This review is part of a Special Issue on PPAR Ligands and Cardiovascular Disorders: Friend or Foe. This Special Issue carries the following articles: Editorial: PPAR Ligands and Cardiovascular Disorders: Friend or Foe
• The involvement of PPARs in the causes, consequences and mechanisms for correction of cardiac lipotoxicity and oxidative stress. • Healing the diabetic heart: modulation of cardiometabolic syndrome through peroxisome proliferator activator receptors (PPARs). • Effects of PPARγ agonists against vascular and renal dysfunction. • Use of clinically available PPAR agonists for heart failure; do the risks outweigh the potential benefits? • Assessment of cardiac safety for PPARγ agonists in rodent models of heart failure: A translational medicine perspective. • Peroxisome proliferator-activated receptorγ (PPARγ) agonists on glycemic control, lipid profile and cardiovascular risk. • Effects of PPARγ ligands on vascular tone. • PPARγ agonists in polycystic kidney disease with frequent development of cardiovascular disorders.
Pitchai Balakumar and Gowraganahalli Jagadeesh Guest Editors
Effects of PPARγ Ligands on Vascular Tone Salvatore Salomone* and Filippo Drago
Department of Clinical and Molecular Biomedicine, Catania University, Catania, Italy Abstract: Peroxisome Proliferator-Activated Receptor γ (PPARγ), originally described as a transcription factor for genes of carbohydrate and lipid metabolism, has been more recently studied in the context of cardiovascular pathophysiology. Here, we review the available data on PPARγ ligands as modulator of vascular tone. PPARγ ligands include: thiazolidinediones (used in the treatment of type 2 diabetes mellitus), glitazars (bind and activate both PPARγ and PPARα), and other experimental drugs (still in development) that exploit the chemistry of thiazolidinediones as a scaffold for PPARγ-independent pharmacological properties. In this review, we examine both short (mostly from in vitro data)- and long (mostly from in vivo data)-term effects of PPARγ ligands that extends from PPARγ-independent vascular effects to PPARγ-dependent gene expression. Because endothelium is a master regulator of vascular tone, we have attempted to differentiate between endothelium-dependent and endothelium-independent effects of PPARγ ligands. Based on available data, we conclude that PPARγ ligands appear to influence vascular tone in different experimental paradigms, most often in terms of vasodilatation (potentially increasing blood flow to some tissues). These effects on vascular tone, although potentially beneficial, must be weighed against specific cardiovascular warnings that may apply to some drugs, such as rosiglitazone.
Peroxisome Proliferator-Activated Receptors (PPAR) were first described as nuclear receptor members of the steroid hormone receptor superfamily of ligand-activated transcription factors, that cause proliferation of peroxisomes in liver of rodents, following challenge with carcinogens [1]. The PPAR family consists of three members, α, β/δ, and γ, which share 60% - 80% homology in their ligand- and DNA-binding domains [2]. Endogenous/natural ligands identified so far include polyunsaturated fatty acids, oxidized phospholipids, lipoprotein lipolytic products, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and forms of oxidized linoleic acid [2]. All these ligands, however, display low affinity, whereas some marketed drugs, such as fibrates and thiazolidinediones, display high affinity for PPARα and PPARγ, respectively [2]. PPARβ/δ is the least well characterized of the three PPAR isoforms, but there is increasing evidence that it acts as an important regulator of skeletal muscle metabolism [3, 4]. No approved drugs acting as PPARβ/δ agonist are currently available; however, some
*Address correspondence to this author at the Department of Clinical and Molecular Biomedicine, Pharmacology Section, Catania University, Viale A. Doria 6, 95125, Catania, Italy; Tel: +39 095 738 4085; Fax: +39 095 738 4238; E-mail: [email protected]
PPARβ/δ agonists are in pre-clinical or clinical development and may prove useful for treating atherosclerosis-related disorders [5, 6].
