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1.  Very Low Density Lipoprotein Cholesterol Associates with Coronary Artery Calcification in Type 2 Diabetes beyond Circulating Levels of Triglycerides 
Atherosclerosis  2014;236(2):244-250.
While recent genomic studies have focused attention on triglyceride (TG) rich lipoproteins in cardiovascular disease (CVD), little is known of very low-density lipoprotein cholesterol (VLDL-C) relationship with atherosclerosis and CVD. We examined, in a high-risk type-2 diabetic population, the association of plasma VLDL-C with coronary artery calcification (CAC).
The Penn Diabetes Heart Study (PDHS) is a cross-sectional study of CVD risk factors in type-2 diabetics (n=2118, mean age 59.1 years, 36.5% female, 34.1% Black). Plasma lipids including VLDL-C were calculated (n=1879) after ultracentrifugation.
In Tobit regression, VLDL-C levels were positively associated with increasing CAC after adjusting for age, race, gender, Framingham risk score, body mass index, C-reactive protein, exercise, medication and alcohol use, hemoglobin A1c, and diabetes duration [Tobit ratio (TR) and 95% confidence interval (CI) 0.38 (0.12–0.65), P=0.005] and even after inclusion of apolipoprotein B data [TR 0.31 (0.03–0.58), P=0.030]. Approximately 3-fold stronger effect was observed in women [TR 0.75 (0.16 – 1.34), P=0.013] than men [TR 0.20 (−0.10–0.50), P=0.189; gender interaction P=0.034]. Plasma VLDL-C was related more strongly to CAC scores than TG levels (e.g., Akaike information criteria of 7263.65 v. 7263.94) and had stronger CAC association in individuals with TGs >150mg/dl (TR 0.80, P=0.010) vs. those with TGs <150 mg/dl (TR 0.27, P=0.185).
In PDHS, VLDL-C is associated with CAC independent of established CVD risk factors, particularly in women, and may have value even beyond apolipoprotein B levels and in patients with elevated TGs.
PMCID: PMC4209900  PMID: 25105581
VLDL-C; triglycerides; subclinical atherosclerosis
2.  Leptin Resistance: A Possible Interface of Inflammation and Metabolism in Obesity-Related Cardiovascular Disease 
Nonstructured Abstract
Leptin is an adipocyte-derived hormone and cytokine that regulates energy balance through a wide range of functions, including several important to cardiovascular health. Increased circulating leptin, a marker of leptin resistance, is common in obesity and independently associated with insulin resistance and cardiovascular disease (CVD) in humans. Mechanisms of leptin resistance include genetic mutation, leptin self regulation, limited tissue access and cellular or circulating molecular regulation. Evidence suggests that central leptin resistance causes obesity and that obesity-induced leptin resistance injures numerous peripheral tissues, including liver, pancreas, platelets, vasculature, and myocardium. This metabolic- and inflammatory-mediated injury may result from either resistance to leptin’s action in selective tissues, or excess leptin action from adiposity associated hyperleptinemia. In this sense, the term “leptin resistance” encompasses a complex pathophysiological phenomenon. The leptin axis has functional interactions with elements of metabolism, such as insulin, and inflammation, including mediators of innate immunity such as interleukin-6. Leptin is even purported to physically interact with C-reactive protein (CRP), resulting in leptin resistance, which is particularly intriguing given CRP’s well-studied relationship to CVD. Given that plasma levels of leptin and inflammatory markers are correlated and also predict cardiovascular risk, it is conceivable that part of this risk may be mediated through leptin-resistance related insulin resistance, chronic inflammation, type II diabetes, hypertension, atherothrombosis and myocardial injury. Leptin resistance and its interactions with metabolic and inflammatory factors, therefore, represent potential novel diagnostic and therapeutic targets in obesity-related cardiovascular disease.
PMCID: PMC4556270  PMID: 18926322
obesity; leptin resistance; inflammation; atherosclerosis; cardiovascular disease
3.  Tissue-specific RNA-Seq in Human Evoked Inflammation Identifies Blood and Adipose LincRNA Signatures of Cardio-metabolic Diseases 
Inappropriate transcriptional activation of innate immunity is a pathologic feature of several cardio-metabolic disorders but little is known of inflammatory modulation of long intergenic non-coding RNAs (lincRNAs) in disease-relevant human tissues.
Approach and Results
We applied deep RNA sequencing (RNA-seq; >500 million filtered reads per sample) to blood and adipose during low-dose experimental endotoxemia (LPS) in a healthy human, with targeted replication in separate individuals undergoing endotoxemia (n=6), to identify inflammatory lincRNAs. A subset of these lincRNAs was examined for expression in adipocytes and monocytes, modulation in adipose in obesity, and overlap with genome-wide association study signals for inflammatory and cardio-metabolic traits. Of a stringent set of 4,284 lincRNAs, about 11–22% were expressed with 201 and 56 lincRNAs modulated by LPS in blood or adipose, respectively. Tissue-specific expression of a subset of six LPS-lincRNAs was replicated with LPS-modulation confirmed for all three expressed in blood and two of four expressed in adipose. The broader generalizabilty of findings in blood of subject A was confirmed by RNA-seq in seven additional subjects. We confirmed adipocytes and monocytes as potential cell sources of selective LPS-regulated lincRNAs and two of these, linc-DMRT2 (P=0.002) and linc-TP53I13 (P=0.01), were suppressed in adipose in obesity. Finally, we provide examples of LPS-modulated lincRNAs that overlap SNPs that are associated with cardiometabolic traits.
