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1.  Adipose Modulation of High-Density Lipoprotein Cholesterol 
Circulation  2011;124(15):1602-1605.
PMCID: PMC3928975  PMID: 21986773
Editorials; high-density lipoprotein cholesterol; lipoproteins; obesity; adipose tissue; Reverse Cholesterol Transport
2.  Cardiovascular Risk Factors and Mitral Annular Calcification in Type 2 Diabetes 
Atherosclerosis  2012;226(2):419-424.
Mitral annular calcification (MAC) is a degenerative process of the mitral fibrous annulus associated with cardiac disease and stroke. Although thought to be more prevalent in type 2 diabetes (T2DM), MAC remains poorly characterized in this population, due to confounding by renal and cardiac disease. Our goal was to study the risk factors for MAC in asample of T2DM subjects without renal and cardiac disease.
The Penn Diabetes Heart Study (PDHS) is a cross-sectional study of diabetic individuals without clinical cardiovascular or renal disease. We quantified and analyzed MAC Agatston scores in baseline cardiac CTs from 1753 individuals. Logistic and tobit regression were used to assess MAC’s relationship with risk factors and coronary artery calcification (CAC).
MAC was present in 12.0% of -subjects, with a median Agatston score of 72.3 [Interquartile range (22.2 256.9)]. Older age, diabetes female gender, Caucasian race, and longer duration were independently associated with both the presence and extent MAC even after controlling for the CAC; hypertension, hyperlipidemia, comorbidities however, tobacco use, CRP levels, and other were not associated. CAC was strongly associated with MAC [OR of 4.0, (95% CI 2.4-6.6)] in multivariable models.
Age, AC female gender, Caucasian race, and diabetes duration were associated with the presence and extent of MAC in T2DM subjects, independent of CAC, which was also strongly associated with MAC. These data suggest that additional mechanisms for MAC formation in diabetics may exist which are distinct from those related to generalized atherosclerosis and deserve further investigation.
PMCID: PMC3568504  PMID: 23273961
Diabetes; Mitral Annular Calcification; Coronary Heart Disease; Risk Factors
3.  PennSeq: accurate isoform-specific gene expression quantification in RNA-Seq by modeling non-uniform read distribution 
Nucleic Acids Research  2013;42(3):e20.
Correctly estimating isoform-specific gene expression is important for understanding complicated biological mechanisms and for mapping disease susceptibility genes. However, estimating isoform-specific gene expression is challenging because various biases present in RNA-Seq (RNA sequencing) data complicate the analysis, and if not appropriately corrected, can affect isoform expression estimation and downstream analysis. In this article, we present PennSeq, a statistical method that allows each isoform to have its own non-uniform read distribution. Instead of making parametric assumptions, we give adequate weight to the underlying data by the use of a non-parametric approach. Our rationale is that regardless what factors lead to non-uniformity, whether it is due to hexamer priming bias, local sequence bias, positional bias, RNA degradation, mapping bias or other unknown reasons, the probability that a fragment is sampled from a particular region will be reflected in the aligned data. This empirical approach thus maximally reflects the true underlying non-uniform read distribution. We evaluate the performance of PennSeq using both simulated data with known ground truth, and using two real Illumina RNA-Seq data sets including one with quantitative real time polymerase chain reaction measurements. Our results indicate superior performance of PennSeq over existing methods, particularly for isoforms demonstrating severe non-uniformity. PennSeq is freely available for download at
PMCID: PMC3919567  PMID: 24362841
4.  Risk Factors for Coronary Artery Calcium Among Patients with Chronic Kidney Disease (From the Chronic Renal Insufficiency Cohort Study) 
The American journal of cardiology  2012;110(12):1735-1741.