Following the discovery that PPARγ ligands modulate insulin sensitivity and glucose metabolism [7], the study of PPARγ biology and pharmacology had received a great momentum, and drugs such as thiazolidinediones, became widely prescribed for the treatment of type 2 diabetes [2]. Following a 10-year wave of enthusiasm, however, the clinical use of thiazolidinediones has been put under scrutiny by the medical community and the regulatory agencies, because some serious adverse reactions have been linked to some members of the class. In particular, troglitazone has been associated with liver failure [8, 9] and rosiglitazone, more recently, to an increased risk of coronary heart disease [10, 11]. On the other hand, basic science studies, together with serendipitous clinical observations and systematic retrospective surveys, pointed on so-called “pleiotropic” effects of thiazolidinediones, indicating effects that are not strictly related to lipid and sugar metabolism, and, in some instances, even not related to PPARγ activity. These effects include inhibition of Vascular Endothelial Growth Factor (VEGF)-induced angiogenesis [12], change in expression levels of some cytokines [13, 14], such as Tumor Necrosis
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Factor α (TNFα) and interleukin-6 (IL-6) [15], some apoptosis-related genes [16], which provides the basis for additional potential therapeutic indications, encompassing sepsis, cancer, atherosclerosis, neurodegeneration [17, 18]. Based on these observations, medicinal chemistry continues to generate ligands with special properties that include in their pharmacological profile a PPARγ agonist activity; among them, worthy of note, dual PPAR ligands such as glitazars (agonist at both PPARα and PPARγ) [19], and some angiotensin II receptor antagonists, such as telmisartan and irbesartan [20, 21].
While basic science studies try to shed light on the molecular mechanisms accounting for novel biological effects of PPARγ stimulation, clinical observations also outline beneficial cardiovascular effects of PPARγ ligands that seem at least in part independent of their metabolic effects [22, 23]. Although in vivo clinical observation are of paramount importance for assessing therapeutic benefits, they do often not allow to precisely discriminate molecular mechanisms that define a pharmacological profile of a compound, in terms of receptor specificity and selectivity. Here we review available evidence for effects of PPARγ ligands such as thiazolidinediones on vascular tone, and data and hypothesis on their potential cellular and molecular mechanisms.
PPARγ-DEPENDENT VERSUS PPARγ-INDEPEN-DENT EFFECTS ON VASCULAR TONE
Smooth muscle contraction is essential for regulating important homeostatic functions in vascular tree. By contracting or relaxing, vascular smooth muscle cells (VSMC) determine the caliber of arteries and thereby downstream perfusion pressure, or, at the organism level, the total resistance and systemic blood pressure. In capacitance vessels (major veins), VSMC contraction determines capacitance volume (vascular volume) and thereby affects the amount of blood reaching the ventricles in diastole (pre-load). A number of approved drugs are intended for reducing vascular tone, in situations like essential hypertension (to reduce tone of resistance arteries), coronary artery disease (venodilatation to reduce cardiac pre-load and/or coronary artery dilatation to increase blood supply to myocardium), peripheral artery disease (peripheral artery dilatation to increase blood supply to skeletal muscle), heart failure (to reduce the sympathetic-driven tone of resistance arteries). Stroke represents a situation where vasodilator mechanisms, if more active on cerebral circulation, may theoretically increase cerebral blood flow and rescue ischemic penumbra. Currently available vasodilators tend to reduce systemic blood pressure, counteracting their potential benefit on brain circulation [24].