Our findings provide novel insights into tissue-level, inflammatory transcriptome regulation in cardio-metabolic diseases. These are complementary to more usual approaches limited to interrogation of DNA variations.
PMCID: PMC3966947  PMID: 24504737
Genomics; cardio-metabolic disease; RNA-seq; alternative splicing; linc-RNA
4.  Omega-3 polyunsaturated fatty acid supplementation and the response to evoked endotoxemia in healthy volunteers 
Fish oil-derived n-3 PUFA may improve cardiometabolic health through modulation of innate immunity; however, findings in clinical studies are conflicting. We hypothesized that n-3 PUFA supplementation would dose-dependently reduce the systemic inflammatory response to experimental endotoxemia in healthy humans.
Methods and Results
The Fenofibrate and omega-3 Fatty Acid Modulation of Endotoxemia (FFAME) study was an 8-week randomized double-blind trial of placebo or n-3 PUFA supplementation (Lovaza 465mg EPA + 375mg DHA) at “low” (1/day, 900mg) or “high” (4/day, 3,600mg) dose in healthy individuals (N=60; age 18–45; BMI 18–30; 43% Female; 65% European-, 20% African-, 15% Asian-ancestry) before a low-dose endotoxin challenge (LPS 0.6ng/kg intravenous bolus). The endotoxemia-induced temperature increase was significantly reduced with high-dose (P =0.03) but not low-dose EPA+DHA compared to placebo. Although there was no statistically significant impact of EPA+DHA on individual inflammatory responses (TNFα, IL-6, MCP-1, IL-1RA, IL-10, CRP, SAA), there was a pattern of lower responses across all biomarkers with high-dose (9 of 9 observed), but not low-dose EPA+DHA.
EPA+DHA at 3,600mg/day, but not 900mg/day, reduced fever and had a pattern of attenuated LPS-induction of plasma inflammatory markers during endotoxemia. Clinically and nutritionally relevant long-chain n-3 PUFA regimens may have specific, dose-dependent, anti-inflammatory actions.
PMCID: PMC3947472  PMID: 24190860
endotoxemia; fish oil; inflammation; LPS; n-3 PUFA
5.  Targeting adipose tissue via systemic gene therapy 
Gene therapy  2014;21(7):653-661.
Adipose tissue plays a critical role in energy and metabolic homeostasis, but it is challenging to adapt techniques to modulate adipose function in vivo. Here we develop an in vivo, systemic method of gene transfer specifically targeting adipose tissue using adeno-associated virus (AAV) vectors. We constructed AAV vectors containing CMV promoter regulated reporter genes, intravenously injected adult mice with vectors using multiple AAV serotypes, and determined that AAV2/8 best targeted adipose tissue. Altering vectors to contain adiponectin promoter/enhancer elements and liver specific microRNA-122 target sites restricted reporter gene expression to adipose tissue. As proof of efficacy, the leptin gene was incorporated into the adipose-targeted expression vector, package into AAV2/8, and administered intravenously to 9-10 week old ob/ob mice. Phenotypic changes were measured over an eight week period. Leptin mRNA and protein were expressed in adipose and leptin protein was secreted into plasma. Mice responded with reversal of weight gain, decreased hyperinsulinemia, and improved glucose tolerance. AAV2/8-mediated systemic delivery of an adipose-targeted expression vector can replace a gene lacking in adipose tissue and correct a mouse model of human disease, demonstrating experimental application and therapeutic potential in disorders of adipose.
PMCID: PMC4342115  PMID: 24830434
Gene Therapy; Adipose; AAV; Leptin; Obesity
6.  Differential Associations of Oral Glucose Tolerance Test–Derived Measures of Insulin Sensitivity and Pancreatic β-Cell Function With Coronary Artery Calcification and Microalbuminuria in Type 2 Diabetes 
Diabetes Care  2013;37(1):124-133.
We evaluated relationships of oral glucose tolerance testing (OGTT)–derived measures of insulin sensitivity and pancreatic β-cell function with indices of diabetes complications in a cross-sectional study of patients with type 2 diabetes who are free of overt cardiovascular or renal disease.
A subset of participants from the Penn Diabetes Heart Study (n = 672; mean age 59 ± 8 years; 67% male; 60% Caucasian) underwent a standard 2-h, 75-g OGTT. Insulin sensitivity was estimated using the Matsuda Insulin Sensitivity Index (ISI), and β-cell function was estimated using the Insulinogenic Index. Multivariable modeling was used to analyze associations between quartiles of each index with coronary artery calcification (CAC) and microalbuminuria.
The Insulinogenic Index and Matsuda ISI had distinct associations with cardiometabolic risk factors. The top quartile of the Matsuda ISI had a negative association with CAC that remained significant after adjusting for traditional cardiovascular risk factors (Tobit ratio −0.78 [95% CI −1.51 to −0.05]; P = 0.035), but the Insulinogenic Index was not associated with CAC. Conversely, the highest quartile of the Insulinogenic Index, but not the Matsuda ISI, was associated with lower odds of microalbuminuria (OR 0.52 [95% CI 0.30–0.91]; P = 0.022); however, this association was attenuated in models that included duration of diabetes.
