It was reported that stable analogs of PGI2 protect myocardial cells from adriamycin-induced apoptosis and hypertonicityinduced renal epithelial cell apoptosis [25,26]. But the mechanism was unclear. It was subsequently noted that PGI2 analogs protect against apoptosis via PPAR receptors. PPAR belong to the nuclear receptor superfamily. Three members: PPAR␣, PPAR␥ and PPAR␦ (homologous to PPAR␤ in chicken cells) have been detected in mammalian cells [27]. Their natural ligands in cells are not entirely clear but are thought to include fatty acid derivatives such as PGI2 . Kliewer et al. and Forman et al. were the first to report that stable PGI2 analogs such as iloprost and carbaprostacyclin (cPGI2 ) bind and activate PPAR␦ and PPAR␣ [28,29]. Liou et al. reported that pretreatment of human umbilical vein EC (HUVEC) with cPGI2 prevents H2 O2 -induced apoptosis and silencing of PPAR␦ abrogates the anti-apoptotic action of cPGI2 [30]. Furthermore, repetitive lowlevel H2 O2 stress was reported to protect HUVEC from apoptosis via PPAR␦ activation [31]. Besides protection against H2 O2 -induced apoptosis, PPAR␦ agonists prevent endothelial dysfunction induced by ␤-amyloid precursor proteins or cigarette smoke [32,33]. These findings are in agreement with the concept that external addition of synthetic PGI2 analogs protect ECs and enhance EC functions through a mechanism involving PPAR␦ [34]. It is interesting to note that the effective anti-apoptotic concentrations of cPGI2 are 50–100 ␮M which are much higher than the effective concentrations in controlling platelet activation and SMC contractility. Due to a short half-life of PGI2 in aqueous solution, it is difficult to determine whether authentic PGI2 binds and activates PPARs by in vitro assays. To provide evidence that endogenously produced PGI2 acts through activation of PPARs, the effect of amplified PGI2 production by gene transfer on EC survival is evaluated. We have reported that transfection of HUVEC with an adenoviral vector containing a bicistronic COX-1 and PGIS construct (Ad-COPI) augments PGI2 production while suppresses the synthesis of other eicosanoids due to metabolic shift to the COX → PGIS pathway [35]. Ad-COPI transfected HUVECs are resistant to H2 O2 -induced apoptosis which is abrogated by silencing of PPAR␦ expression with specific siRNA [30]. Ad-COPI transfection into renal cells defends against gentamicin-induced renal cell apoptosis via PPAR␣ [36]. These results suggest that authentic PGI2 is effective in protecting ECs from apoptosis via PPAR␦ and/or PPAR␣. A majority of PGI2 produced by Ad-COPI are secreted into extracellular milieu. It is possible that a fraction of the PGI2 produced enter nucleus to bind and activate PPAR␦ and/or PPAR␣. PGI2 released into the extracellular milieu acts in a paracrine manner to protect surrounding ECs from apoptosis. It remain unclear how the extracellular PGI2 or PGI2 agonists penetrates plasma membrane and traverses cytoplasm to reach nucleus to target PPAR␦ and/or PPAR␣. Activation of PPAR␦ in ECs by synthetic agonists not only protects EC from apoptosis but also promotes EC proliferation and angiogenesis [37]. Furthermore, it reduces stress-induced expression of VCAM-1 and E-selectin and increases the expression of superoxide dismutase 1, catalase and thioredoxin and consequently suppresses reactive oxygen species (ROS) generation [38]. It is unclear how PGI2 controls the expression of pro-inflammatory genes and increases the expression of anti-oxidant enzymes.