Cardiovascular disease is the leading cause of death in patients with chronic kidney disease (CKD). We examined the cross-sectional association between novel risk factors and coronary artery calcium (CAC) measured by electron-beam computed tomography or multidetector computed tomography among 2,018 patients with CKD. Based on total Agatston scores, participants were classified as no (0), moderate (>0–100) or high (>100) CAC. After adjustment for age, sex, race, study sites, cigarette smoking, prior cardiovascular disease, hypertension, and diabetes, use of lipid-lowering drugs, body-mass index, waist circumference, and cystatin C, several novel risk factors were significantly associated with high CAC. For example, odds ratios (95% confidence interval) of high CAC associated with one standard deviation higher levels of risk factors were 1.20 (1.04, 1.38) for serum calcium, 1.21 (1.04, 1.41) for serum phosphate, 0.83 (0.71, 0.97) for log (total parathyroid hormone), 1.21 (1.03, 1.43) for log (HOMA-insulin resistance), and 1.23 (1.04, 1.45) for hemoglobin A1c. Additionally, the multivariable-adjusted odds ratio for one standard deviation higher level of cystatin C was 1.31 (1.14, 1.50). Serum high-sensitive C-reactive protein, interleukin-6, tumor necrosis factor-α, and homocysteine were not statistically significantly associated with high CAC. In conclusion, these data indicate that abnormal calcium and phosphate metabolism, insulin resistance, and declined kidney function were associated with the prevalence of high CAC independent of traditional risk factors in patients with CKD. Further studies are warranted to examine the causal effect of these risk factors on CAC in CKD patients.
PMCID: PMC3511639  PMID: 22980963
calcium; chronic kidney disease; coronary artery calcium; cystatin C; insulin-resistance; phosphate; total parathyroid hormone
5.  Fibroblast growth factor 23 is not associated with and does not induce arterial calcification 
Kidney international  2013;83(6):1159-1168.
Elevated fibroblast growth factor 23 (FGF23) is associated with cardiovascular disease in patients with chronic kidney disease. As a potential mediating mechanism, FGF23 induces left ventricular hypertrophy; however, its role in arterial calcification is less clear. In order to study this we quantified coronary artery and thoracic aorta calcium by computed tomography in 1501 patients from the Chronic Renal Insufficiency Cohort (CRIC) study within a median of 376 days (interquartile range 331 to 420 days) of baseline. Baseline plasma FGF23 was not associated with prevalence or severity of coronary artery calcium after multivariable adjustment. In contrast, higher serum phosphate levels were associated with prevalence and severity of coronary artery calcium, even after adjustment for FGF23. Neither FGF23 nor serum phosphate were consistently associated with thoracic aorta calcium. We could not detect mRNA expression of FGF23 or its co-receptor, klotho, in human or mouse vascular smooth muscle cells, or normal or calcified mouse aorta. Whereas elevated phosphate concentrations induced calcification in vitro, FGF23 had no effect on phosphate uptake or phosphate-induced calcification regardless of phosphate concentration or even in the presence of soluble klotho. Thus, in contrast to serum phosphate, FGF23 is not associated with arterial calcification and does not promote calcification experimentally. Hence, phosphate and FGF23 promote cardiovascular disease through distinct mechanisms.
PMCID: PMC3672330  PMID: 23389416
phosphate; fibroblast growth factor 23; vascular calcification; vascular smooth muscle; chronic kidney disease
6.  Abnormal Lipoprotein Particles and Cholesterol Efflux Capacity in Patients with Psoriasis 
Atherosclerosis  2012;224(1):218-221.
Psoriasis is a Th-1/17 mediated inflammatory disease associated with increased risk of cardiovascular disease (CVD). Inflammation may modulate lipoprotein particle number and directly impair HDL functions, in particular reverse cholesterol transport (RCT). We sought to study how chronic in vivo inflammation modulates lipoprotein particle composition using nuclear magnetic resonance spectroscopy (NMR) and HDL efflux in psoriasis.