Lower β-cell function is associated with microalbuminuria, a microvascular complication, while impaired insulin sensitivity is associated with higher CAC, a predictor of macrovascular complications. Despite these pathophysiological insights, the Matsuda ISI and Insulinogenic Index are unlikely to be translated into clinical use in type 2 diabetes beyond established clinical variables, such as obesity or duration of diabetes.
PMCID: PMC3867998  PMID: 23949560
7.  Importance of Evaluating Cell Cholesterol Influx With Efflux in Determining the Impact of Human Serum on Cholesterol Metabolism and Atherosclerosis 
Cholesterol efflux relates to cardiovascular disease but cannot predict cellular cholesterol mass changes. We asked whether influx and net flux assays provide additional insights.
Approach and Results
Adapt a bidirectional flux assay to cells where efflux has clinical correlates and examine the association of influx, efflux, and net flux to serum triglycerides (TGs). Apolipoprotein B–depleted (high-density lipoprotein-fraction) serum from individuals with unfavorable lipids (median [interquartile range]; high-density lipoprotein-cholesterol=39 [32–42], low-density lipoprotein-cholesterol=109 [97–137], TGs=258 [184–335] mg/dL; n=13) promoted greater ATP-binding cassette transporter A1–mediated [1,2-3H] cholesterol efflux (3.8±0.3%/4 hour versus 1.2±0.4%/4 hour; P<0.0001) from cyclic 3’,5’-amp(CTP-amp)-treated J774 macrophages than from individuals with favorable lipids (high-density lipoprotein-cholesterol=72 [58–88], low-density lipoprotein-cholesterol=111 [97–131], TGs=65 [56–69] mg/dL; n=10). Thus, high TGs associated with more ATP-binding cassette transporter A1 acceptors. Efflux of cholesterol mass (µg free cholesterol/mg cell protein per 8 hour) to serum was also higher (7.06±0.33 versus 5.83±0.48; P=0.04). However, whole sera from individuals with unfavorable lipids promoted more influx (5.14±0.65 versus 2.48±0.85; P=0.02) and lower net release of cholesterol mass (1.93±0.46 versus 3.36±0.47; P=0.04). The pattern differed when mass flux was measured using apolipoprotein B–depleted serum rather than serum. Although individuals with favorable lipids tended to have greater influx than those with unfavorable lipids, efflux to apolipoprotein B–depleted serum was markedly higher (6.81±0.04 versus 2.62±0.14; P<0.0001), resulting in an efflux:influx ratio of ≈3-fold. Thus both serum and apolipoprotein B–depleted serum from individuals with favorable lipids promoted greater net cholesterol mass release despite increased ATP-binding cassette transporter A1–mediated efflux in samples of individuals with high TGs/unfavorable lipids.
When considering the efficiency of serum specimens to modulate cell cholesterol content, both influx and efflux need to be measured.
PMCID: PMC4005807  PMID: 24202308
coronary artery disease; high-density lipoprotein-1
8.  Candidate Gene Association Study of Coronary Artery Calcification in Chronic Kidney Disease: Findings from the Chronic Renal Insufficiency Cohort Study 
To identify loci for coronary artery calcification (CAC) in patients with chronic kidney disease (CKD).
CKD is associated with increased CAC and subsequent coronary heart disease (CHD) but the mechanisms remain poorly defined. Genetic studies of CAC in CKD may provide a useful strategy for identifying novel pathways in CHD.
We performed a candidate gene study (~2,100 genes; ~50,000 SNPs) of CAC within the Chronic Renal Insufficiency Cohort (CRIC) Study (n=1,509; 57% European, 43% African ancestry). SNPs with preliminary evidence of association with CAC in CRIC were examined for association with CAC in PennCAC (n=2,560) and Amish Family Calcification Study (AFCS; n=784) samples. SNPs with suggestive replication were further analyzed for association with myocardial infarction (MI) in the Pakistan Risk of Myocardial Infarction study (PROMIS) (n=14,885).
Of 268 SNPs reaching P <5×10−4 for CAC in CRIC, 28 SNPs in 23 loci had nominal support (P <0.05 and in same direction) for CAC in PennCAC or AFCS. Besides chr9p21 and COL4A1, known loci for CHD, these included SNPs having reported GWAS association with hypertension (e.g., ATP2B1). In PROMIS, four of the 23 suggestive CAC loci (chr9p21, COL4A1, ATP2B1 and ABCA4) had significant associations with MI consistent with their direction of effect on CAC.
We identified several loci associated with CAC in CKD that also relate to MI in a general population sample. CKD imparts a high risk of CHD and may provide a useful setting for discovery of novel CHD genes and pathways.
PMCID: PMC3953823  PMID: 23727086
Coronary artery calcification (CAC); chronic kidney disease (CKD); Chronic Renal Insufficiency Cohort Study (CRIC); myocardial infarction (MI); risk factors; candidate genes; single nucleotide polymorphisms (SNPs)
9.  C-Reactive Protein Modifies the Association of Plasma Leptin With Coronary Calcium in Asymptomatic Overweight Individuals 
Obesity (Silver Spring, Md.)  2011;20(4):856-861.