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PPAR␣ proteins are detected in ECs. It was reported that PPAR␣ activation by synthetic agonists protects HUVEC from apoptosis [39]. In fact, PPAR␣ activation by agonists protect against apoptosis in a number of cell types including renal cells [36], cardiomyocytes [40] and hepatocytes [41]. Genetic deletion of PPAR␣ in mice renders EC dysfunctional and unable to protect against ischemia–reperfusion injury [42]. The reported data suggest that PPAR␦ and PPAR␣ are functionally important in protecting ECs which appear to be depend on the type of insulting agents. 5. PGI2 controls vascular SMC apoptosis via PPAR␣ SMC apoptosis not only reduces cell mass and weakens atherosclerotic plaques but also induces inflammatory responses [43]. Furthermore, vascular SMC apoptosis is associated with increased SMC migration, proliferation and extracellular matrix production [44]. Vascular MSC apoptosis is induced by reactive oxygen species and pro-inflammatory cytokines. PGI2 was reported to protect SMC from oxidant-induced apoptosis [44]. cPGI2 as well as Ad-COPI transfection increases resistance to H2 O2 -induced apoptosis of neonatal rat aortic SMC [45]. It is interesting that the anti-apoptotic effect of PGI2 is abolished by PPAR␣ inhibitors but not PPAR␦ or PPAR␥ inhibitors. Importance of PPAR␣ in defending against apoptosis is supported by experimental results which show that PPAR␣ agonists protect SMCs from apoptosis. PPAR␣ overexpression in SMCs by transient transfection renders SMCs resistant to H2 O2 -induced apoptosis. Thus, in contrast with the predominate role of PPAR␦ in controlling EC apoptosis, PPAR␣ is pivotal in suppressing stress-induced SMC apoptosis. It is unclear why PGI2 targets different PPAR isoforms in EC vs SMC. One possible reason is differential quantitative expression of PPAR␣ and PPAR␦ in these two vascular cells. Current available data are insufficient to address this possibility. It is important to recognize that PPAR activation is regulated by IP receptor signaling. It has been reported that IP receptor activation by PGI2 analogs upregulates PPAR␦ in adipocytes [46,47]. Although PGI2 and its analogs do not directly activate PPAR␥, they activate PPAR␥ by IP receptor activation and the consequent cyclic AMP-PKA signaling [48]. In vascular SMCs, iloprost was reported to bind IP receptors and activate PKA whereby it induces COX-2 expression and PGI2 production [49]. It was proposed that the endogenously produced PGI2 activates PPAR␣ or ␦ to protect vascular SMC survival and contractile property [50]. Thus, there is a close relationship between IP receptor activation and PPAR activation in cells expressing both receptors such as vascular SMCs. It remains to be investigated whether IP receptor-mediated posttranslational modification of PPAR␣ or ␦ alters its selection of transcriptional targets. 6. PPAR␦-mediated 14-3-3␧ upregulation confers resistance to apoptosis Many genes harbor PPAR response elements (PPRE) in the promoter region. Activated PPAR␦ or PPAR␣ forms heterodimer complex with retinoid X receptor (RXR) [51] which binds to PPRE and activate or repress the expression of diverse classes of genes. Gene expression profiling by microarray assay has revealed a complex set of gene expression regulated by PPAR␣ or PPAR␦ [52]. However, only a few reports have identified relevant genes which are involved in cell survival and anti-apoptosis. Di-Poi et al. reported that PPAR␦ activation in keratinocytes upregulates ILK (integrin-linked kinase) and PDK-1 (phosphoinositide-dependent kinase-1) which activate Akt [53]. Akt protects cell survival by increasing sequestration of the pro-apoptotic Bcl-2 family proteins. To identify downstream target of PPAR␦, we screened a number of anti-apoptotic genes in HUVEC and found that 14-3-3␧ is

Please cite this article in press as: Chu L-y, et al. Prostacyclin protects vascular integrity via PPAR/14-3-3 pathway. Prostaglandins Other Lipid Mediat (2015), http://dx.doi.org/10.1016/j.prostaglandins.2015.04.006