Methods and Results
We prospectively enrolled a consecutive sample of patients with psoriasis (n=122) and compared lipoprotein and metabolic risk factors to patients without psoriasis (n=134). Fasting lipids, insulin, glucose were measured by standard assays, and lipoprotein concentration and size were measured by NMR. In a random subset (n=100 each group), HDL efflux capacity was quantified using a validated ex vivo system involving the incubation of macrophages with apolipoprotein B-depleted serum from patients. Traditional lipid concentrations were similar in both groups except for HDL concentration which was lower in psoriasis (43 mg/dL (36–58) vs 50 (42–62), p<0.01). However, NMR showed an atherogenic profile in psoriasis similar to that observed in diabetes, with significant increase in LDL particle concentration [1210.5 (1002–1498) vs 1115 (935–1291), p=0.03] with decrease in LDL size [20.6 (20.3–21.1) vs 21.3 (20.6–21.1), p<0.001] beyond CV risk factors and HOMA-IR (p=0.001). Finally, HDL efflux capacity was lower in psoriasis compared to controls in fully adjusted models (beta −0.14, p=0.001).
These data support a more atherogenic lipoprotein profile by NMR and decreased HDL efflux capacity in psoriasis patients compared to controls beyond CVD risk factors. The abnormal lipoprotein particle composition and HDL efflux capacity in psoriasis may provide a link between psoriasis and CVD.
PMCID: PMC3693845  PMID: 22858285
inflammation; atherosclerosis; HDL efflux; cholesterol; lipoprotein particles
7.  Emerging diagnostic and therapeutic molecular imaging applications in vascular disease 
Assessment of vascular disease has evolved from mere indirect and direct measurements of luminal stenosis to sophisticated imaging methods to depict millimeter structural changes of the vasculature. In the near future, the emergence of multimodal molecular imaging strategies may enable robust therapeutic and diagnostic (‘theragnostic’) approaches to vascular diseases that comprehensively consider structural, functional, biological and genomic characteristics of the disease in individualized risk assessment, early diagnosis and delivery of targeted interventions. This review presents a summary of recent preclinical and clinical developments in molecular imaging and theragnostic applications covering diverse atherosclerosis events such as endothelial activation, macrophage infammatory activity, plaque neovascularization and arterial thrombosis. The main focus is on molecular targets designed for imaging platforms commonly used in clinical medicine including magnetic resonance, computed tomography and positron emission tomography. A special emphasis is given to vascular ultrasound applications, considering the important role this imaging platform plays in the clinical and research practice of the vascular medicine specialty.
PMCID: PMC3750985  PMID: 21310769
atherosclerosis plaque; imaging based therapeutic delivery systems; molecular imaging; theragnostics; thrombosis; vascular ultrasound
8.  Evaluating the Impact of Sequencing Depth on Transcriptome Profiling in Human Adipose 
PLoS ONE  2013;8(6):e66883.
Recent advances in RNA sequencing (RNA-Seq) have enabled the discovery of novel transcriptomic variations that are not possible with traditional microarray-based methods. Tissue and cell specific transcriptome changes during pathophysiological stress in disease cases versus controls and in response to therapies are of particular interest to investigators studying cardiometabolic diseases. Thus, knowledge on the relationships between sequencing depth and detection of transcriptomic variation is needed for designing RNA-Seq experiments and for interpreting results of analyses. Using deeply sequenced Illumina HiSeq 2000 101 bp paired-end RNA-Seq data derived from adipose of a healthy individual before and after systemic administration of endotoxin (LPS), we investigated the sequencing depths needed for studies of gene expression and alternative splicing (AS). In order to detect expressed genes and AS events, we found that ∼100 to 150 million (M) filtered reads were needed. However, the requirement on sequencing depth for the detection of LPS modulated differential expression (DE) and differential alternative splicing (DAS) was much higher. To detect 80% of events, ∼300 M filtered reads were needed for DE analysis whereas at least 400 M filtered reads were necessary for detecting DAS. Although the majority of expressed genes and AS events can be detected with modest sequencing depths (∼100 M filtered reads), the estimated gene expression levels and exon/intron inclusion levels were less accurate. We report the first study that evaluates the relationship between RNA-Seq depth and the ability to detect DE and DAS in human adipose. Our results suggest that a much higher sequencing depth is needed to reliably identify DAS events than for DE genes.