Evidence suggests putative interactions of leptin and C-reactive protein (CRP) in the pathogenesis of adiposity-related atherosclerotic cardiovascular disease (CVD). Therefore, we investigated whether CRP levels modify the relationship of leptin levels with coronary artery calcium (CAC). We examined 1,460 asymptomatic individuals from two community-based cross-sectional studies coordinated at a single, university-based research center. We focused on subjects who were overweight or obese (BMI ≥25) given greater biologic plausibility in this setting. In multivariable CAC models, we analyzed the interaction of log-transformed plasma leptin levels with higher CRP levels as defined by three cut-points: two clinically based (2 mg/l, 3 mg/l) and one dataset specific (sex-specific upper quartile). The association of plasma leptin with CAC was modified by higher CRP regardless of cut-point (interaction term P values all <0.01 in fully adjusted models). Leptin levels were associated with CAC in those with high, but not low CRP levels (e.g., tobit ratio for a 1 unit increase in ln(leptin) (95% CI): 2.18 (1.29–3.66) if CRP level ≥3 mg/l; N = 461 vs. 0.94 (0.67–1.31) if CRP levels <3 mg/l; N = 999) in fully adjusted models. No interaction with CRP was present in control analyses with adiponectin, BMI and waist circumference. In conclusion, in asymptomatic overweight and obese adults, increased leptin levels were independently associated with increased CAC in the presence of high, but not low CRP levels, supporting a leptin-CRP interface in atherosclerosis risk.
PMCID: PMC4005808  PMID: 21738237
10.  Monocyte Mayhem: Do Subtypes Modulate Distinct Atherosclerosis Phenotypes? 
PMCID: PMC3670688  PMID: 22337925
editorial; monocytes; cardiovascular outcomes; inflammation
11.  Inflammation Modulates Human HDL Composition and Function in vivo 
Atherosclerosis  2012;222(2):390-394.
Inflammation may directly impair HDL functions, in particular reverse cholesterol transport (RCT), but limited data support this concept in humans.
Methods and Results
We employed low-dose human endotoxemia to assess the effects of inflammation on HDL and RCT-related parameters in vivo. Endotoxemia induced remodelling of HDL with depletion of pre-β1a HDL particles determined by 2-D gel electrophoresis (-32.2 ± 9.3% at 24h, p<0.05) as well as small (-23.0 ± 5.1%, p<0.01, at 24h) and medium (-57.6 ± 8.0% at 16h, p<0.001) HDL estimated by nuclear magnetic resonance (NMR). This was associated with induction of class II secretory phospholipase A2 (~36 fold increase) and suppression of lecithin:cholesterol acyltransferase activity (-20.8 ± 3.4% at 24h, p<0.01) and cholesterol ester transfer protein mass (-22.2 ± 6.8% at 24h, p<0.001). The HDL fraction, isolated following endotoxemia, had reduced capacity to efflux cholesterol in vitro from SR-BI and ABCA1, but not ABCG1 transporter cell models.
These data support the concept that “atherogenic-HDL dysfunction” and impaired RCT occur in human inflammatory syndromes, largely independent of changes in plasma HDL-C and ApoA-I levels.
PMCID: PMC3361641  PMID: 22456230
inflammation; atherosclerosis; cholesterol; lipoproteins; macrophages
13.  ABO Blood Groups and Cardiovascular Diseases 
ABO blood groups have been associated with various disease phenotypes, particularly cardiovascular diseases. Cardiovascular diseases are the most common causes of death in developed countries and their prevalence rate is rapidly growing in developing countries. There have been substantial historical associations between non-O blood group status and an increase in some cardiovascular disorders. Recent GWASs have identified ABO as a locus for thrombosis, myocardial infarction, and multiple cardiovascular risk biomarkers, refocusing attention on mechanisms and potential for clinical advances. As we highlight in this paper, more recent work is beginning to probe the molecular basis of the disease associations observed in these observational studies. Advances in our understanding of the physiologic importance of various endothelial and platelet-derived circulating glycoproteins are elucidating the mechanisms through which the ABO blood group may determine overall cardiovascular disease risk. The role of blood group antigens in the pathogenesis of various cardiovascular disorders remains a fascinating subject with potential to lead to novel therapeutics and prognostics and to reduce the global burden of cardiovascular diseases.
PMCID: PMC3485501  PMID: 23133757
14.  Peroxisome Proliferator–Activated Receptor-α Agonism With Fenofibrate Does Not Suppress Inflammatory Responses to Evoked Endotoxemia 
Data conflict with regard to whether peroxisome proliferator–activated receptor-α agonism suppresses inflammation in humans. We hypothesized that in healthy adults peroxisome proliferator–activated receptor-α agonism with fenofibrate would blunt the induced immune responses to endotoxin (lipopolysaccharide [LPS]), an in vivo model for the study of cardiometabolic inflammation.
Methods and Results
In the Fenofibrate and omega-3 Fatty Acid Modulation of Endotoxemia (FFAME) trial, 36 healthy volunteers (mean age 26±7 years, mean body mass index 24±3 kg/m2, 44% female, 72% white) were randomized to fenofibrate 145 mg or placebo daily. After 6 to 8 weeks of treatment, subjects underwent a low-dose LPS challenge. Clinical and blood measurements were collected at randomization, before LPS administration, and serially for 24 hours after LPS administration. We examined area under the curve for evoked responses by treatment group. Compared to placebo, but before LPS challenge, fenofibrate reduced total cholesterol and tended to decrease triglycerides, consistent with achieved therapeutic plasma levels of fenofibric acid. In the placebo group, LPS induced a modest inflammatory response with increased cytokines and chemokines (2- to 4-hour post-LPS 8-fold increase in tumor necrosis factor-α, 9-fold increase in interleukin-6, 9-fold increase in interleukin-10, and 10-fold increase in monocyte chemotactic protein-1; all P<0.001) and acute-phase reactants (24-hour post-LPS 15-fold increase in serum amyloid A and 9-fold increase in C-reactive protein; both P<0.001). Compared to placebo, however, fenofibrate did not significantly attenuate LPS-induced levels of plasma cytokines, chemokines, or acute-phase proteins.