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upregulated by PPAR␦ [30]. 14-3-3␧ is a member of the 14-3-3 family which comprises seven members in mammalian cells. All seven isoforms of 14-3-3 proteins are detected in HUVEC. PGI2 analogs and Ad-COPI selectively upregulate 14-3-3␧ expression [30]. Activation of PPAR␦ with L-165041, a synthetic PPAR␦ agonist, also selectively upregulates 14-3-3␧. Furthermore, PGI2 -induced 143-3␧ upregulation is abolished by silencing of PPAR␦ with siRNA. To confirm that PGI2 upregulates 14-3-3␧ at the transcriptional level through induction of PPAR␦ binding to 14-3-3␧, we searched human 14-3-3␧ promoter region for PPAR␦ binding motifs. Human 14-3-3␧ gene harbors several putative PPREs. We found that PPAR␦-RXR binds to PPRE sites located at -1426 to -1477 of human 14-3-3␧. Deletion of this region abolished PPAR␦-mediated 14-33␧ upregulation without affecting the basal 14-3-3␧ expression [30]. These findings indicate that PGI2 and PPAR␦ agonists activate PPAR␦ which is complexed with RXR and binds to the 14-3-3␧ promoter region to enhance 14-3-3␧ transcription. 14-3-3 proteins function as scaffold proteins to facilitate biochemical reactions, protein interactions or sequestration. The 14-3-3 family proteins share sequence homology, functional characteristics and structural similarities [54]. However, their expressions vary in different cell types which influence their physiological roles. A major function of 14-3-3 proteins is to bind phosphorylated Bad or Bax, sequester them in the cytosol and thereby reduce Bad/Bax-induced apoptosis [55,56]. In HUVEC at basal state, constitutively expressed 14-3-3␧ binds and sequesters Bad in the cytosol with a small amount of Bad detected in mitochondria. H2 O2 treatment results in a large increase of Bad translocation to mitochondria where it triggers apoptosis by perturbing mitochondrial membrane potential. PGI2 is capable of cutting down H2 O2 -induced Bad translocation to mitochondria by enhancing Bad sequestration in cytosol through PPAR␦-mediated 14-3-3␧ upregulation and Akt activation. It appears that selective increase in cytosolic 14-3-3␧ proteins is sufficient to exert a significant augmentation of Bad binding and sequestration in the cytosol. Selective COX-2 inhibitors (coxibs) and a number of nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with cardiovascular complications [57,58]. The exact mechanism by which COX-2 inhibition increases the risk of myocardial infarction has not been clearly elucidated but is thought to be related to shutdown of PGI2 production. Liou et al. reported that NSAIDs such as sulindac induces endothelial apoptosis by inhibiting the expression of PPAR␦ and 14-3-3␧ [59]. Neither PGI2 nor synthetic PPAR␦ agonists can rescue HUVEC because of deficiency of PPAR␦. These results may explain why NSAIDs increase risk of myocardial infarction (MI). Furthermore, they provide additional evidence to support the crucial role that the PPAR␦ → 14-3-3␧ pathway plays in mediating the anti-apoptotic effect of PGI2. The transcriptional pathway via which PGI2 confers resistance to apoptosis in ECs is summarized in Fig. 2. In brief, within the nucleus, PGI2 binds and activates PPAR␦ which forms a heterodimer with RXR. PPAR␦-RXR complex binds to specific sites on 14-3-3␧ promoter to enhance 14-3-3␧ transcription resulting in an increase in cytosolic 14-3-3␧ proteins. H2 O2 triggers Bad mobilization to mitochondria in HUVECs where Bad binds and inactivates Bcl-2 resulting in apoptosis via the mitochondrial pathway. PGI2 suppresses the pro-apoptotic effect of Bad by activation of Akt and upregulation of 14-3-3␧ which increases Bad sequestration in cytosol.

Endothelial Cells Nucleus

Cytosol PGI2 PPARδ

PDK-1 and ILK Akt

PPARδ-RXR

p-Bad

14-3-3ε transcripon 14-3-3ε/Bad Bad sequestraon mitochondria damage apoptosis

Fig. 2. Transcriptional pathways via which PGI2 protects against endothelial cell apoptosis. Dashed line denotes the potential pathway for increased expression of PDK-1 and ILK. Abbreviations: PDK-1, phosphoinositide-dependent kinase-1; ILK, integrin-linked kinase; RXR, retinoid X receptor.