PMCID: PMC3691247  PMID: 23826166
9.  Monocyte Mayhem: Do Subtypes Modulate Distinct Atherosclerosis Phenotypes? 
PMCID: PMC3670688  PMID: 22337925
editorial; monocytes; cardiovascular outcomes; inflammation
10.  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
11.  Inflammation induces fibrinogen nitration in experimental human endotoxemia 
Free radical biology & medicine  2009;47(8):1140-1146.
Elevated plasma fibrinogen is a prothrombotic risk factor for cardiovascular disease (CVD). Recent small studies report that fibrinogen oxidative modifications, specifically tyrosine residue nitration, can occur in inflammatory states and may modify fibrinogen function. HDL cholesterol is inversely related to CVD and suggested to reduce the oxidation of LDL cholesterol, but whether these antioxidant functions extend to fibrinogen modifications is unknown. We used a recently validated ELISA to quantify nitrated fibrinogen during experimental human endotoxemia (N=23) and in a cohort of healthy adults (N=361) who were characterized for inflammatory and HDL parameters as well as subclinical atherosclerosis measures, carotid artery intima-medial thickness (IMT) and coronary artery calcification (CAC). Fibrinogen nitration increased following endotoxemia and directly correlated with accelerated ex vivo plasma clotting velocity. In the observational cohort, nitrated fibrinogen was associated with levels of CRP and serum amyloid A. Nitrated fibrinogen levels were not lower with increasing HDL cholesterol and did not associate with IMT and CAC. In humans, fibrinogen nitration was induced during inflammation and was correlated with markers of inflammation and clotting function but not HDL cholesterol or subclinical atherosclerosis in our modest sample. Inflammation-induced fibrinogen nitration may be a risk factor for promoting CVD events.
PMCID: PMC3651370  PMID: 19631267
Fibrinogen; Nitration; Intima-medial thickness; Coronary artery calcification; High-density lipoprotein (HDL)
12.  Race and gender variation in response to evoked inflammation 
Race- and gender-variation in innate immunity may contribute to demographic differences in inflammatory and cardiometabolic disease; yet their influence on dynamic responses during inflammatory stress is poorly understood. Our objective was to examine race and gender influence on the response to experimental endotoxemia.
The Genetics of Evoked Responses to Niacin and Endotoxemia (GENE) study was designed to investigate regulation of inflammatory and metabolic responses during low-grade endotoxemia (LPS 1 ng/kg intravenously) in healthy individuals (median age 24, IQR=7) of European (EA; n=193, 47% female) and African ancestry (AA; n=101, 59% female).
Baseline clinical, metabolic, and inflammatory biomarkers by race and gender were consistent with epidemiological literature; pre-LPS cytokines (e.g. median (IQR) IL-6, 2.7 (2) vs.2.1 (2) pg/ml, P=0.001) were higher in AA than EA. In contrast, acute cytokine responses during endotoxemia were lower in AA than EA (e.g. median (IQR) peak IL-1RA, 30 (38) vs.43 (45) ng/ml P=0.002) as was the induction of hepatic acute-phase proteins (e.g. median (IQR) peak CRP 12.9 (9) vs.17.4 (12) mg/L P=0.005). Further, baseline levels of cytokines were only weakly correlated with peak inflammatory responses (all rs <0.2) both in AA and in EA. There were less pronounced and less consistent differences in the response by gender, with males having a higher AUC for CRP response compared to females (median (IQR) AUC: 185 (112) vs. 155 (118), P=0.02).
