Scott Gleim

Acceleration of Cardiovascular Disease by a Dysfunctional Prostacyclin Receptor Mutation Potential Implications for Cyclooxygenase-2 Inhibition

Recent increased adverse cardiovascular events observed with selective cyclooxygenase-2 inhibition led to the withdrawal of rofecoxib (Vioxx) and valdecoxib (Bextra), but the mechanisms underlying these atherothrombotic events remain unclear. Prostacyclin is the major end product of cyclooxygenase-2 in vascular endothelium. Using a naturally occurring mutation in the prostacyclin receptor, we report for the first time that a deficiency in prostacyclin signaling through its G protein–coupled receptor contributes to atherothrombosis in human patients. We report that a prostacyclin receptor variant (R212C) is defective in adenylyl cyclase activation in both patient blood and in an in vitro COS-1 overexpression system. This promotes increased platelet aggregation, a hallmark of atherothrombosis. Our analysis of patients in 3 separate white cohorts reveals that this dysfunctional receptor is not likely an initiating factor in cardiovascular disease but that it accelerates the course of disease in those patients with the greatest risk factors. R212C was associated with cardiovascular disease only in the high cardiovascular risk cohort (n=980), with no association in the low-risk cohort (n=2293). In those at highest cardiovascular risk, both disease severity and adverse cardiovascular events were significantly increased with R212C when compared with age- and risk factor–matched normal allele patients. We conclude that for haploinsufficient mutants, such as the R212C, the enhanced atherothrombotic phenotype is likely dependent on the presence of existing atherosclerosis or injury (high risk factors), analogous to what has been observed in the cyclooxygenase-2 inhibition studies or prostacyclin receptor knockout mice studies. Combining both biochemical and clinical approaches, we conclude that diminished prostacyclin receptor signaling may contribute, in part, to the underlying adverse cardiovascular outcomes observed with cyclooxygenase-2 inhibition.

Apelin-APJ Signaling Is a Critical Regulator of Endothelial MEF2 Activation in Cardiovascular Development

Rationale: The peptide ligand apelin and its receptor APJ constitute a signaling pathway with numerous effects on the cardiovascular system, including cardiovascular development in model organisms such as xenopus and zebrafish. Objective: This study aimed to characterize the embryonic lethal phenotype of the Apj−/− mice and to define the involved downstream signaling targets. Methods and Results: We report the first characterization of the embryonic lethality of the Apj−/− mice. More than half of the expected Apj−/− embryos died in utero because of cardiovascular developmental defects. Those succumbing to early embryonic death had markedly deformed vasculature of the yolk sac and the embryo, as well as poorly looped hearts with aberrantly formed right ventricles and defective atrioventricular cushion formation. Apj−/− embryos surviving to later stages demonstrated incomplete vascular maturation because of a deficiency of vascular smooth muscle cells and impaired myocardial trabeculation and ventricular wall development. The molecular mechanism implicates a novel, noncanonical signaling pathway downstream of apelin-APJ involving G&agr;13, which induces histone deacetylase (HDAC) 4 and HDAC5 phosphorylation and cytoplasmic translocation, resulting in activation of myocyte enhancer factor 2. Apj−/− mice have greater endocardial Hdac4 and Hdac5 nuclear localization and reduced expression of the myocyte enhancer factor 2 (MEF2) transcriptional target Krüppel-like factor 2. We identify a number of commonly shared transcriptional targets among apelin-APJ, G&agr;13, and MEF2 in endothelial cells, which are significantly decreased in the Apj−/− embryos and endothelial cells. Conclusions: Our results demonstrate a novel role for apelin-APJ signaling as a potent regulator of endothelial MEF2 function in the developing cardiovascular system.