14-3-3 expression and regulation in VSMCs, we analyzed 14-3-3 expression with Western blotting. All the 14-3-3 family proteins except 14-3-3␴ are detected. Ad-PGIS transfection or cPGI2 treatment results in upregulation predominantly of 14-3-3␤ with a lesser increase in 14-3-3␪ and 14-3-3␧ [45]. PPAR␣ agonists such as WY14643 and GW9578 upregulates primarily 14-3-3␤ with a minor increase in 14-3-3␧. Thus, PGI2 and PPAR␣ agonists predominantly increase 14-3-3␤ protein expression. Interestingly, H2 O2 treatment of VSMCs results in 14-3-3␤ degradation by caspase 3 and PGI2 rescues 14-3-3␤ through PPAR␣-induced 14-3-3␤ upregulation. It is surprising that 14-3-3␧ does not play a significant role in the anti-apoptotic action of PGI2 in SMCs. 14-3-3␤ upregulation increases Bad binding and sequestration and consequently retards Bad translocation to mitochondria. H2 O2 -induced caspase 3 in VSMCs is suppressed by 14-3-3␤ overexpression but not 14-3-3␧ or 14-3-3␪ overexpression. Conversely, 14-3-3␤ siRNA abrogates the anti-apoptotic effect of PGI2 . Thus, PGI2 protects VSMCs via a different PPAR → 14-3-3 transcriptional pathway. As illustrated in Fig. 3, H2 O2 induces 14-3-3␤ degradation which weakens Bad sequestration and consequently enhances Bad-induced apoptosis. It is likely that PGI2 -activated PPAR␣ forms heterodimer with RXR and binds to PPAR response elements at the promoter region of 14-3-3␤ to augment 14-3-3␤ expression. Robust 14-3-3␤ expression overwhelms caspase-3 induced 14-33␤ degradation and raises 14-3-3␤ protein levels. 14-3-3␤ binds and sequesters Bad and attenuates Bad-induced apoptosis.

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7. PGI2 protects VSMCs from H2 O2 -induced apoptosis by activating PPAR␣ → 14-3-3␤ transcriptional pathway In view of the involvement of PPAR␦ → 14-3-3␧ in regulating EC apoptosis, we were curious whether this pathway is operative in regulating apoptosis in SMCs. Since little was known about

Bad sequestraon Bad-induced apoptosis Fig. 3. PGI2 protects against H2 O2 -induced apoptosis in vascular SMC via PPAR␣/143-3␤.

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8. PGI2 prevents vascular cell apoptosis through multiple mechanisms The mechanisms by which PGI2 defends vascular cells against apoptosis are not restricted to the Akt and the PPAR → 14-3-3 pathways. It was reported that PGI2 increases XIAP proteins in ECs as a result of blocking XIAP ubiquitination and proteasome degradation [60]. XIAP is a member of the IAP family proteins which inhibit caspase 9, caspase 7 and caspase 3. It is well characterized that the cellular XIAP level is tightly regulated by degradation via ubiquitin-proteasome system. Prevention of XIAP degradation by PGI2 preserves XIAP level to defend against apoptosis. PGI2 was reported to protect VSMCs via transforming growth factors ␤ (TGF␤) pathway. Kim et al. reported that PPAR␦ activation by ligand GW501516 attenuates VSMC apoptosis induced by oxidized low-density lipoprotein (oxLDL) by a mechanism involving extracellular matrix (ECM) [61]. They show that activated PPAR␦ binds and activates the promoters of two collagen genes, i.e. COL3A1 and COL1A1, which depends on TGF␤ → Smad 3 pathway. Smad 3 binds to the collagen promoter and together with PPAR␦ increases ECM production. Interestingly, collagen and ECM accumulation plays an essential role in protecting VSMCs from oxLDL and elastase-induced apoptosis, as silencing of COL3A1 or 1A1 abrogates the protective effect of GW501516. Pro-inflammatory cytokines notably tumor necrosis factor ␣ (TNF␣) induce apoptosis and convert EC from a protective phenotype to inflammatory phenotype. Pro-inflammatory mediators trigger oxidative pathways to generate ROS. ROS damage cells and cause apoptosis as well as cell death via necrosis. PPAR␦ agonists were reported to suppress EC inflammatory switch and ROS production. PGI2 may suppress EC ROS production and inflammation via PPAR␦ activation, which further contributes to protection against vascular cell death. Apoptotic cell debris is cleared primarily by macrophages. PPAR␦ was reported to be critical for clearance of apoptotic cells by macrophages [62]. In a murine model with PPAR␦ deletion, macrophages are found to be dysfunctional and defective in clearing apoptosis cells as well as production of pro-inflammatory cytokines. Defect in clearing apoptotic cells leads to accumulation of cell debris which induce immune and inflammatory responses, further aggregating cell damage and apoptosis. PPAR␦ agonists rescue the clearance of apoptotic cells by macrophages and hence avoid the dire consequence of inflammation and cell death.