These data suggest a lack of systemic antiinflammatory properties of fenofibrate at clinically relevant dosing in humans.
Clinical Trial Registration
URL: Unique identifier: NCT01048502. (J Am Heart Assoc. 2012;1:e002923 doi: 10.1161/JAHA.112.002923.)
PMCID: PMC3487364  PMID: 23130172
clinical trials; cytokines; endotoxemia; fenofibrate; inflammation
15.  Fractalkine Is a Novel Human Adipochemokine Associated With Type 2 Diabetes 
Diabetes  2011;60(5):1512-1518.
Leukocyte infiltration of adipose is a critical determinant of obesity-related metabolic diseases. Fractalkine (CX3CL1) and its receptor (CX3CR1) comprise a chemokine system involved in leukocyte recruitment and adhesion in atherosclerosis, but its role in adipose inflammation and type 2 diabetes is unknown.
CX3CL1 mRNA and protein were quantified in subcutaneous adipose and blood during experimental human endotoxemia and in lean and obese human adipose. CX3CL1 cellular source was probed in human adipocytes, monocytes, and macrophages, and CX3CL1-blocking antibodies were used to assess its role in monocyte-adipocyte adhesion. The association of genetic variation in CX3CR1 with metabolic traits was determined in a community-based sample. Finally, plasma CX3CL1 levels were measured in a case-control study of type 2 diabetes.
Endotoxemia induced adipose CX3CL1 mRNA (32.7-fold, P < 1 × 10−5) and protein (43-fold, P = 0.006). Obese subjects had higher CX3CL1 levels in subcutaneous adipose compared with lean (0.420 ± 0.387 vs. 0.228 ± 0.187 ng/mL, P = 0.04). CX3CL1 was expressed and secreted by human adipocytes and stromal vascular cells. Inflammatory cytokine induction of CX3CL1 in human adipocytes (27.5-fold mRNA and threefold protein) was completely attenuated by pretreatment with a peroxisome proliferator–activated receptor-γ agonist. A putative functional nonsynonymous single nucleotide polymorphism (rs3732378) in CX3CR1 was associated with adipose and metabolic traits, and plasma CX3CL1 levels were increased in patients with type 2 diabetes vs. nondiabetics (0.506 ± 0.262 vs. 0.422 ± 0.210 ng/mL, P < 0.0001).
CX3CL1-CX3CR1 is a novel inflammatory adipose chemokine system that modulates monocyte adhesion to adipocytes and is associated with obesity, insulin resistance, and type 2 diabetes. These data provide support for CX3CL1 as a diagnostic and therapeutic target in cardiometabolic disease.
PMCID: PMC3292325  PMID: 21525510
16.  Identification of ADAMTS7 as a novel locus for coronary atherosclerosis and association of ABO with myocardial infarction in the presence of coronary atherosclerosis: two genome-wide association studies 
Lancet  2011;377(9763):383-392.
We tested whether genetic factors distinctly contribute to either development of coronary atherosclerosis or, specifically, to myocardial infarction in existing coronary atherosclerosis.
We did two genome-wide association studies (GWAS) with coronary angiographic phenotyping in participants of European ancestry. To identify loci that predispose to angiographic coronary artery disease (CAD), we compared individuals who had this disorder (n=12 393) with those who did not (controls, n=7383). To identify loci that predispose to myocardial infarction, we compared patients who had angiographic CAD and myocardial infarction (n=5783) with those who had angiographic CAD but no myocardial infarction (n=3644).
In the comparison of patients with angiographic CAD versus controls, we identified a novel locus, ADAMTS7 (p=4·98×10−13). In the comparison of patients with angiographic CAD who had myocardial infarction versus those with angiographic CAD but no myocardial infarction, we identified a novel association at the ABO locus (p=7·62×10−9). The ABO association was attributable to the glycotransferase-deficient enzyme that encodes the ABO blood group O phenotype previously proposed to protect against myocardial infarction.
Our findings indicate that specific genetic predispositions promote the development of coronary atherosclerosis whereas others lead to myocardial infarction in the presence of coronary atherosclerosis. The relation to specific CAD phenotypes might modify how novel loci are applied in personalised risk assessment and used in the development of novel therapies for CAD.
The PennCath and MedStar studies were supported by the Cardiovascular Institute of the University of Pennsylvania, by the MedStar Health Research Institute at Washington Hospital Center and by a research grant from GlaxoSmithKline. The funding and support for the other cohorts contributing to the paper are described in the webappendix.
PMCID: PMC3297116  PMID: 21239051
17.  Gender Differences in the Association of C-Reactive Protein with Coronary Artery Calcium in Type-2 Diabetes 
Clinical endocrinology  2011;74(1):44-50.
Plasma C-reactive protein (CRP) is associated with cardiovascular disease (CVD) but effects may vary by gender and degree of CVD risk. Whether CRP has value as a CVD risk marker in type-2 diabetes (T2DM) is unclear. We examined whether CRP has gender differences in the association of coronary artery calcium (CAC) in diabetic and non diabetic samples without clinical CVD.
