Manikandan Panchatcharam

Autotaxin/Lysopholipase D and lysophosphatidic acid regulate murine hemostasis and thrombosis

The lipid mediator lysophosphatidic acid (LPA) is a potent regulator of vascular cell function in vitro, but its physiologic role in the cardiovasculature is largely unexplored. To address the role of LPA in regulating platelet function and thrombosis, we investigated the effects of LPA on isolated murine platelets. Although LPA activates platelets from the majority of human donors, we found that treatment of isolated murine platelets with physiologic concentrations of LPA attenuated agonist-induced aggregation. Transgenic overexpression of autotaxin/lysophospholipase D (Enpp2), the enzyme necessary for production of the bulk of biologically active LPA in plasma, elevated circulating LPA levels and induced a bleeding diathesis and attenuation of thrombosis in mice. Intravascular administration of exogenous LPA recapitulated the prolonged bleeding time observed in Enpp2-Tg mice. Enpp2+/- mice, which have ∼50% normal plasma LPA levels, were more prone to thrombosis. Plasma autotaxin associated with platelets during aggregation and concentrated in arterial thrombus, and activated but not resting platelets bound recombinant autotaxin/lysoPLD in an integrin-dependent manner. These results identify a novel pathway in which LPA production by autotaxin/lysoPLD regulates murine hemostasis and thrombosis and suggest that binding of autotaxin/lysoPLD to activated platelets may provide a mechanism to localize LPA production.

Roles of lysophosphatidic acid in cardiovascular physiology and disease

The bioactive lipid mediator lysophosphatidic acid (LPA) exerts a range of effects on the cardiovasculature that suggest a role in a variety of critical cardiovascular functions and clinically important cardiovascular diseases. LPA is an activator of platelets from a majority of human donors identifying a possible role as a regulator of acute thrombosis and platelet function in atherogenesis and vascular injury responses. Of particular interest in this context, LPA is an effective phenotypic modulator of vascular smooth muscle cells promoting the de-differentiation, proliferation and migration of these cells that are required for the development of intimal hyperplasia. Exogenous administration of LPA results in acute and systemic changes in blood pressure in different animal species, suggesting a role for LPA in both normal blood pressure regulation and hypertension. Advances in our understanding of the molecular machinery responsible for the synthesis, actions and inactivation of LPA now promise to provide the tools required to define the role of LPA in cardiovascular physiology and disease. In this review we discuss aspects of LPA signaling in the cardiovasculature focusing on recent advances and attempting to highlight presently unresolved issues and promising avenues for further investigation.

Lysophosphatidic Acid Receptors 1 and 2 Play Roles in Regulation of Vascular Injury Responses but Not Blood Pressure

Phenotypic modulation of vascular smooth muscle cells (SMCs) is essential for the development of intimal hyperplasia. Lysophosphatidic acid (LPA) is a serum component that can promote phenotypic modulation of cultured SMCs, but an endogenous role for this bioactive lipid as a regulator of SMC function in vivo has not been established. Ligation injury of the carotid artery in mice increased levels in the vessel of both autotaxin, the lysophospholipase D enzyme responsible for generation of extracellular LPA, and 2 LPA responsive G protein–coupled receptors 1 (LPA1) and 2 (LPA2). LPA1−/−2−/− mice were partially protected from the development of injury-induced neointimal hyperplasia, whereas LPA1−/− mice developed larger neointimal lesions after injury. Growth in serum, LPA-induced extracellular signal-regulated protein kinase activation, and migration to LPA and serum were all attenuated in SMCs isolated from LPA1−/−2−/− mice. In contrast, LPA1−/− SMCs exhibited enhanced migration resulting from an upregulation of LPA3. However, despite their involvement in intimal hyperplasia, neither LPA1 nor LPA2 was required for dedifferentiation of SMCs following vascular injury or dedifferentiation of isolated SMCs in response to LPA or serum in vitro. Similarly, neither LPA1 nor LPA2 was required for LPA to elicit a transient increase in blood pressure following intravenous administration of LPA to mice. These results identify a role for LPA1 and LPA2 in regulating SMC migratory responses in the context of vascular injury but suggest that additional LPA receptor subtypes are required for other LPA-mediated effects in the vasculature.