9. Prostacyclin analogs protect endothelial barrier function via cyclic AMP pathway Vascular endothelium possesses tight intercellular junctions to restrict the passage of proteins, circulating cells and small molecules into the vascular wall. Vascular permeability at the intercellular junctions is highly regulated at the level of adherens junction (AJ) and tight junction. The barrier is one of the fundamental functions of endothelial cells. Disruption of the barrier by environmental insults such as hypoxia, LPS and TNF␣ results in drastic changes in the blood vessel including subendothelial edema, expression of adhesive molecules on EC luminal surface which facilitate transendothelial migration of blood cells and vascular wall inflammation [58]. Severe vascular changes lead to endothelial detachment which exposes the subendothelial tissues to circulating platelets, coagulation factors with consequent thrombosis and intimal hyperplasia. Prostacyclin analogs were reported to enhance endothelial barrier function by upregulating VE-cadherin in a cyclic AMPdependent manner [63]. VE-cadherin is a key constituent of the AJ protein complex. VE-cadherin upregulation increases EC cell

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VE-cadherin Vascular permeability

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to cell contact and enhances the barrier function. VEGF, LPS and TNF␣ disrupt the barrier function by causing degradation of VEcadherin and thereby increase the permeability to allow passage of blood proteins and cells to the subendothelial region [64]. Iloprost, a PGI2 analog was reported to prevent LPS-induced disintegration of VE-cadherin and preserve the barrier function in endothelial cells by a cAMP-dependent suppression of NF-␬B activation [65]. Hypoxia also causes barrier disruption and increases endothelial permeability through generation of ROS which trigger transendothelial migration of polymorphonuclear cells and inflammation [66]. Inflammation in turn increases endothelial permeability via cytokines such as IL-6 [67]. Continuous hypoxia induces acute endothelial damage as manifested by vascular wall edema, and inflammation which eventually leads to endothelial detachment and denudation. Loss of PGI2 production aggravates the vascular damage. Increase in PGI2 production by transfer of PGI synthase gene protects vascular endothelium and inhibits neointimal formation in animal models [68,69]. The reported data suggest that PGI2 produced by endothelial cells plays an important role in maintaining the physiological state of barrier function and protecting the barrier from disruption by environmental insults and pro-inflammatory cytokines. Continuous exposure to severe insults results in endothelial barrier disruption and the consequent vascular wall edema, expression of pro-inflammatory adhesive molecules on endothelial cells and endothelial detachment. Loss of endothelium and progressive inflammation leads to platelet thrombosis, intimal hyperplasia, and the consequent tissue damage and organ infarction. Supply of PGI2 either by infusion of PGI2 analogs or by gene transfer at an early stage of barrier disruption may rescue the barrier function and prevents acute vascular damage and endothelial detachment via a cyclic AMP-dependent mechanism (Fig. 4). 10. Endothelial damage predisposes heart and brain to ischemia reperfusion injury Ischemia–reperfusion (IR) is a major cause of severe human diseases including MI and ischemic stroke. IR is due to temporary occlusion of an artery such as coronary artery followed by opening of the arterial lumen and reperfusion of myocardial tissues. During the arterial occlusion period, the arterial endothelium suffers from severe hypoxia and ischemia and loses the endothelial barrier function resulting in an increase in vascular permeability