First evidence of effects of PPARγ ligands on blood pressure came in 1993 [25]. Since then, several observations pointed on effects of PPARγ ligands on vascular tone and hypothetical mechanisms proposed to explain these effects are discussed below. A first question is, whether or not the effects of PPARγ ligands are PPARγ-dependent, i.e., as a result of activation of PPARγ and subsequent changes in
gene expression. Looking at phenotypes associated with disruption or modification of the PPARγ gene may help in answering this question. Human phenotypes associated with PPARγ polymorphisms have been described [26], and some of them include cardiovascular phenotypes [27-29], however the direct demonstration of a role of PPARγ in regulating vascular tone comes from genetic manipulation in animal models, although they provide some conflicting data. In some studies, targeted disruption of PPARγ in mouse endothelium does not seem to affect blood pressure; however, these mice become hypertensive if fed with a high fat diet or treated with DOCA-salt [30, 31]. In these animal models, PPARγ ligands decrease blood pressure relative to wild type, but are ineffective in mice with targeted disruption of PPARγ in endothelium [30, 31]. The mechanisms responsible for the actions of endothelial PPARγ on blood pressure are unclear. PPARγ deletion in endothelium causes enhanced vasoconstriction to phenylephrine, angiotensin II (ATII), and high KCl in isolated arteries without altering endothelium-dependent acetylcholine (ACh)-induced vasorelaxation accompanied by changes in the clock gene Bmal1 in blood vessel, which may affect regulation of cardiovascular rhythms [32]. In contrast with these studies, Kleinhenz et al. [33] reported that mice with endothelial disruption of PPARγ showed increased blood pressure and endothelial dysfunction (response to ACh was impaired in isolated aorta). Expression of dominant negative PPARγ in endothelial cells again produces a subtle phenotype, i.e., lack of baseline alteration of vascular tone, but increased endothelial dysfunction following prolonged high fat diet [34]. Expression of a dominant negative PPARγ in VSMC causes hypertension in mice with impairment of relaxation in isolated blood vessels in response to ACh, sodium nitroprusside and to a cyclic GMP (cGMP) analog, suggesting a dysregulation in the downstream of cGMP signaling pathway [35]. In contrast, a more recent study demonstrated that VSMC-selective PPARγ deficiency led to hypotension [36], accompanied by increased β2-adrenergic receptor expression and increased vasodilatation in response to norepinephrine. Based on these data, PPARγ-dependent effects of PPARγ-ligands on vascular tone could be obviously hypothesized with the expectation that they would have to be slow-developing and long lasting, consistently with the kinetic of PPARγ-driven gene expression, as well as with half lives of related mRNAs and proteins. As discussed below, some of the effects of PPARγ ligands on vascular tone are fast and reversible, suggesting an underlying mechanism not involving gene expression (assumption seldom confirmed by PPARγ antagonism and/or PPARγ gene deletion). Medicinal chemistry and quantitative structure-activity relationship (QSAR) studies show that thiazolidinediones, the most commonly used PPARγ ligands, also have other pharmacological properties [37]. Molecules containing the thiazolidinedione moiety have been synthesized and screened for binding to diverse molecular targets. Some of these targets are protein kinases and phosphatases. In in vitro experimental settings, this implies that kinetics of PPARγ-independent responses to thiazolidinediones might be faster (within minutes) than PPARγ-dependent gene expression-related effects [38]. For example, class I phosphoinositide 3-kinases (PI3Ks), in particular PI3Kγ, have become potential targets of
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thiazolidinedione-related compounds for inflammatory and autoimmune conditions [39]. Importantly, in cardiomyocytes as well as in endothelium and VSMC, PI3Kγ is also involved in cardiovascular pathophysiology [40, 41] through Akt/protein kinase B stimulation that affects nitric oxide synthase (NOS) and endothelium-dependent vasodilatation [42]. Other PPARγ-independent fast effects of PPARγ ligands may occur as a result of binding to ion channels in VSMC (Fig. 1).