Glucose and collagen regulate human platelet activity through aldose reductase induction of thromboxane

Diabetes mellitus is associated with platelet hyperactivity, which leads to increased morbidity and mortality from cardiovascular disease. This is coupled with enhanced levels of thromboxane (TX), an eicosanoid that facilitates platelet aggregation. Although intensely studied, the mechanism underlying the relationship among hyperglycemia, TX generation, and platelet hyperactivity remains unclear. We sought to identify key signaling components that connect high levels of glucose to TX generation and to examine their clinical relevance. In human platelets, aldose reductase synergistically modulated platelet response to both hyperglycemia and collagen exposure through a pathway involving ROS/PLCγ2/PKC/p38α MAPK. In clinical patients with platelet activation (deep vein thrombosis; saphenous vein graft occlusion after coronary bypass surgery), and particularly those with diabetes, urinary levels of a major enzymatic metabolite of TX (11-dehydro-TXB2 [TX-M]) were substantially increased. Elevated TX-M persisted in diabetic patients taking low-dose aspirin (acetylsalicylic acid, ASA), suggesting that such patients may have underlying endothelial damage, collagen exposure, and thrombovascular disease. Thus, our study has identified multiple potential signaling targets for designing combination chemotherapies that could inhibit the synergistic activation of platelets by hyperglycemia and collagen exposure.

Versatility and Differential Roles of Cysteine Residues in Human Prostacyclin Receptor Structure and Function

Prostacyclin plays important roles in vascular homeostasis, promoting vasodilatation and inhibiting platelet thrombus formation. Previous studies have shown that three of six cytoplasmic cysteines, particularly those within the C-terminal tail, serve as important lipidation sites and are differentially conjugated to palmitoyl and isoprenyl groups (Miggin, S. M., Lawler, O. A., and Kinsella, B. T. (2003) J. Biol. Chem. 278, 6947-6958). Here we report distinctive roles for extracellular- and transmembrane-located cysteine residues in human prostacyclin receptor structure-function. Within the extracellular domain, all cysteines (4 of 4) appear to be involved in disulfide bonding interactions (i.e. a highly conserved Cys-92-Cys-170 bond and a putative non-conserved Cys-5-Cys-165 bond), and within the transmembrane (TM) region there are several cysteines (3 of 8) that maintain critical hydrogen bonding interactions (Cys-118 (TMIII), Cys-251 (TMVI), and Cys-202 (TMV)). This study highlights the necessity of sulfhydryl (SH) groups in maintaining the structural integrity of the human prostacyclin receptor, as 7 of 12 extracellular and transmembrane cysteines studied were found to be differentially indispensable for receptor binding, activation, and/or trafficking. Moreover, these results also demonstrate the versatility and reactivity of these cysteine residues within different receptor environments, that is, extracellular (disulfide bonds), transmembrane (H-bonds), and cytoplasmic (lipid conjugation).

Hospital Patterns of Use of Positive Inotropic Agents in Patients with Heart Failure

OBJECTIVES This study sought to determine hospital variation in the use of positive inotropic agents in patients with heart failure. BACKGROUND Clinical guidelines recommend targeted use of positive inotropic agents in highly selected patients, but data are limited and the recommendations are not specific. METHODS We analyzed data from 376 hospitals including 189,948 hospitalizations for heart failure from 2009 through 2010. We used hierarchical logistic regression models to estimate hospital-level risk-standardized rates of inotrope use and risk-standardized in-hospital mortality rates. RESULTS The risk-standardized rates of inotrope use ranged across hospitals from 0.9% to 44.6% (median: 6.3%, interquartile range: 4.3% to 9.2%). We identified various hospital patterns based on the type of agents: dobutamine-predominant (29% of hospitals), dopamine-predominant (25%), milrinone-predominant (1%), mixed dobutamine and dopamine pattern (32%), and mixed pattern including all 3 agents (13%). When studying the factors associated with interhospital variation, the best model performance was with the hierarchical generalized linear models that adjusted for patient case mix and an individual hospital effect (receiver operating characteristic curves from 0.77 to 0.88). The intraclass correlation coefficients of the hierarchical generalized linear models (0.113 for any inotrope) indicated that a noteworthy proportion of the observed variation was related to an individual institutional effect. Hospital rates or patterns of use were not associated with differences in length of stay or risk-standardized mortality rates. CONCLUSIONS We found marked differences in the use of inotropic agents for heart failure patients among a diverse group of hospitals. This variability, occurring in the context of little clinical evidence, indicates an urgent need to define the appropriate use of these medications.