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with the development of subendothelial edema, inflammation and endothelial detachment. Endothelial damage is aggravated by ROS and inflammation produced by reperfusion. Time course experiments in animal models have revealed that endothelial damage preceded myocardial damage following left anterior descending coronary artery occlusion and reperfusion [70–72]. Morphological examination of endothelium with transmission electron microscopy showed evidence of endothelial damage [70,71]. These results suggest that endothelial damage and its consequent loss of vasodilating and vasoprotective eicosanoids during early phase of IR plays an important role in IR-induced MI. Arterial occlusion-reperfusion causes endothelial damage by several possible mechanisms including mechanical stresses due to turbulent blood flow at the occlusion-reperfusion site, generation of reactive oxygen species and pro-inflammatory cytokines. It is possible that endothelial damage leads to EC apoptosis and consequently endothelial denudation. Loss of EC barrier function and protective molecules results in subendothelial inflammation and extracellular matrix accumulation. Pro-inflammatory cytokines and chemokines and growth factors attract SMCs to the intima which contribute to intimal hyperplasia and inflammation. SMC apoptosis due to hypoxia, ROS or inflammation aggravates inflammation and increases SMC proliferation and migration. Robust cytokine/chemokine productions generate systemic inflammation which damages tissues and is thought to be a major pathogenetic factor for post-MI heart failure [73]. Large amounts of ROS are generated during the vicious cycle of inflammation and tissue damage. ROS represents a master trigger of endothelial dysfunction and apoptosis and a key mediator of cellular and tissue damage. Tabernero et al. reported that endothelial and myocardial PPAR␣ are vital for controlling ROS-induced endothelial damage by promoting expression of anti-oxidant enzymes [42]. As PGI2 ligates PPAR␣, it is possible that PGI2 protects endothelium against oxidant-induced damage and endothelial dysfunction by PPAR␣ as well as PPAR␦.

11. PGI2 and 15d-PGJ2 protect against ischemia–reperfusion injury Arterial occlusion followed by reperfusion creates complex biochemical changes which cause damage to the tissues supplied by the involved arteries. IR not only causes tissue infarction but also damages the downstream arterial endothelium resulting in loss of protective molecules including PGI2 [74]. A number of laboratories have reported beneficial effects of stable PGI2 analogs on protecting against cerebral ischemia in animal models [75,76]. Augmentation of PGI2 production by administration of Ad-COPI to a rat stroke model via intracerebral ventricle infusion shows increased brain levels of 6-keto-PGF1␣ (a degradation product of PGI2 ) and suppression of thromboxane, and leukotrienes [77]. It reduced IRinduced cerebral infarct size. These experimental data prove the principle that restoration of PGI2 production is effective in alleviating IR injury. PGI2 protects against IR injury not only by preventing endothelial and neuronal apoptosis but also by control of vasoconstriction and promoting angiogenesis. PGI2 defends against IR-induced EC and tissue damage via several transcriptional pathways. Besides the 14-3-3 upregulation, it was reported that PPAR␦ agonist GW0742 attenuates I/R induced myocardial infarction size by suppressing NF-␬B, COX-2, inducible nitric oxide synthase as well as the glycogen synthase kinase-3␤ (GSK-3␤) pathway [78]. Chen et al. [79] reported that PGI2 protects against I/R-induced renal damage via PPAR␣. They demonstrated that PGI2 reduces NF-␬␤ activation resulting in reduction of TNF␣ and apoptosis [79]. I/R-induced renal damage worsens in PPAR␣ knockout mice whereas PPAR␣ agonists alleviate the I/R renal damage. These findings are consistent with the interpretation that PGI2