We performed cross-sectional analyses of gender influence on CRP association with CAC in the Penn Diabetes Heart Study (N = 1299 with T2DM), the Study of Inherited Risk of Coronary Atherosclerosis (N = 860 non diabetic subjects), and a combined sample.
Female gender was associated with higher plasma CRP in diabetic and non-diabetic samples after adjustment for covariates. There was a strong interaction by gender in the association of CRP with CAC (interaction p < 0.001). In diabetic women, CRP was associated with higher CAC even after further adjustment for age, race, medications, metabolic syndrome, Framingham risk score, and body mass index [Tobit ratio 1.60, 95% CI (1.03-2.47)]. Although this relationship was attenuated in non diabetic women, the combined sample maintained this association in fully adjusted models [1.44, 95% CI (1.13-1.83)]. There was no association of CRP with CAC in either diabetic or non diabetic men.
CRP may be a useful marker of cardiovascular risk in women, particularly in diabetic women who otherwise have no known CVD. Prospective studies are needed to better assess gender differences in CRP utility and the use of CRP in T2DM.
PMCID: PMC3005137  PMID: 20874770
Coronary artery calcium; C-reactive protein; Diabetes; Gender
18.  Resistin gene variation is associated with systemic inflammation but not plasma adipokine levels, metabolic syndrome or coronary atherosclerosis in nondiabetic Caucasians 
Clinical endocrinology  2008;70(5):698-705.
Resistin causes insulin resistance and diabetes in mice whereas in humans it is linked to inflammation and atherosclerosis. Few human genetic studies of resistin in inflammation and atherosclerosis have been performed. We hypothesized that the −420C>G putative gain-of-function resistin variant would be associated with inflammatory markers and atherosclerosis but not with metabolic syndrome or adipokines in humans.
Design and methods
We examined the association of three resistin polymorphisms, −852A>G, −420C>G and +157C>T, and related haplotypes with plasma resistin, cytokines, C-reactive protein (CRP), adipokines, plasma lipoproteins, metabolic syndrome and coronary artery calcification (CAC) in nondiabetic Caucasians (n = 851).
Resistin levels were higher, dose-dependently, with the −420G allele (CC 5·9 ± 2·7 ng/ml, GC 6·5 ± 4·0 ng/ml and GG 7·2 ± 4·8 ng/ml, trend P = 0·04) after age and gender adjustment [fold higher for GC + GG vs. CC; 1·07 (1·00–1·15), P < 0·05)]. The −852A>G single nucleotide polymorphism (SNP) was associated with higher soluble tumour necrosis factor-receptor 2 (sol-TNFR2) levels in fully adjusted models [1·06 (95% CI 1·01–1·11), P = 0·01)]. The estimated resistin haplotype (GGT) was associated with sol-TNFR2 (P = 0·04) and the AGT haplotype was related to CRP (P = 0·04) in the fully adjusted models. Resistin SNPs and haplotypes were not associated with body mass index (BMI), fasting glucose, insulin resistance, metabolic syndrome, adipokines or CAC scores.
Despite modest associations with plasma resistin and inflammatory biomarkers, resistin 5′ variants were not associated with metabolic parameters or coronary calcification. This suggests that resistin is an inflammatory cytokine in humans but has little influence on adiposity, metabolic syndrome or atherosclerosis.
PMCID: PMC3108432  PMID: 18710472
19.  Adipose Inflammation, Insulin Resistance, and Cardiovascular Disease 
Adiposity-associated inflammation and insulin resistance are strongly implicated in the development of type 2 diabetes and atherosclerotic cardiovascular disease. This article reviews the mechanisms of adipose inflammation, because these may represent therapeutic targets for insulin resistance and for prevention of metabolic and cardiovascular consequences of obesity. The initial insult in adipose inflammation and insulin resistance, mediated by macrophage recruitment and endogenous ligand activation of Toll-like receptors, is perpetuated through chemokine secretion, adipose retention of macrophages, and elaboration of pro-inflammatory adipocytokines. Activation of various kinases modulates adipocyte transcription factors, including peroxisome proliferator-activated receptor-γ and NFκB, attenuating insulin signaling and increasing adipocytokine and free fatty acid secretion. Inflammation retards adipocyte differentiation and further exacerbates adipose dysfunction and inflammation. Paracrine and endocrine adipose inflammatory events induce a local and systemic inflammatory, insulin-resistant state promoting meta-bolic dyslipidemia, type 2 diabetes, and cardiovascular disease. Developing therapeutic strategies that target both adipose inflammation and insulin resistance may help to prevent type 2 diabetes and cardiovascular disease in the emerging epidemic of obesity.
PMCID: PMC3088110  PMID: 18974244
type 2 diabetes; insulin resistance; adiposity-associated inflammation; adipose tissue; insulin; fatty acids
20.  Atheroprotective lipoprotein effects of a niacin-simvastatin combination compared to low- and high-dose simvastatin monotherapy 
American heart journal  2009;157(4):687.e1-687.e8.
Niacin has multiple lipoprotein effects that may provide cardiovascular benefit when added to statin monotherapy.
In this randomized, placebo-controlled trial (n = 75) of magnetic resonance imaging of carotid atherosclerosis, we performed a secondary comparison of combination niacin-statin (simvastatin 20 mg/Niacin-ER 2G [S20/N]) to monotherapy with moderate (20 mg [S20]) and high-dose (80 mg [S80]) simvastatin on lipids, apolipoproteins (apo), low density lipoprotein (LDL) and high density lipoprotein (HDL) particle subclasses, and inflammatory markers.