We observed lower levels of evoked inflammation in response to endotoxin in AA compared with EA, despite similar or higher baseline levels of inflammatory markers in AA. Our data also suggest that levels of inflammatory biomarkers measured in epidemiological settings might not predict the degree of acute stress-response or risk of diseases characterized by activation of innate immunity.
Trial registration
FDA registration number NCT00953667
PMCID: PMC3636014  PMID: 23497455
13.  Translational studies of lipoprotein-associated phospholipase A2 in inflammation and atherosclerosis 
To examine the role of lipoprotein-associated phospholipase A2 (Lp-PLA2/PLA2G7) in human inflammation and coronary atherosclerosis.
Lp-PLA2 has emerged as a potential therapeutic target in coronary heart disease (CHD). Data supporting Lp-PLA2 are indirect and confounded by species differences; whether Lp-PLA2 is causal in CHD remains in question.
We examined inflammatory regulation of Lp-PLA2 during experimental endotoxemia in human, probed the source of Lp-PLA2 in human leukocytes under inflammatory conditions, and assessed the relationship of variation in PLA2G7, the gene encoding Lp-PLA2, with coronary artery calcification (CAC).
In contrast to circulating TNFα and CRP, blood and monocyte Lp-PLA2 mRNA decreased transiently, and plasma Lp-PLA2 mass declined modestly during endotoxemia. In vitro, Lp-PLA2 expression increased dramatically during human monocyte to macrophage differentiation and further in inflammatory macrophages and foam like-cells. Despite only a marginal association of SNPs in PLA2G7 with Lp-PLA2 activity or mass, numerous PLA2G7 SNPs were associated with CAC. In contrast, several SNPs in CRP were significantly associated with plasma CRP levels but had no relation with CAC.
Circulating Lp-PLA2 did not increase during acute phase response in human, while inflammatory macrophages and foam cells, but not circulating monocytes, are major leukocyte sources of Lp-PLA2. Common genetic variation in PLA2G7 is associated with sub-clinical coronary atherosclerosis. These data link Lp-PLA2 to atherosclerosis in human while highlighting the challenge in using circulating Lp-PLA2 as a biomarker of Lp-PLA2 actions in the vasculature.
PMCID: PMC3285416  PMID: 22340269
14.  Mixed Modeling of Meta-Analysis P-Values (MixMAP) Suggests Multiple Novel Gene Loci for Low Density Lipoprotein Cholesterol 
PLoS ONE  2013;8(2):e54812.
Informing missing heritability for complex disease will likely require leveraging information across multiple SNPs within a gene region simultaneously to characterize gene and locus-level contributions to disease phenotypes. To this aim, we introduce a novel strategy, termed Mixed modeling of Meta-Analysis P-values (MixMAP), that draws on a principled statistical modeling framework and the vast array of summary data now available from genetic association studies, to test formally for locus level association. The primary inputs to this approach are: (a) single SNP level p-values for tests of association; and (b) the mapping of SNPs to genomic regions. The output of MixMAP is comprised of locus level estimates and tests of association. In application of MixMAP to summary data from the Global Lipids Gene Consortium, we suggest twelve new loci (PKN, FN1, UGT1A1, PPARG, DMDGH, PPARD, CDK6, VPS13B, GAD2, GAB2, APOH and NPC1) for low-density lipoprotein cholesterol (LDL-C), a causal risk factor for cardiovascular disease and we also demonstrate the potential utility of MixMAP in small data settings. Overall, MixMAP offers novel and complementary information as compared to SNP-based analysis approaches and is straightforward to implement with existing open-source statistical software tools.
PMCID: PMC3566142  PMID: 23405096
15.  Genetic Basis of Atherosclerosis: Insights from Mice and Humans 
Circulation Research  2012;110(2):337-355.