SHORT-TERM EFFECTS OF PPARγ LIGANDS ON VASCULAR TONE
Contractions of smooth muscle cells are due to membrane depolarization, that may spread from cell to cell
via gap junctions, and/or to signaling cascades triggered by neurohormonal agonists acting on membrane receptors. Potential short-term effects of PPARγ ligands are summarized in Fig. (1). Depolarization induces Ca2+ entry, via voltage-dependent L-type Ca2+ channels, while agonists that couple to phospholipase C signaling cascades release Ca2+ from the sarcoplasmic reticulum via inositol 1,4,5 trisphosphate (IP3) generation [43]. Because L-type Ca2+ channels are mainly responsible for Ca2+ entry into VSMC, drugs that inhibit L-type Ca2+ channels, such as calcium antagonists, are widely used since the ‘80s as antihypertensive agents [44]. First evidence of a direct, Ca2+ opposing effect of PPARγ ligand on vascular tone was observed in 1995 [45]. More recently, PPARγ ligands have been shown to directly interact in a PPARγ-independent
Fig. (1). Potential short-term effects of PPARγ ligands on vascular tone. EC, endothelial cell, VSMC, vascular smooth muscle cell. A Shows PPARγ-dependent effects, including those that are consequence of PPARγ-dependent gene expression as depicted in Fig. (2A), but may affect rapid changes of tone; these include reduced release of endothelin-1 (ET-1) as consequence of reduced expression of preproET-1 and increased NO release as consequence of endothelial nitric oxide synthase (eNOS) upregulation. B PPARγ-independent effects, include effects on ion channels on VSMC and effects on eNOS from potential PI3K inhibition. Thick arrows indicate increase/stimulation (upward) or decrease/inhibition (downward) of a mediator or a function; thick right-angle arrows from DNA indicate induced (or repressed, crossed arrows) gene expression; dotted arrows indicate potential mechanistic relationships. Numbers in square parentheses indicate references.
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manner with voltage-gated Ca2+ channels in neurons [46] and with Transient Receptor Potential (TRP) channels over-expressed in HEK 293 cells [47]. In both channels, PPAR ligands inhibited Ca2+ entry in the micromolar range. More importantly, a direct inhibition of L-type Ca2+ channel has been shown in VSMC. Thiazolidinediones have been reported to inhibit Ca2+ currents in VSMC in a dose-dependent manner (troglitazone > pioglitazone = rosiglitazone) [48]. Non-thiazolidinedione PPARγ ligands, such as tyrosine-derived PPARγ agonists have also been shown to inhibit L-type Ca2+ channels more potently than pioglitazone with IC50s of 2-5 and 10 µM, respectively [49]. The wide range in potency (up to 2 orders of magnitude) of PPARγ ligands suggests that interaction with L-type Ca2+ channels is not a “class effect” (i.e., a property of any PPARγ ligand), but rather an ancillary property of individual compounds.
Besides direct interaction with L-type Ca2+ channels that contribute to vascular tone, indirect effect on Ca2+ channels may produce effects on membrane potential through interaction with K+ channels (Fig. 1B). In general, K+ channel opening leads to cell hyperpolarization, which reduces opening of voltage-dependent L-type Ca2+ channels, whereas K+ channel blockade induces depolarization and subsequent opening of L-type Ca2+ channels. K+ channel subtypes are many and are differentially expressed in different cell types. The detailed discussion of K+ channel subtypes in regulation of vascular tone is beyond the scope of this paper considering that their expression may vary along the vascular tree [50]. Several studies document modulation of K+ currents by PPARγ ligands in different cell types. Modulation may involve PPARγ-dependent gene expression of K+ channels, signaling proteins that modulate K+ channels, and direct interaction of PPARγ ligands with the K+channels [51, 52]. In rat pituitary GH3 cells, ciglitazone increases whereas troglitazone decreases the amplitude of the Ca2+-dependent K+ currents [53]. Studies in clonal hamster insulinoma cell line have shown that troglitazone and 15d-PGJ2 inhibit KATP channel activity in micromolar range correlating with their ability in displacing [3H]glibenclamide (a reference ligand for K+
ATP channel) binding [54]. Some structure-activity similarity between ligands interacting with K+ channels (sulfonylureas, glinides) and ligands interacting with PPARγ has been noted, first in silico and later confirmed in biological assays [55]. In endothelial cells (EC), rosiglitazone significantly inhibited VEGF-induced angiogenesis via a maxi-K+ channel opening subsequent to PPARγ-induced NO production [56]. In VSMC from guinea pig mesenteric artery, troglitazone (1 µM) inhibited both voltage-gated K+ currents as well as Ca2+-dependent K+ currents, whereas rosiglitazone and pioglitazone were almost ineffective at concentrations up to 100 µM (rosiglitazone slightly stimulated Ca2+-dependent K+ currents) [48]. Another study showed that troglitazone and rosiglitazone recover the function of K+
ATP channels in part through their antioxidant property [57]. While a more recent study [58] showed that pioglitazone relaxed norepinephrine-preconstricted rat aorta in a manner dependent on both voltage-dependent and inward rectifying K+ channels, but was insensitive to PPARγ antagonists. The latter two studies are therefore compatible with the properties of at least some
PPARγ ligands in restoring vasodilatation through an effect on electro-mechanical coupling (Fig. 1B).
