Isha Agarwal

LC/MS analysis of cellular RNA reveals NAD-linked RNA

We developed a general method to detect cellular small molecule-RNA conjugates that does not rely on the reactivity of the small molecule, revealing NAD-linked RNA in E. coli and S. venezuelae. Subsequent characterization shows NAD is a 5’ modification of RNA, cannot be installed in vitro through aberrant transcriptional initiation, is only found among smaller cellular RNAs, and is present at a surprisingly high abundance of ~3000 copies per cell.

A chemical screen for biological small molecule–RNA conjugates reveals CoA-linked RNA

Compared with the rapidly expanding set of known biological roles for RNA, the known chemical diversity of cellular RNA has remained limited primarily to canonical RNA, 3′-aminoacylated tRNAs, nucleobase-modified RNAs, and 5′-capped mRNAs in eukaryotes. We developed two methods to detect in a broad manner chemically labile cellular small molecule–RNA conjugates. The methods were validated by the detection of known tRNA and rRNA modifications. The first method analyzes small molecules cleaved from RNA by base or nucleophile treatment. Application to Escherichia coli and Streptomyces venezuelae RNA revealed an RNA-linked hydroxyfuranone or succinyl ester group, in addition to a number of other putative small molecule–RNA conjugates not previously reported. The second method analyzes nuclease-generated mononucleotides before and after treatment with base or nucleophile and also revealed a number of new putative small molecule–RNA conjugates, including 3′-dephospho-CoA and its succinyl-, acetyl-, and methylmalonyl-thioester derivatives. Subsequent experiments established that these CoA species are attached to E. coli and S. venezuelae RNA at the 5′ terminus. CoA-linked RNA cannot be generated through aberrant transcriptional initiation by E. coli RNA polymerase in vitro, and CoA-linked RNA in E. coli is only found among smaller (≲200 nucleotide) RNAs that have yet to be identified. These results provide examples of small molecule-RNA conjugates and suggest that the chemical diversity of cellular RNA may be greater than previously understood.

Fibroblast Growth Factor–23 and Cardiac Structure and Function

Background Fibroblast growth factor–23 (FGF‐23) is a phosphaturic factor previously associated with left ventricular hypertrophy and systolic dysfunction among individuals with chronic kidney disease. Whether FGF‐23 acts directly to induce left ventricular hypertrophy, potentially independent of its klotho coreceptor, remains uncertain. We investigated associations of FGF‐23 with cardiac structural abnormalities among individuals with a broad range of kidney function and explored potential biological mechanisms using cardiac magnetic resonance imaging and histology in klotho‐null mice, an established model of constitutively elevated FGF‐23. Methods and Results Among 887 participants with coronary artery disease in the Heart and Soul Study, FGF‐23 was modestly associated with worse left ventricular ejection fraction (−1.0% per standard deviation increase in lnFGF‐23; standard error, 0.4%), but was not associated with the overall prevalence of concentric hypertrophy (odds ratio, 1.5; CI, 0.9 to 2.4) or eccentric hypertrophy (odds ratio, 1.1; CI, 0.9 to 1.3). FGF‐23 was only associated with concentric hypertrophy among individuals with diminished kidney function (eGFR <60 mL/min per 1.73 m2; odds ratio, 2.3; CI, 1.0 to 5.3; P‐interaction=0.28). Comparing klotho‐null with wild‐type mice, null mice did not have greater left ventricular mass (P=0.37) or a lower ejection fraction (P=0.94). Conclusions Together, our results suggest that FGF‐23 is unlikely to have major effects on cardiovascular structure and function among patients free of substantial chronic kidney disease, and these effects may not be independent of the klotho coreceptor.

Novel metabolic biomarkers of cardiovascular disease

Coronary heart disease (CHD) accounts for one in every six deaths in US individuals. Great advances have been made in identifying important risk factors for CHD, such as hypertension, diabetes mellitus, smoking and hypercholesterolaemia, which have led to major developments in therapy. In particular, statins represent one of the greatest successes in the prevention of CHD. While these standard risk factors are important, an obvious opportunity exists to take advantage of ongoing scientific research to better risk-stratify individuals and to identify new treatment targets. In this Review, we summarize ongoing scientific research in a number of metabolic molecules or features, including lipoproteins, homocysteine, calcium metabolism and glycaemic markers. We evaluate the current state of the research and the strength of evidence supporting each emerging biomarker. We also discuss whether the associations with CHD are strong and consistent enough to improve current risk stratification metrics, and whether these markers enhance our understanding of the underlying biology of CHD and thus point towards new treatment options.