New Insights into Structural Determinants for Prostanoid Thromboxane A2 Receptor- and Prostacyclin Receptor-G Protein Coupling

ABSTRACT G protein-coupled receptors (GPCRs) interact with heterotrimeric G proteins and initiate a wide variety of signaling pathways. The molecular nature of GPCR-G protein interactions in the clinically important thromboxane A2 (TxA2) receptor (TP) and prostacyclin (PGI2) receptor (IP) is poorly understood. The TP activates its cognate G protein (Gαq) in response to the binding of thromboxane, while the IP signals through Gαs in response to the binding of prostacyclin. Here, we utilized a combination of approaches consisting of chimeric receptors, molecular modeling, and site-directed mutagenesis to precisely study the specificity of G protein coupling. Multiple chimeric receptors were constructed by replacing the TP intracellular loops (ICLs) with the ICL regions of the IP. Our results demonstrate that both the sequences and lengths of ICL2 and ICL3 influenced G protein specificity. Importantly, we identified a precise ICL region on the prostanoid receptors TP and IP that can switch G protein specificities. The validities of the chimeric technique and the derived molecular model were confirmed by introducing clinically relevant naturally occurring mutations (R60L in the TP and R212C in the IP). Our findings provide new molecular insights into prostanoid receptor-G protein interactions, which are of general significance for understanding the structural basis of G protein activation by GPCRs in basic health and cardiovascular disease.

An Eicosanoid-Centric View of Atherothrombotic Risk Factors

Cardiovascular disease is the foremost cause of morbidity and mortality in the Western world. Atherosclerosis followed by thrombosis (atherothrombosis) is the pathological process underlying most myocardial, cerebral, and peripheral vascular events. Atherothrombosis is a complex and heterogeneous inflammatory process that involves interactions between many cell types (including vascular smooth muscle cells, endothelial cells, macrophages, and platelets) and processes (including migration, proliferation, and activation). Despite a wealth of knowledge from many recent studies using knockout mouse and human genetic studies (GWAS and candidate approach) identifying genes and proteins directly involved in these processes, traditional cardiovascular risk factors (hyperlipidemia, hypertension, smoking, diabetes mellitus, sex, and age) remain the most useful predictor of disease. Eicosanoids (20 carbon polyunsaturated fatty acid derivatives of arachidonic acid and other essential fatty acids) are emerging as important regulators of cardiovascular disease processes. Drugs indirectly modulating these signals, including COX-1/COX-2 inhibitors, have proven to play major roles in the atherothrombotic process. However, the complexity of their roles and regulation by opposing eicosanoid signaling, have contributed to the lack of therapies directed at the eicosanoid receptors themselves. This is likely to change, as our understanding of the structure, signaling, and function of the eicosanoid receptors improves. Indeed, a major advance is emerging from the characterization of dysfunctional naturally occurring mutations of the eicosanoid receptors. In light of the proven and continuing importance of risk factors, we have elected to focus on the relationship between eicosanoids and cardiovascular risk factors.

New insights into human prostacyclin receptor structure and function through natural and synthetic mutations of transmembrane charged residues

The human prostacyclin receptor (hIP), a G‐protein coupled receptor (GPCR) expressed mainly on platelets and vascular smooth muscle cells, plays important protective roles in the cardiovascular system. We hypothesized that significant insights could be gained into the structure and function of the hIP through mutagenesis of its energetically unfavourably located transmembrane charged residues.

Phosphorylation of GATA-6 is required for vascular smooth muscle cell differentiation after mTORC1 inhibition.