When [Ca2+]i rises in VSMC, it binds calmodulin and activates myosin light chain kinase (MLCK), which then phosphorylates myosin light chains (LC20). Activation of myosin MgATPase activity is dependent on the phosphorylation of LC20 bound to the head–neck junction of the myosin heavy chains (MHC). The main pathways regulating phospho-LC20 level and, thereby, crossbridge cycling in smooth muscle involve: (i) Ca2+/calmodulin-dependent phosphorylation of LC20 at ser19 by MLCK, and (ii) the inhibition of myosin light chain phosphatase (MLCP)-mediated dephosphorylation of LC20 [59]. Alternative Ca2+- independent pathways of LC20 phosphorylation and increased myosin ATPase activity have also been described [59, 60]. During periods of sustained vascular tone, LC20 phosphorylation levels often decline and force is maintained by slowly cycling cross-bridges. Upon stimulus cessation, [Ca2+]i declines by plasmalemmal Ca2+ extrusion (via the Ca2+-ATPase and Na+-Ca2+ exchanger) and sequestration by intracellular organelles such as the sarcoplasmic reticulum and mitochondria. Subsequent to this, LC20 is dephosphorylated by MLCP resulting in vasorelaxation [59, 61]. A cellular signaling pathway involving Rho kinase (ROK)-mediated suppression of LC20 dephosphorylation may contribute to the regulation of arterial diameter in the myogenic response [62]. Phosphorylation of myosin phosphatase target subunit 1 (MYPT1) by ROK is a well-established mechanism of MLCP inhibition and increased force generation in agonist-evoked VSMC contraction [59]. Thus, ROK is considered a pharmacological target, whose inhibition may, for example, be beneficial in increasing collateral blood flow in experimental brain ischemia [24]. PPARγ ligands have been reported to lower systolic blood pressure in spontaneously hypertensive rats and inhibit Rho/ROK pathway in their aortic tissues, by inducing the expression of protein tyrosine phosphatase SHP-2 (Fig. 2A) [63]. This was based on a 4-week in vivo rat treatment with pioglitazone and the results were compatible with the kinetics of PPARγ-mediated gene transcription. In contrast, a more recent study suggests that pioglitazone causes a rapid inhibition of MYPT1 phosphorylation in a ROK-independent manner (Fig. 1A) [64]. The intriguing finding of this latter study is that the effect of pioglitazone on MYPT1 is very fast (occurring within 15 min) but still seems to be PPARγ-dependent (inhibited by GW9662) [64]. Because such a fast rate is not compatible with gene-expression mediated effects, if we exclude potential non-specific (PPARγ-unrelated) effects of GW9662, then we must assume that a fast PPARγ-mediated signaling mechanism (for ex. activation/inhibition of specific protein kinase and/or phosphatases) is involved. Classical activation of PPARγ has also been shown to affect Ca2+ sensitization in primary rat VSMCs, where pioglitazone promotes the activation of MLCP, thus reducing phosphorylation of myosin light chain [64], while pioglitazone and troglitazone suppress AT II-stimulated ROK activity [63]. Beside ROK, other still poorly defined mechanisms may reduce the sensitivity of contractile proteins to Ca2+ in a PPARγ-dependent manner, as suggested by the observation that aorta isolated from mice expressing
Vascular Effects of PPARγ Ligands Current Molecular Pharmacology, 2012, Vol. 5, No. 2 5
dominant negative PPARγ under control of smooth muscle specific promoter exhibits a increased vasoconstriction to endothelin-1 (ET-1) and 5-hydroxytryptamine (5-HT) [35]. This enhancement of responsiveness to vasoconstrictors is likely to be a post-receptor event, perhaps at the level or below cyclic GMP-dependent protein kinase [35].