At baseline, average age was 71, 72% were male, 62.5% used statins, and average LDL-cholesterol was 111 mg/dL. At 12 months, S20/N, compared to S80, significantly reduced apoB (−36.6% vs −11.9%; P = .05) and lipoprotein(a) (−18% vs +3.5%; P = .001) and had at least an equivalent effect on LDL-cholesterol (−39.3% vs −24.3%; P = .24). The combination reduced the proportion of subjects with atherogenic LDL pattern-B (50% to 11.5%) compared to S80 (56% to 56%) (P = .01). Despite increases in plasma free fatty acids (+62.4%; F = 5.65, P = .005 vs S20 and S80), plasma triglycerides (−29.4%; F = 6.88, P = .002 vs S20 and S80), and very-low-density lipoprotein (−44.2%; F = 7.94, P < .001 vs S20 and S80), levels were reduced by S20/N. S20/N increased HDL-cholesterol levels (+18.1%) as compared to S20 (0%) and S80 (+5.9%) (P < .001 vs both statin arms), largely due to an increase in HDL particle size (+4.6%; P = .01 vs both statin arms).
We demonstrate that full-dose niacin/moderate-dose simvastatin combination has sustained benefits on atherogenic apoB lipoproteins, at least comparable to high-dose simvastatin, while also raising HDL-cholesterol. Results of large clinical trials will inform whether niacin-statin combinations reduce cardiovascular disease events.
PMCID: PMC3088112  PMID: 19332196
21.  Adipocyte Modulation of High-Density Lipoprotein Cholesterol 
Circulation  2010;121(11):1347-1355.
Adipose harbors a large depot of free cholesterol. However, a role for adipose in cholesterol lipidation of HDL in vivo is not established. We present the first evidence that adipocytes support transfer of cholesterol to HDL in vivo as well as in vitro and implicate ABCA1 and SR-BI, but not ABCG1, cholesterol transporters in this process.
Methods and Results
Cholesterol efflux from wild-type (WT), ABCA1−/−, SR-BI−/− and ABCG1−/− adipocytes to apoA-I and HDL3 were measured in vitro. 3T3L1-adipocytes, labeled with 3H-cholesterol, were injected intraperitoneally (IP) into WT, apoA-I transgenic and apoA-I−/− mice and tracer movement onto plasma HDL monitored. Identical studies were performed with labeled WT, ABCA1−/− or SR-BI−/− mouse-embryonic-fibroblast (MEF) adipocytes. The effect of TNFα on transporter expression and cholesterol efflux was monitored during adipocyte differentiation. Cholesterol efflux to apoA-I and HDL3 was impaired in ABCA1−/− and SR-BI−/− adipocytes respectively, with no effect observed in ABCG1−/− adipocytes. Injection IP of labeled 3T3L1-adipocytes resulted in increased HDL-associated 3H-cholesterol in apoA-I transgenic mice but reduced levels in apoA-I −/− animals. Injection IP of labeled ABCA1−/− or SR-BI−/− adipocytes reduced plasma counts relative to their respective controls. TNFα reduced both ABCA1 and SR-BI expression and impaired cholesterol efflux from partially-differentiated adipocytes.
These data suggest a novel metabolic function of adipocytes in promoting cholesterol transfer to HDL in vivo and implicate adipocyte SR-BI and ABCA1, but not ABCG1, in this process. Further, adipocyte modulation of HDL may be impaired in adipose inflammatory disease states such as type-2 diabetes.
PMCID: PMC2925122  PMID: 20212278
cholesterol; lipoproteins; adipocytes; atherosclerosis; inflammation
22.  Association of Lower Plasma Fetuin-A Levels With Peripheral Arterial Disease in Type 2 Diabetes 
Diabetes Care  2009;33(2):408-410.
Fetuin-A is an inhibitor of vascular calcification and a mediator of insulin resistance. This study evaluated the association of plasma fetuin-A and peripheral arterial disease (PAD).
A total of 738 individuals with type 2 diabetes (mean age 58.7 years, 37.1% female) without known cardiovascular or kidney disease were included in this cross-sectional analysis.
Subjects with PAD had a significantly lower fetuin-A (264.3 vs. 293.4 ng/dl, P < 0.001). In multivariable analysis, a 1-SD decrease in fetuin-A increased the odds of PAD (odds ratio 1.6, P = 0.02). Subgroup analysis revealed an increased odds even in subjects with glomerular filtration rate >80 (odds ratio 1.9, P = 0.05) or high-sensitivity C-reactive protein <3 mg/dl (odds ratio 2.7, P = 0.002).
Lower circulating fetuin-A is associated with PAD in type 2 diabetes beyond traditional and novel cardiovascular risk factors. Our findings suggest a potentially unique role for fetuin-A deficiency as a biomarker of PAD in patients with type 2 diabetes.
PMCID: PMC2809293  PMID: 19910501
23.  Experimental Endotoxemia Induces Adipose Inflammation and Insulin Resistance in Humans 
Diabetes  2009;59(1):172-181.
An emerging model of metabolic syndrome and type 2 diabetes is of adipose dysfunction with leukocyte recruitment into adipose leading to chronic inflammation and insulin resistance (IR). This study sought to explore potential mechanisms of inflammatory-induced IR in humans with a focus on adipose tissue.