Increasing the activity of the transcription factor GATA-6 prevents excessive proliferation of vascular smooth muscle cells in injured blood vessels. Blocking proliferation to keep blood vessels open A treatment option for clearing blood vessels narrowed by atherosclerotic plaques is angioplasty and stenting, the insertion of a tube to help keep the blood vessel open. However, stents can trigger proliferation of the vascular smooth muscle cells resulting in renarrowing of the vessel. To avoid this complication, stents may contain the drug rapamycin, which is released to limit the proliferation and promote the differentiation of vascular smooth muscle cells. Xie et al. found that these effects of rapamycin on vascular smooth muscle cells required phosphorylation of the transcription factor GATA-6, which increased its stability and function. Various experiments indicated that Akt2 may be the kinase that phosphorylated GATA-6, and injured arteries in Akt2-null mice developed thicker blood vessel walls than those in control mice. Overexpression of a phosphorylation-mimetic form of GATA-6 prevented thickening of the blood vessel walls in Akt2-null mice after injury to a greater extent than did a phosphorylation-deficient mutant. Vascular smooth muscle cells (VSMCs) undergo transcriptionally regulated reversible differentiation in growing and injured blood vessels. This dedifferentiation also contributes to VSMC hyperplasia after vascular injury, including that caused by angioplasty and stenting. Stents provide mechanical support and can contain and release rapamycin, an inhibitor of the mechanistic target of rapamycin complex 1 (mTORC1). Rapamycin suppresses VSMC hyperplasia and promotes VSMC differentiation. We report that rapamycin-induced differentiation of VSMCs required the transcription factor GATA-6. Inhibition of mTORC1 stabilized GATA-6 and promoted the nuclear accumulation of GATA-6, its binding to DNA, its transactivation of promoters encoding contractile proteins, and its inhibition of proliferation. These effects were mediated by phosphorylation of GATA-6 at Ser290, potentially by Akt2, a kinase that is activated in VSMCs when mTORC1 is inhibited. Rapamycin induced phosphorylation of GATA-6 in wild-type mice, but not in Akt2−/− mice. Intimal hyperplasia after arterial injury was greater in Akt2−/− mice than in wild-type mice, and the exacerbated response in Akt2−/− mice was rescued to a greater extent by local overexpression of the wild-type or phosphomimetic (S290D) mutant GATA-6 than by that of the phosphorylation-deficient (S290A) mutant. Our data indicated that GATA-6 and Akt2 are involved in the mTORC1-mediated regulation of VSMC proliferation and differentiation. Identifying the downstream transcriptional targets of mTORC1 may provide cell type–specific drug targets to combat cardiovascular diseases associated with excessive proliferation of VSMCs.

Comprehensive biochemical analysis of rare prostacyclin receptor variants: Study of association of signaling with coronary artery obstruction

Currently, pharmacogenetic studies are at an impasse as the low prevalence (<2%) of most variants hinder their pharmacogenetic analysis with population sizes often inadequate for sufficiently powered studies. Grouping rare mutations by functional phenotype rather than mutation site can potentially increase sample size. Using human population-based studies (n = 1,761) to search for dysfunctional human prostacyclin receptor (hIP) variants, we recently discovered 18 non-synonymous mutations, all with frequencies less than 2% in our study cohort. Eight of the 18 had defects in binding, activation, and/or protein stability/folding. Mutations (M113T, L104R, and R279C) in three highly conserved positions demonstrated severe misfolding manifested by impaired binding and activation of cell surface receptors. To assess for association with coronary artery disease, we performed a case-control study comparing coronary angiographic results from patients with reduced cAMP production arising from the non-synonymous mutations (n = 23) with patients with non-synonymous mutations that had no reduction in cAMP (n = 17). Major coronary artery obstruction was significantly increased in the dysfunctional mutation group in comparison with the silent mutations. We then compared the 23 dysfunctional receptor patients with 69 age- and risk factor-matched controls (1:3). This verified the significantly increased coronary disease in the non-synonymous dysfunctional variant cohort. This study demonstrates the potential utility of in vitro functional characterization in predicting clinical phenotypes and represents the most comprehensive characterization of human prostacyclin receptor genetic variants to date.