We have recently observed in vitro, that troglitazone rapidly and reversibly blocked contraction of vascular smooth muscle induced by either K+-dependent depolarization or α1-adrenoceptor stimulation [65]. This is because the effect of troglitazone occurred 30 min post-incubation, it does not seem to be consistent with the latency of PPARγ-activated gene expression. The latter response requires at least 2 h for significantly changing mRNAs [38]. Furthermore, troglitazone-induced inhibition of vasoconstriction was rapidly reversible (as early as 30 min), which again shows that PPARγ stimulation is the unlikely underlying mechanism. Because reversibility would imply more time needed for the turnover of PPARγ-induced mRNAs and proteins. Furthermore, the lack of effect of GW9662 ruled out the involvement of PPARγ. Taken together, these and other [66] observations suggest that it is a class effect for thiazolidinediones. Because this PPARγ-independent mechanism is likely to have an impact on vascular tone in in vivo, it may have a clinical significance in patients with type 2 diabetes who are at high cardiovascular risk and/or have already developed cardiovascular diseases.
LONG-TERM EFFECTS OF PPARγ LIGANDS ON VASCULAR TONE
Atherosclerosis is a slow developing condition where the artery wall changes not only its physical properties (thickness, stiffness), but also the physiology of its cell constituents, including VSMC proliferation and apoptosis with subsequent remodeling, and endothelial dysfunction. These pathophysiological changes, although slow to develop, have consequences on fast regulation of vascular tone (reduced capability to vasodilate, tendency to vasospasm). This further increases cardiovascular risk as a result of development of stiffer and narrower than normal blood vessels. An hallmark of atherosclerosis is the inflammatory activation of the transcription factor NF-κB in endothelium in response to diverse stimuli such as hyperglycemia, oxidized low density lipoprotein (LDL) and shear stress [67]. The subsequent upregulation of adhesion molecules in ECs recruits circulating leukocytes in the vessel wall [67]. Activated leukocytes generate and release reactive oxygen species (ROS) that scavenge nitric oxide, thereby contributing to dysregulation of vascular tone. PPARγ is expressed not only in the two main constituents of vessel wall, ECs and VSMCs, but also in other inflammatory cell types that may be found in the context of vascular atherosclerosis [13, 14, 68, 69]. Potential long-term effects of PPARγ ligands are summarized in Fig. (2). Overall, PPARγ activity exerts anti-inflammatory effects in vascular atherosclerosis. Expression of constitutively active PPARγ in ECs reduces adhesion molecule expression and leukocyte recruitment via suppression of NF-κB and AP1 activation (Fig. 2A) [70]. PPARγ also inhibits the inflammatory responses to cytokines such as interferon-γ (IFNγ) and TNFα
[71]. In VSMCs, inhibition of the protein kinase C pathway may represent an anti-inflammatory target of PPARγ [72] and, in some instances, might follow toll like receptor 4 (TLR4) signaling blockade [73]. Activation of PPARγ in ECs inhibits IFNγ–induced expression of chemokines such as IP-10, Mig, and I-TAC, thereby decreasing vascular recruitment of activated T cells [71]. In human microvascular ECs, Lombardi et al. reported another faster mechanism for rosiglitazone that contributes to inhibition of TNFα and IFNγ inflammatory effects [74], which involves phosphorylation and activation of extracellular signal-regulated kinases (ERK1/2) [74]. A cross talk between fast (contractile tone) and long-term (chronic remodeling) effects on vascular pathophysiology is resumed by mediators such as ATII (Fig. 1 and 2). In blood vessels, PPARγ ligands have been shown to