Dominant-Negative Loss of PPARγ Function Enhances Smooth Muscle Cell Proliferation, Migration, and Vascular Remodeling

Objective—The peroxisome proliferator activated receptor-gamma (PPAR&ggr;) protein is a nuclear transcriptional activator with importance in diabetes management as the molecular target for the thiazolidinedione (TZD) family of drugs. Substantial evidence indicates that the TZD family of PPAR&ggr; agonists may retard the development of atherosclerosis. However, recent clinical data have suggested that at least one TZD may increase the risk of myocardial infarction and death from cardiovascular disease. In this study, we used a genetic approach to disrupt PPAR&ggr; signaling to probe the proteins role in smooth muscle cell (SMC) responses that are important for atherosclerosis. Methods and Results—SMC isolated from transgenic mice harboring the dominate-negative P465L mutation in PPAR&ggr; (PPAR&ggr;L/+) exhibited greater proliferation and migration then did wild-type cells. Upregulation of ETS-1, but not ERK activation, correlated with enhanced proliferative and migratory responses PPAR&ggr;L/+ SMCs. After arterial injury, PPAR&ggr;L/+ mice had a ≈4.3-fold increase in the development of intimal hyperplasia. Conclusion—These findings are consistent with a normal role for PPAR&ggr; in inhibiting SMC migration and proliferation in the context of restenosis or atherosclerosis.

CARDIOPROTECTIVE EFFECTS OF CURCUMIN

Curcumin, a major active component of turmeric, is extracted from the powdered dry rhizome of Curcuma longa Linn (Zingiberaceae) and it has been used for centuries in indigenous medicine. We have shown that curcumin has a protective role against myocardial necrosis in rats. The antioxidant activity of curcumin could be attributed to the phenolic and methoxy groups in conjunction with the 1,3-diketone-conjugated diene system, for scavenging of the oxygen radicals. In addition, curcumin is shown to enhance the activities of detoxifying enzymes such as glutathione-S-transferase in vivo. We have also shown that oxygen free radicals exacerbate cardiac damage and curcumin induces cardioprotective effect and it also inhibits free-radical generation in myocardial ischemia in rats. This chapter on the cardioprotective effects of curcumin covers the following aspects: (1) the history of curcumin and its discovery as a potent drug with relevance to cardiovascular diseases; (2) mechanistic role of curcumin in vitro, emphasizing the antiplatelet and anticoagulant effects; (3) cardiovascular properties of curcumin; (4) application of curcumin in different animal models (viz. myocardial ischemia, myocardial infarction, cardiomyopathy, and arrhythmia in vitro and in vivo); (5) curcumin free-radical scavenging activity, particularly against O2 radical and depletion of the oxidative stress.

Mechanism of rapid elimination of lysophosphatidic acid and related lipids from the circulation of mice

Lysophosphatidic acid (LPA) is a bioactive lipid mediator. Concentrations of the major LPA species in mouse plasma decreased uniformly following administration of a potent selective inhibitor of the LPA-generating lysophospholipase D autotaxin, identifying an active mechanism for removal of LPA from the circulation. LPA, akylglycerol phosphate (AGP), sphingosine 1-phosphate (S1P), and a variety of structural mimetics of these lipids, including phosphatase-resistant phosphonate analogs of LPA, were rapidly eliminated (t1/2 < 30 s) from the circulation of mice following intravenous administration of a single bolus dose without significant metabolism in situ in the blood. These lipids accumulated in the liver. Elimination of intravenously administered LPA was blunted by ligation of the hepatic circulation, and ∼90% of LPA administered through the portal vein was accumulated by the isolated perfused mouse liver at first pass. At early times following intravenous administration, more LPA was associated with a nonparenchymal liver cell fraction than with hepatocytes. Primary cultures of nonparenchymal liver cells rapidly assimilated exogenously provided LPA. Our results identify hepatic uptake as an important determinant of the bioavailability of LPA and bioactive lysophospholipid mimetics and suggest a mechanism to explain changes in circulating LPA levels that have been associated with liver dysfunction in humans.

Regulation of blood and vascular cell function by bioactive lysophospholipids

Summary.  Lysophosphatidic acid (LPA), its sphingolipid homolog sphingosine 1‐phosphate (S1P) and several other related molecules constitute a family of bioactive lipid phosphoric acids that function as receptor‐active mediators with roles in cell growth, differentiation, inflammation, immunomodulation, apoptosis and development. LPA and S1P are present in physiologically relevant concentrations in the circulation. In isolated cell culture systems or animal models, these lipids exert a range of effects that suggest that S1P and LPA could play important roles in maintaining normal vascular homeostasis and in vascular injury responses. LPA and S1P act on a series of G protein‐coupled receptors, and LPA may also be an endogenous regulator of PPARγ activity. In this review, we discuss potential roles for lysolipid signaling in the vasculature and mechanisms by which these bioactive lipids could contribute to cardiovascular disease.