is capable of controlling apoptosis and tissue damage by multiple signaling and transcriptional pathways. 15-Deoxy-12,14 PGJ2 (15d-PGJ2 ) is a non-enzymatic degradation product of PGD2 . It binds PPAR␥ and activates PPAR␥ via which it confers anti-inflammatory protection [80]. Lin et al. reported that Ad-COX-1 administration to the rat stroke model increases PGD2 and 15d-PGJ2 which might cooperate with PGI2 to reduce IR-induced brain infarction and neuronal apoptosis [77]. Direct infusion of 15d-PGJ2 reduced the IR-induced cerebral infarction size [81]. Rosiglitazone, a synthetic PPAR␥ agonist protects brain from IR-induced infarction which was abrogated by PPAR␥ siRNA [82]. Proteomic analysis identified 14-3-3␧ as the key target of PPAR␥. Rosiglitazone treatment resulted in more than 5-fold increase in 14-3-3␧ proteins in the rat brain. In vitro studies reveal that rosiglitazone increased 14-3-3␧ protein expression in neuronal cells which confers resistance to apoptosis via binding and sequestering Bad. Thus PPAR␥ activation, like PPAR␦ activation in ECs enhances 14-3-3␧ promoter activity and increases 14-3-3␧ protein expression which contributes to protection against apoptosis. Rosiglitazone was reported to protect mitochondrial membrane potential and upregulate the expression of Bcl-2 which augment the protection against IR-induced brain infarction [83]. These reported data indicate that 15d-PGJ2 protects against IR injury via PPAR␥ and the protective effect of PPAR␥ may be mediated by 143-3 upregulation. 14-3-3 upregulation may be one of the universal mechanism to protect cells from apoptosis. 12. Physiological and therapeutic considerations Endothelial integrity is vital to healthy blood vessels. Disruption of endothelial integrity leads to vascular dysfunction, endothelial cell apoptosis and endothelial detachment which set the stage for serious vascular diseases, such as atherosclerosis and restenosis. Vascular endothelial integrity is protected by multiple molecules whose production is coupled to stress signals. Blood flow shear stress maintains basal vascular homeostasis by stimulating the production of PGI2 , and NO [84,85]. Based on in vitro cell experiments, it has been presumed that endothelium is resilient and able to withstand diverse insults and stresses by rapid responses to the external stress signals resulting in robust production of protective molecules such as PGI2 via induction of COX-2 expression [14]. These presumptions are validated in part by gene transfer experiments but to a large extent unproven. The quantitative and temporal relationship between the severity of environmental stresses and the extent of vasoprotective molecule production remains to be determined. Environmental stresses such as LPS and cytokines alter the EC properties converting it from protective to pro-inflammatory phenotypes. Pro-inflammatory eicosanoids notably leukotrienes and 20-HETE are produced which act against PGI2 . It is unclear how the balance between protective eicosanoids notably PGI2 and possibly EETs (epoxyeicosatrienoic acid) and pro-inflammatory eicosanoids is regulated. The temporal changes in this balance are also not well understood. Understanding of these issues will advance the use of PGI2 stable analogs in prevention and treatment of vascular diseases and tissue damage. PGI2 and its stable prostacyclin analogs were reported to be effective in treating human pulmonary artery hypertension (PAH) in 1980s and remain a mainstay in the treatment of PAH [86–88]. Several PGI2 analogs including iloprost, beraprost, epoprostenol and treprostinil are available for clinical treatment of this serious vascular disease. Their clinical effects are attributed to control of pulmonary arterial SMC proliferation and vascular remodeling [89,90]. By contrast, PGI2 analogs have not been proven to be effective in alleviating other types of vascular diseases [91]. Previous clinical trials on peripheral vascular diseases were designed to treat advanced diseases focusing on demonstration of improvement in