inhibit cell growth, inflammation, rise in blood pressure, and the structural, functional, and molecular changes induced by ATII [75]. More recently, in apolipoprotein E-deficient mice, pretreatment with rosiglitazone has been shown to inhibit ATII-induced expression of AT1 receptor and inflammatory mediators (E-selectin, TNFα and IL-6) in association with reduction of development and rupture of experimental aortic aneurysms [76]. Reduction of ICAM-1 and VCAM-1 expression by troglitazone or 15d-PGJ2 that potentially lead to reduced monocyte/macrophage recruitment and accumulation has also been found in a mouse model of atherosclerosis [77, 78]. Data on apoptosis in endothelium by PPARγ ligands are somewhat conflicting, possibly because of variable expression levels of PPARγ in different cells [79-81]. Particularly abundant (and growing) is the literature on PPARγ and angiogenesis. Initially, PPARγ activators were shown to inhibit VEGF receptor 1 and 2 expression, reduce endothelial tube formation in vitro and limit angiogenesis [82]. One of the mechanisms in the anti-angiogenic effect of PPARγ ligands is upregulation of PTEN, a phosphatase that functions as a negative regulator of PI3K/Akt signaling (Fig. 2B) [83, 84]. However, may other mechanisms have also been proposed [85]. Because neovascularization is involved in complications of atherosclerosis (plaque progression, aneurysm formation and intraplaque hemorrhage) and diabetes (proliferative retinopathy), and is a major target in anti-cancer therapy, PPARγ ligands are considered as potentially beneficial in these conditions [86-88]. A potential caveat in atherosclerosis and diabetes, however is that inhibition of angiogenesis may negatively impact on the development of collateral circles in ischemic areas.
Besides long-term structural effects on vascular wall, PPARγ-dependent gene expression may also directly affect mediators that are responsible for short-term effects on vascular tone such as NO, ET-1 and ATII (Fig. 1A). The potential beneficial effects of PPARγ ligands are increasingly studied in this context [89]. The overall pattern of endothelial vasoactive genes affected by PPARγ would be toward vasodilatation, because it is characterized by inhibition of preproET-1 expression [90] and by stimulation of a transcriptional mechanism unrelated to eNOS expression that determines increased NO release [91]. PPARγ ligands enhance NO production [92] through a PPARγ-dependent increase in heat shock protein 90-eNOS interaction and eNOS ser1177 phosphorylation, whereas
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disruption of PPARγ causes a reduction in endothelium-dependent vasodilatation [33]. Moreover, PPARγ ligands potentially reduce NO scavenging by ROS by decreasing expression of the NADPH oxidase subunits nox1, gp91phox, and nox4 and by increasing activity and expression of copper/zinc superoxide dismutase (Fig. 2A) [93]. Besides modulation of NO levels, PPARγ ligands can affect vascular tone through suppression of ET-1 synthesis in ECs [90, 91]. In DOCA-salt hypertensive rats, a hypertensive model associated with enhanced expression of preproET-1, rosiglitazone inhibits the increase of preproET-1 mRNA in
mesenteric blood vessels, the hypertrophic remodeling and the increase in blood pressure [94].
Long-term effects of PPARγ ligands on VSMCs, related to PPARγ-dependent gene expression, may also have important consequences on vascular tone. Vascular tone may vary because of vascular remodeling, particularly in relation to VSMC number, and changes in the expression of vasoconstriction and/or vasodilatation signaling molecules. In proliferative VSMCs, PPARγ ligands upregulate smooth muscle myosin heavy chain and smooth muscle α-actin, the two specific markers of differentiated VSMCs [95].