Atherosclerosis is a complex and heritable disease involving multiple cell types and the interactions of many different molecular pathways. The genetic and molecular mechanisms of atherosclerosis have in part been elucidated by mouse models; at least 100 different genes have been shown to influence atherosclerosis in mice. Importantly, unbiased genome-wide association studies have recently identified a number of novel loci robustly associated with atherosclerotic coronary artery disease (CAD). Here we review the genetic data elucidated from mouse models of atherosclerosis, as well as significant associations for human CAD. Furthermore, we discuss in greater detail some of these novel human CAD loci. The combination of mouse and human genetics has the potential to identify and validate novel genes that influence atherosclerosis, some of which may be candidates for new therapeutic approaches.
PMCID: PMC3357004  PMID: 22267839
CAD; Lipids; Mice; GWAS; Genome-wide
17.  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
18.  Genome-wide association study for serum urate concentrations and gout among African Americans identifies genomic risk loci and a novel URAT1 loss-of-function allele 
Human Molecular Genetics  2011;20(20):4056-4068.
Serum urate concentrations are highly heritable and elevated serum urate is a key risk factor for gout. Genome-wide association studies (GWAS) of serum urate in African American (AA) populations are lacking. We conducted a meta-analysis of GWAS of serum urate levels and gout among 5820 AA and a large candidate gene study among 6890 AA and 21 708 participants of European ancestry (EA) within the Candidate Gene Association Resource Consortium. Findings were tested for replication among 1996 independent AA individuals, and evaluated for their association among 28 283 EA participants of the CHARGE Consortium. Functional studies were conducted using 14C-urate transport assays in mammalian Chinese hamster ovary cells. In the discovery GWAS of serum urate, three loci achieved genome-wide significance (P< 5.0 × 10−8): a novel locus near SGK1/SLC2A12 on chromosome 6 (rs9321453, P= 1.0 × 10−9), and two loci previously identified in EA participants, SLC2A9 (P= 3.8 × 10−32) and SLC22A12 (P= 2.1 × 10−10). A novel rare non-synonymous variant of large effect size in SLC22A12, rs12800450 (minor allele frequency 0.01, G65W), was identified and replicated (beta −1.19 mg/dl, P= 2.7 × 10−16). 14C-urate transport assays showed reduced urate transport for the G65W URAT1 mutant. Finally, in analyses of 11 loci previously associated with serum urate in EA individuals, 10 of 11 lead single-nucleotide polymorphisms showed direction-consistent association with urate among AA. In summary, we identified and replicated one novel locus in association with serum urate levels and experimentally characterize the novel G65W variant in URAT1 as a functional allele. Our data support the importance of multi-ethnic GWAS in the identification of novel risk loci as well as functional variants.
PMCID: PMC3177647  PMID: 21768215
19.  Relationship of Estimated GFR and Coronary Artery Calcification in the (CRIC) Chronic Renal Insufficiency Cohort Study 
Coronary artery calcification (CAC) is associated with increased mortality risk in the general population. Although individuals with chronic kidney disease (CKD) are at markedly increased mortality risk, the incidence, prevalence, and prognosis of CAC in CKD is not well-understood.
Study Design
Cross-sectional observational study.
Setting and Participants
Analysis of 1,908 participants who underwent coronary calcium scanning as part of the multi-ethnic CRIC (Chronic Renal Insufficiency Cohort) Study.
Estimated glomerular filtration rate (eGFR) computed using the Modification of Diet in Renal Disease (MDRD) Study equation, stratified by race, sex and diabetic status. eGFR was treated as a continous variable and a categorical variable compared to the reference range of >60 ml/min/1.73 m2
CAC detected using CT scans using either an Imatron C-300 electron beam computed tomography scanner or multi-detector CT scanner. CAC was computed using the Agatston score, as a categorical variable. Analyses were performed using ordinal logistic regression.