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blood flow and relief of ischemic symptoms. Given the new findings that PGI2 protects vascular integrity, the therapeutic strategy should be shifted to prevention and early treatment to protect endothelial integrity. Earlier studies with gene transfer of PGI2 synthetic enzymes have provided evidence that enrichment of PGI2 at early arterial injury is effective in preventing arterial thrombosis [92] and attenuating IR-induced cerebral infarction [77] in animal models. The experimental data from animal experiments provide proof of principle but their translation to human therapy is challenging. One strategy is using biomarkers to identify and select patients with evidence of early arterial wall injury for PGI2 gene transfer therapy or chemoprevention. This remains an extremely difficult challenge since there are no reliable biomarkers available. Although gene therapy for hereditary diseases is promising, the application to vascular diseases required additional experiments. Chemoprevention of chronic vascular diseases such as atherosclerosis and restenosis with PGI2 and its analogs has been hampered by lacking efficacious PGI2 analogs that can be taken orally for a long time without adverse effects.

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endothelial cells via different signaling pathways. It is not entirely clear whether production and actions of those molecules are coordinated. A new therapeutic approach based on combination of the diverse group of vasoprotective molecules should be considered. Conflict of interest We did not receive payment or services at any time from a third party for any aspect of the submitted work. We declare no financial relationships with entities that could be perceived to influence, or that give the appearance of potentially influencing. Author and contributions Ling-Yun Chu: preparing and final approval of the manuscript, Jun-Yang Liou: preparing and final approval of the manuscript and Kenneth K. Wu: drafting, revising and final approval of the manuscript. Acknowledgement

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Endothelial cells are highly responsive to the environmental insults. When attacked by chemical or physical insults, EC arachidonic metabolism is activated resulting in generation of protective eicosanoids such as PGI2 . PGI2 plays multiple roles in protecting endothelial survival and vascular integrity through interaction with two types of receptors: IP receptors and PPAR (␣ and ␦). PGI2 and several synthetic analogs defend against vascular thrombosis and vasoconstriction via IP receptors while protects EC and SMC from apoptosis primarily via PPAR␦ and PPAR␣. We have identified 143-3 upregulation as an important target of PPAR␣ and ␦. PPARs complex with RXR and bind to PPAR response elements on 143-3␧ in ECs and 14-3-3␤ in SMC to promote the expression of these two types of 14-3-3 isoform proteins in EC and SMC, respectively. Elevated 14-3-3␧ or ␤ facilitates Bad sequestration in the cytosol. Through protecting EC and SMC survival, PGI2 is pivotal in maintaining vascular integrity thereby defending against stressinduced vascular damage. Once endothelium is damaged and ECs are defective in generating the protective molecules such as PGI2 , inflammatory cell accumulation, smooth muscle cell migration and proliferation and lipid deposition occur resulting in development of atherosclerosis, intimal hyperplasia and restenosis. EC damage by ischemia–reperfusion injury contributes to tissue damage and organ infarction. Preservation of EC function and its ability to produce PGI2 is crucial in preventing vascular diseases and I/R-induced organ infarction such as myocardial infarction, and ischemic stroke. There have been numerous attempts to treat vascular diseases and I/R-induced organ infarction with stable PGI2 analogs [86]. Several PGI2 analogs were found to be efficient in treating human pulmonary artery hypertension (PAH) and are now used in clinical treatment of PAH patients. Although the effects of PGI2 analogs are attributed to control VSMC proliferation, preservation of endothelial function is an important attribute. Treatment of other vascular diseases such as peripheral artery disease with PGI2 analogs has not produced consistent results and the adverse effects of the drugs have hampered their clinical use. A new perspective is to target protection of vascular cell survival and vascular integrity. There are major challenges ahead with this approach. We need precise biomarkers to predict early vascular injury and safe oral PGI2 analogs in order to selective appropriate patients for chemoprevention and/or early treatment of vascular diseases and I/R tissue damage. Besides PGI2 , ECs produce nitric oxide, EETs and other metabolites such as 5-methoxytryptophan (5-MTP) [93] which protect

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