Individual Heterogeneity in Platelet Response to Lysophosphatidic Acid. Evidence for a Novel Inhibitory Pathway

Objective—The bioactive lipid lysophosphatidic acid (LPA) stimulates platelet actin reorganization, adhesion, shape change, and aggregation. LPA is present in blood and exposure or release of LPA after atherosclerotic plaque rupture has been proposed to trigger platelet thrombus formation. Methods and Results—In this report, we characterize heterogeneity in LPA responses among individuals. Platelets isolated from approximately 20% of healthy donors consistently failed to aggregate in response to LPA. Our studies indicate that, rather than lacking stimulatory pathways, platelets from “nonresponsive” donors respond to LPA by triggering inhibitory pathway(s) to block platelet aggregation. Consistent with this observation, LPA-induced aggregation could be partially restored to “nonresponsive” platelets by pharmacological inhibition of cAMP generation. LPA “nonresponsiveness” may be related to heightened platelet expression of LPA receptor 4 and PPARγ. Among 70 patients with stable coronary artery disease (CAD), only 1 (1.4%) was identified as an LPA nonresponder. Moreover, in 33 patients presenting for diagnostic catheterization, CAD was identified as having a bivariate association with platelet LPA responder/nonresponder status. Conclusions—Platelet LPA signaling may involve stimulatory and inhibitory pathways, with the inhibitory pathway predominating in ≈20% of individuals. Diseases such as CAD that affect platelet reactivity may attenuate this inhibitory pathway in platelets.

Enhanced proliferation and migration of vascular smooth muscle cells in response to vascular injury under hyperglycemic conditions is controlled by β3 integrin signaling

Atheroma formation and restenosis following percutaneous vascular intervention involve the growth and migration of vascular smooth muscle cells (SMCs) into neointimal lesions, in part due to changes in the extracellular matrix. While some clinical studies have suggested that, in comparison to non-diabetics, beta3 integrin inhibition in diabetic patients confers protection from restenosis, little is known regarding the role of beta3 integrin inhibition on SMC responses in this context. To understand the molecular mechanisms underlying integrin-mediated regulation of SMC function in diabetes, we examined SMC responses in diabetic mice deficient in integrin beta3 and observed that the integrin was required for enhanced proliferation, migration and extracellular regulated kinase (ERK) activation. Hyperglycemia-enhanced membrane recruitment and catalytic activity of PKCbeta in an integrin beta3-dependent manner. Hyperglycemia also promoted SMC filopodia formation and cell migration, both of which required alphaVbeta3, PKCbeta, and ERK activity. Furthermore, the integrin-kinase association was regulated by the alphaVbeta3 integrin ligand thrombospondin and the integrin modulator Rap1 under conditions of hyperglycemia. These results suggest that there are differences in SMC responses to vascular injury depending on the presence or absence of hyperglycemia and that SMC response under hyperglycemic conditions is largely mediated through beta3 integrin signaling.

Mice With Targeted Inactivation of Ppap2b in Endothelial and Hematopoietic Cells Display Enhanced Vascular Inflammation and Permeability

Objective—Lipid phosphate phosphatase 3 (LPP3), encoded by the PPAP2B gene, is an integral membrane enzyme that dephosphorylates, and thereby terminates, the G-protein–coupled receptor–mediated signaling actions of lysophosphatidic acid (LPA) and sphingosine-1-phosphate. LPP3 is essential for normal vascular development in mice, and a common PPAP2B polymorphism is associated with increased risk of coronary artery disease in humans. Herein, we investigate the function of endothelial LPP3 to understand its role in the development and human disease. Approach and Results—We developed mouse models with selective LPP3 deficiency in endothelial and hematopoietic cells. Tyrosine kinase Tek promoter–mediated inactivation of Ppap2b resulted in embryonic lethality because of vascular defects. LPP3 deficiency in adult mice, achieved using a tamoxifen-inducible Cre transgene under the control of the Tyrosine kinase Tek promoter, enhanced local and systemic inflammatory responses. Endothelial, but not hematopoietic, cell LPP3 deficiency led to significant increases in vascular permeability at baseline and enhanced sensitivity to inflammation-induced vascular leak. Endothelial barrier function was restored by pharmacological or genetic inhibition of either LPA production by the circulating lysophospholipase D autotaxin or of G-protein–coupled receptor–dependent LPA signaling. Conclusions—Our results identify a role for the autotaxin/LPA-signaling nexus as a mediator of endothelial permeability in inflammation and demonstrate that LPP3 limits these effects. These findings have implications for therapeutic targets to maintain vascular barrier function in inflammatory states.