Fig. (2). Potential long-term effects of PPARγ ligands on vascular tone. EC, endothelial cell, VSMC, vascular smooth muscle cell. A PPARγ-dependent effects, B PPARγ-independent effects. Note the effects on inflammatory response, angiogenesis and cell cycling, because these affect vascular physiology in the long term and thereby, indirectly, vascular tone. Thick arrows indicate increase/stimulation (upward) or decrease/inhibition (downward) of a mediator or a function; thick right-angle arrows from DNA indicate induced (or repressed, crossed arrows) gene expression; dotted arrows indicate potential mechanistic relationships. Numbers in square parentheses indicate references.
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Interestingly, intimal VSMCs seems to have increased functional PPARγ relative to medial VSMCs, which may reflect a distinct differentiation/proliferation phenotype of VSMCs at these locations [96]. PPARγ ligands inhibit VSMC proliferation through increased expression of the cyclin-dependent kinase inhibitor p27 and decreased phosphorylation of the retinoblastoma protein, thus leading to cell-cycle arrest (Fig. 2A) [97-99]. Moreover, they induce caspase-mediated apoptosis of VSMCs closely correlated with an upregulation of growth arrest and DNA damage-inducible gene 45 (GADD45) [100]. PPARγ ligands inhibit the expression of the AT1 receptor in VSMC resulting in inhibition of neointimal formation and inhibition of vascoconstriction to ATII [101].
As mentioned above, besides EC and VSMC, other cell types that may be found in vessel wall do express PPARγ; these include monocyte/macrophages and lymphocytes. In these cell types, PPARγ ligands exert anti-inflammatory effects and regulate macrophage lipid homeostasis [102], which may contribute to their antiatherogenic action. The detailed discussion of these effects and mechanisms is beyond the scope of this article. As far as effects on vascular tone are considered, the inhibition of inducible NOS (iNOS or NOS II) by PPARγ ligands in monocytes is worthy of consideration [14], This is because increased NO production following iNOS induction may be responsible, at least in theory, for vasodilation and fall in systemic blood pressure.
CONCLUDING REMARKS
Based on available data, PPARγ ligands appear to influence vascular tone, in different experimental paradigms, most often in terms of vasodilatation (potentially increasing blood flow to some tissues and/or reducing systemic blood pressure). Long-term vascular effects of PPARγ ligands mostly depend on PPARγ-regulated gene expression, and seem to have beneficial impact on atherosclerosis (on the inflammatory component as well as on accompanying VSMC remodeling and endothelial dysfunction). In contrast, short-term effects of PPARγ ligands are likely to be, in most instances, PPARγ-independent, relying on modulation of kinases and phosphatases and/or ion channels that are involved in the mechanisms of excitation-contraction coupling in VSMC or in the production and release of vasodilating agents from ECs. These effects on vascular tone may theoretically be beneficial in patients with cardiovascular risk, their clinical significance, however,remains to be determined and carefully weighed against cardiovascular warnings, that may specifically apply to isolated drugs, such as rosiglitazone.
Finally, other hemodynamic effects of PPARγ ligands also deserve consideration. In particular, edema and water retention, that are relatively common side effects of PPARγ ligands. The most relevant mechanism involved in sodium retention and plasma volume expansion associated with PPARγ ligands depends on PPARγ stimulation in the epithelium of the renal collecting duct [103]. PPARγ activation also increases vascular permeability, which may contribute to plasma extravasation [104]. Modification of vascular tone, that if occurring in capacitance vessels may contribute to edema, has not been directly linked to this
phenomenon; however, single nucleotide polymorphisms in genes that affect vascular tone, such as renin and ET-1, have been associated with PPARγ-dependent edema [105].
ACKNOWLEDGEMENTS
Supported by a grant of Italian Ministry of University, PRIN 2007SXKWSA_001.
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