Clinical Research Studies

Site for Will reducing inflammation prevent a second heart attack? This application propose s a mechanism - based study designed to assess the impact of low - dose methotrexate (LD MTX, dose 15 - 20 mg weekly) on markers of disrupted cholesterol transport and metabolism i n post - MI patients enrolled in the Cardiovascular Infl ammation Reduction Trial (CIRT, HL101422) . Access to CIRT study subjects represents a unique opportunity to deepen our understanding of the inflammatory hypothesis of atherothrombosis . CIRT, funded by N HLBI , is a randomized, double blind, placebo - controlled clinical trial designed to determine whether LD MTX will reduce recurrent cardiovascular (CV) events in 7,000 stable post - myocardial infarction (MI) patients with type 2 diabetes (DM) or metabolic syndrome ( 1 ) . We and others have shown that alteration s in cholesterol transport gene and protein expression provide a reliable signature of MTX responsiveness and related anti - atherogenic effect ( 2, 3 ). Despite the potential signifi cance of studies demonstrating that MTX can restore cholesterol transport protein expression in vitro , the effect and mechanism of MTX therapy in humans at risk for CV events in vivo is not known. T he present study will explore whether e xtent of change in these transport proteins with LD MTX provide s a n indicator of efficacy. I mprovement in cholesterol handling may indicate that the individual is a responder and should be continued on MTX . The pathogenesis of atherosclerosis involves interplay among cholesterol transport, cholesterol metabolism and inflammatory pathways. Current research is focused on this in terplay and how it might be regulat ed ( 4 ). Our laboratory has shown that MTX has potent atheroprotective properties that exert influence at multiple points in cholesterol transport and metabolism and that i nflammation plays a significant role not only in autoimmune disorders , but in atherosclerosis and DM (1) . Anti - inflammator y effects of MTX are largely mediated through release of adenosine, an endogenous purine nucleoside that suppresses inflammatory cytokines shown to be elevated in DM and metabolic syndrome (5, 6). Physiological effects of adenosine are triggered by engage ment of 4 cla sses of G protein - coupled receptors: A 1 , A 2A , A 2B and A 3 . We have shown that the A 2A R potentiates cholesterol removal by modifying expression of cholesterol transport proteins (7). Adenosine, via interaction with the A 2A R, may be responsible f or the capacity of MTX to reduce death rates from atherosclerotic CV disease (ASCVD) in the autoimmune disorder rheumatoid arthritis (RA) (8). MTX spurs adenosine release, leading to A 2A R ligation, anti - inflammatory effects and improved cholesterol balance . H YPOTHESIS 1 : MTX will correct impairment of cholesterol balance induced by the inflammatory state in DM and metabolic syndrome. H YPOTHESIS 2 : Adenosine release by MTX will shift cholesterol metabolism toward a less atherogenic state via a mechanism involving activation of the adenosine A 2A receptor. To test our hypotheses, the following 2 aims are proposed: S PECIFIC A IM 1: Examine the impact of LD MTX on cholesterol transport gene s in peripheral blood mononuclear cells (PBMC) from post - MI patients with DM or metabolic syndrome. In PBMC retrieved from the same subject before and after LD MTX, we will measure changes in cholesterol flux genes (immunoblot, quantitative real - time reverse transcription - polymerase chain reaction [QRT - PCR]). To gain a better understanding of the link among MTX, adenosine and regulation of cholesterol transport, we will compare adenosine receptor subtype expression (QRT - PCR) and distribution pattern ( immunocy tochemistry ) in PBMC obtained from DM and met abolic syndrome patients before and after MTX treatment. S PECIFIC A IM 2: Investigate the effect of LD MTX on the atherogenic properties of plasma from CIRT subjects with DM and metabolic syndrome. To evaluate t he impact of plasma milieu on cholesterol metaboli sm , we will assess differences in cholesterol transport protein expression in naïve monocytes/macrophages cultu red in plasma obtained pre - / post - MTX or placebo. We will document the effect of plasma on foam cell transformation (oil red O), neutral and polar lipid content (Nile red, enzymatic assay) and cholesterol efflux ( CholeFlux assay ).