We found a strong and graded relationship between lower eGFR and increasing CAC. In unadjusted models, ORs increased from 1.68 (95% CI, 1.23–2.31) for eGFR from 50–59 to 2.82 (95% CI, 2.06–3.85) for eGFR of <30. Multivariable adjustment only partially attenuated the results (OR, 1.53; 95% CI, 1.07–2.20) for eGFR<30.
Use of eGFR rather than measured GFR.
We demonstrated a graded relationship between severity of CKD and CAC, independent of traditional risk factors. These findings supports recent guidelines that state that if vascular calcification is present, it should be considered as a complementary component to be included in the decision making required for individualizing treatment of CKD.
PMCID: PMC3183168  PMID: 21783289
20.  Ridge Regression for Longitudinal Biomarker Data 
Technological advances facilitating the acquisition of large arrays of biomarker data have led to new opportunities to understand and characterize disease progression over time. This creates an analytical challenge, however, due to the large numbers of potentially informative markers, the high degrees of correlation among them, and the time-dependent trajectories of association. We propose a mixed ridge estimator, which integrates ridge regression into the mixed effects modeling framework in order to account for both the correlation induced by repeatedly measuring an outcome on each individual over time, as well as the potentially high degree of correlation among possible predictor variables. An expectation-maximization algorithm is described to account for unknown variance and covariance parameters. Model performance is demonstrated through a simulation study and an application of the mixed ridge approach to data arising from a study of cardiometabolic biomarker responses to evoked inflammation induced by experimental low-dose endotoxemia.
PMCID: PMC3202941  PMID: 22049265
biomarkers; cardiovascular disease (CVD); mixed effects; repeated measures; ridge regression
21.  A second independent locus within DMRT1 is associated with testicular germ cell tumor susceptibility 
Human Molecular Genetics  2011;20(15):3109-3117.
Susceptibility to testicular germ cell tumors (TGCT) has a significant heritable component, and genome-wide association studies (GWASs) have identified association with variants in several genes, including KITLG, SPRY4, BAK1, TERT, DMRT1 and ATF7IP. In our GWAS, we genotyped 349 TGCT cases and 919 controls and replicated top hits in an independent set of 439 cases and 960 controls in an attempt to find novel TGCT susceptibility loci. We identified a second marker (rs7040024) in the doublesex and mab-3-related transcription factor 1 (DMRT1) gene that is independent of the previously described risk allele (rs755383) at this locus. In combined analysis that mutually conditions on both DMRT1 single nucleotide polymorphism markers, TGCT cases had elevated odds of carriage of the rs7040024 major A allele [per-allele odds ratio (OR) = 1.48, 95% confidence interval (CI) 1.23, 1.78; P = 2.52 × 10−5] compared with controls, while the association with rs755383 persisted (per allele OR = 1.26, 95% CI 1.08, 1.47, P = 0.0036). In similar analyses, the association of rs7040024 among men with seminomatous tumors did not differ from that among men with non-seminomatous tumors. In combination with KITLG, the strongest TGCT susceptibility locus found to date, men with TGCT had greatly elevated odds (OR = 14.1, 95% CI 5.12, 38.6; P = 2.98 × 10−7) of being double homozygotes for the risk (major) alleles at DMRT (rs7040024) and KITLG (rs4474514) when compared with men without TGCT. Our findings continue to corroborate that genes influencing male germ cell development and differentiation have emerged as the major players in inherited TGCT susceptibility.
PMCID: PMC3131044  PMID: 21551455
22.  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
23.  A human model of inflammatory cardio-metabolic dysfunction; a double blind placebo-controlled crossover trial 
Chronic inflammation may contribute to insulin resistance (IR), metabolic syndrome and atherosclerosis although evidence of causality is lacking in humans. We hypothesized that very low-dose experimental endotoxemia would induce adipose tissue inflammation and systemic IR during a low-grade but asymptomatic inflammatory response and thus provide an experimental model for future tests of pharmacologic and genomic modulation of cardio-metabolic traits in humans.