We performed a 60-h endotoxemia protocol (3 ng/kg intravenous bolus) in healthy adults (n = 20, 50% male, 80% Caucasian, aged 27.3 ± 4.8 years). Before and after endotoxin, whole-blood sampling, subcutaneous adipose biopsies, and frequently sampled intravenous glucose tolerance (FSIGT) testing were performed. The primary outcome was the FSIGT insulin sensitivity index (Si). Secondary measures included inflammatory and metabolic markers and whole-blood and adipose mRNA and protein expression.
Endotoxemia induced systemic IR as demonstrated by a 35% decrease in Si (3.17 ± 1.66 to 2.06 ± 0.73 × 10−4 [μU · ml−1 · min−1], P < 0.005), while there was no effect on pancreatic β-cell function. In adipose, endotoxemia suppressed insulin receptor substrate-1 and markedly induced suppressor of cytokine signaling proteins (1 and 3) coincident with local activation of innate (interleukin-6, tumor necrosis factor) and adaptive (monocyte chemoattractant protein-1 and CXCL10 chemokines) inflammation. These changes are known to attenuate insulin receptor signaling in model systems.
We demonstrate, for the first time in humans, that acute inflammation induces systemic IR following modulation of specific adipose inflammatory and insulin signaling pathways. It also provides a rationale for focused mechanistic studies and a model for human proof-of-concept trials of novel therapeutics targeting adipose inflammation in IR and related consequences in humans.
PMCID: PMC2797919  PMID: 19794059
24.  Gene Profiling of Human Adipose Tissue During Evoked Inflammation In Vivo 
Diabetes  2009;58(10):2211-2219.
Adipose inflammation plays a central role in obesity-related metabolic and cardiovascular complications. However, few human adipose-secreted proteins are known to mediate these processes. We hypothesized that microarray mRNA profiling of human adipose during evoked inflammation could identify novel adipocytokines.
Healthy human volunteers (n = 14) were treated with intravenous endotoxin (3 ng/kg lipopolysaccharide [LPS]) and underwent subcutaneous adipose biopsies before and after LPS. On Affymetrix U133Plus 2.0 arrays, adipose mRNAs modulated >1.5-fold (with P < 0.00001) were selected. SignalP 3.0 and SecretomeP 2.0 identified genes predicted to encode secreted proteins. Of these, 86 candidates were chosen for validation in adipose from an independent human endotoxemia protocol (N = 7, with 0.6 ng/kg LPS) and for exploration of cellular origin in primary human adipocytes and macrophages in vitro.
Microarray identified 776 adipose genes modulated by LPS; 298 were predicted to be secreted. Of detectable prioritized genes, 82 of 85 (96% [95% CI 90–99]) were upregulated (fold changes >1.0) during the lower-dose (LPS 0.6 ng/kg) validation study and 51 of 85 (59% [49–70]) were induced greater than 1.5-fold. Treatment of primary adipocytes with LPS and macrophage polarization to M1 proinflammatory phenotype increased expression by 1.5-fold for 58 and 73% of detectable genes, respectively.
We demonstrate that evoked inflammation of human adipose in vivo modulated expression of multiple genes likely secreted by adipocytes and monocytes. These included established adipocytokines and chemokines implicated in recruitment and activation of lymphocytes, adhesion molecules, antioxidants, and several novel genes with unknown function. Such candidates may represent biomarkers and therapeutic targets for obesity-related complications.
PMCID: PMC2750231  PMID: 19581417
25.  Apolipoprotein B but not LDL Cholesterol Is Associated With Coronary Artery Calcification in Type 2 Diabetic Whites 
Diabetes  2009;58(8):1887-1892.
Evidence favors apolipoprotein B (apoB) over LDL cholesterol as a predictor of cardiovascular events, but data are lacking on coronary artery calcification (CAC), especially in type 2 diabetes, where LDL cholesterol may underestimate atherosclerotic burden. We investigated the hypothesis that apoB is a superior marker of CAC relative to LDL cholesterol.
We performed cross-sectional analyses of white subjects in two community-based studies: the Penn Diabetes Heart Study (N = 611 type 2 diabetic subjects, 71.4% men) and the Study of Inherited Risk of Coronary Atherosclerosis (N = 803 nondiabetic subjects, 52.8% men) using multivariate analysis of apoB and LDL cholesterol stratified by diabetes status.
In type 2 diabetes, apoB was associated with CAC after adjusting for age, sex, and medications [Tobit regression ratio of increased CAC for 1-SD increase in apoB; 1.36 (95% CI 1.06–1.75), P = 0.016] whereas LDL cholesterol was not [1.09 (0.85–1.41)]. In nondiabetic subjects, both were associated with CAC [apoB 1.65 (1.38–1.96), P < 0.001; LDL cholesterol 1.56 (1.30–1.86), P < 0.001]. In combined analysis of diabetic and nondiabetic subjects, apoB provided value in predicting CAC scores beyond LDL cholesterol, total cholesterol, the total cholesterol/HDL cholesterol and triglyceride/HDL cholesterol ratios, and marginally beyond non-HDL cholesterol.
Plasma apoB, but not LDL cholesterol, levels were associated with CAC scores in type 2 diabetic whites. ApoB levels may be particularly useful in assessing atherosclerotic burden and cardiovascular risk in type 2 diabetes.
PMCID: PMC2712798  PMID: 19491209

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