Ten healthy, human volunteers (50% male, 90% Caucasian, mean age 22.7 ± 3.8) were randomized in a double-masked, placebo-controlled, crossover study to separate 36-hour inpatient visits (placebo versus intravenous-LPS 0.6 ng/kg). We measured clinical symptoms via the McGill pain questionnaire and serial vital signs. Plasma and serum were collected for measurement of cytokines, C-reactive protein, insulin and glucose, serial whole blood & subcutaneous adipose tissue mRNA expression were measured by real-time PCR. HOMA-IR, a well-validated measure of IR was calculated to estimate insulin resistance, and frequently sampled intravenous glucose tolerance testing (FSIGTT) was performed to confirm an insulin resistant state. We performed ANOVA and within subject ANOVA to understand the differences in cytokines, adipose tissue inflammation and IR before and after LPS or placebo.
There was no significant difference between placebo and LPS in clinical responses of symptom scores, body temperature or heart rate. However, low-dose endotoxemia induced a rapid and transient 25-fold induction of plasma TNF-alpha and 100-fold increase in plasma IL-6 (Figure 1B) (p < 0.001 for both) both peaking at two hours, followed by modest inflammation in adipose tissue with increases in mRNA levels of several inflammatory genes known to modulate adipose and systemic insulin resistance. Adipose tissue mRNA levels of IL-6 (peak 6-fold, ANOVA F = 27.5, p < 0.001) and TNF-alpha (peak 1.8-fold, F = 2.9, p = 0.01) increased with MCP-1 (peak 10-fold, F = 5.6, p < 0.01) and fractalkine (CX3CL1) (peak 15-fold, F = 13.3, p < 0.001). Finally, HOMA-IR was 32% higher following LPS compared to placebo (p < 0.01) and insulin sensitivity declined by 21% following LPS compared to placebo (p < 0.05).
We present a low dose human endotoxemia model of inflammation which induces adipose tissue inflammation and systemic insulin resistance in the absence of overt clinical response. Such a model has the potential for broad and safe application in the study of novel therapeutics and genomic influences in cardio-metabolic disease.
PMCID: PMC3477112  PMID: 22709547
Inflammation; Obesity; Atherosclerosis; Insulin resistance
24.  Metabolic Syndrome, Components, and Cardiovascular Disease Prevalence in Chronic Kidney Disease: Findings from the Chronic Renal Insufficiency Cohort (CRIC) Study 
American Journal of Nephrology  2011;33(6):477-484.
Metabolic syndrome may increase the risk for incident cardiovascular disease (CVD) and all-cause mortality in the general population. It is unclear whether, and to what degree, metabolic syndrome is associated with CVD in chronic kidney disease (CKD). We determined metabolic syndrome prevalence among individuals with a broad spectrum of kidney dysfunction, examining the role of the individual elements of metabolic syndrome and their relationship to prevalent CVD.
We evaluated four models to compare metabolic syndrome or its components to predict prevalent CVD using prevalence ratios in the Chronic Renal Insufficiency Cohort (CRIC) Study.
Among 3,939 CKD participants, the prevalence of metabolic syndrome was 65% and there was a significant association with prevalent CVD. Metabolic syndrome was more common in diabetics (87.5%) compared with non-diabetics (44.3%). Hypertension was the most prevalent component, and increased triglycerides the least prevalent. Using the bayesian information criterion, we found that the factors defining metabolic syndrome, considered as a single interval-scaled variable, was the best of four models of metabolic syndrome, both for CKD participants overall and for diabetics and non-diabetics separately.
The predictive value of this model for future CVD outcomes will subsequently be validated in longitudinal analyses.
PMCID: PMC3095834  PMID: 21525746
Cardiovascular disease; Chronic kidney disease; Chronic Renal Insufficiency Cohort (CRIC) Study; Metabolic syndrome
25.  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

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