Predictive value of updating framingham risk scores with novel risk markers in the U.S. general population

Background According to population-based cohort studies CT coronary calcium score (CTCS), carotid intima-media thickness (cIMT), high-sensitivity C- reactive protein (CRP), and ankle-brachial index (ABI) are promising novel risk markers for improving cardiovascular risk assessment. Their impact in the U.S. general population is however uncertain. Our aim was to estimate the predictive value of four novel cardiovascular risk markers for the U.S. general population. Methods and Findings Risk profiles, CRP and ABI data of 3,736 asymptomatic subjects aged 40 or older from the National Health and Nutrition Examination Survey (NHANES) 2003–2004 exam were used along with predicted CTCS and cIMT values. For each subject, we calculated 10-year cardiovascular risks with and without each risk marker. Event rates adjusted for competing risks were obtained by microsimulation. We assessed the impact of updated 10-year risk scores by reclassification and C-statistics. In the study population (mean age 56±11 years, 48% male), 70% (80%) were at low (<10%), 19% (14%) at intermediate (≥10–<20%), and 11% (6%) at high (≥20%) 10-year CVD (CHD) risk. Net reclassification improvement was highest after updating 10-year CVD risk with CTCS: 0.10 (95%CI 0.02–0.19). The C-statistic for 10-year CVD risk increased from 0.82 by 0.02 (95%CI 0.01–0.03) with CTCS. Reclassification occurred most often in those at intermediate risk: with CTCS, 36% (38%) moved to low and 22% (30%) to high CVD (CHD) risk. Improvements with other novel risk markers were limited. Conclusions Only CTCS appeared to have significant incremental predictive value in the U.S. general population, especially in those at intermediate risk. In future research, cost-effectiveness analyses should be considered for evaluating novel cardiovascular risk assessment strategies.

Fibrosis-Related Biomarkers and Risk of Total and Cause-Specific Mortality The Cardiovascular Health Study

Fibrosis has been implicated in diverse diseases of the liver, kidney, lungs, and heart, but its importance as a risk factor for mortality remains unconfirmed. We determined the prospective associations of 2 complementary biomarkers of fibrosis, transforming growth factor-β (TGF-β) and procollagen type III N-terminal propeptide (PIIINP), with total and cause-specific mortality risks among community-living older adults in the Cardiovascular Health Study (1996-2010). We measured circulating TGF-β and PIIINP levels in plasma samples collected in 1996 and ascertained the number of deaths through 2010. Both TGF-β and PIIINP were associated with elevated risks of total and pulmonary mortality after adjustment for sociodemographic, clinical, and biochemical risk factors. For total mortality, the hazard ratios per doubling of TGF-β and PIIINP were 1.09 (95% confidence interval (CI): 1.01, 1.17; P = 0.02) and 1.14 (CI: 1.03, 1.27; P = 0.01), respectively. The corresponding hazard ratios for pulmonary mortality were 1.27 (CI: 1.01, 1.60; P = 0.04) for TGF-β and 1.52 (CI: 1.11, 2.10; P = 0.01) for PIIINP. Associations of TGF-β and PIIINP with total and pulmonary mortality were strongest among individuals with higher C-reactive protein concentrations (P for interaction < 0.05). Our findings provide some of the first large-scale prospective evidence that circulating biomarkers of fibrosis measured late in life are associated with death.

Fibrosis-Related Biomarkers and Incident Cardiovascular Disease in Older Adults: The Cardiovascular Health Study

Background—Fibrotic changes in the heart and arteries have been implicated in a diverse range of cardiovascular diseases (CVD), but whether circulating biomarkers that reflect fibrosis are associated with CVD is unknown. Methods and Results—We determined the associations of 2 biomarkers of fibrosis, transforming growth factor- &bgr; (TGF-&bgr;), and procollagen type III N-terminal propeptide (PIIINP), with incident heart failure, myocardial infarction, and stroke among community-living older adults in the Cardiovascular Health Study. We measured circulating TGF-&bgr; (n=1371) and PIIINP (n=2568) from plasma samples collected in 1996 and ascertained events through 2010. Given TGF-&bgr;’s pleiotropic effects on inflammation and fibrogenesis, we investigated potential effect modification by C-reactive protein in secondary analyses. After adjustment for sociodemographic, clinical, and biochemical risk factors, PIIINP was associated with total CVD (hazard ratio [HR] per SD=1.07; 95% confidence interval [CI], 1.01–1.14) and heart failure (HR per SD=1.08; CI, 1.01–1.16) but not myocardial infarction or stroke. TGF-&bgr; was not associated with any CVD outcomes in the full cohort but was associated with total CVD (HR per SD=1.16; CI, 1.02–1.31), heart failure (HR per SD=1.16; CI, 1.01–1.34), and stroke (HR per SD=1.20; CI, 1.01–1.42) among individuals with C-reactive protein above the median, 2.3 mg/L (P interaction <0.05). Conclusions—Our findings provide large-scale, prospective evidence that circulating biomarkers of fibrosis, measured in community-living individuals late in life, are associated with CVD. Further research on whether TGF-&bgr; has a stronger fibrogenic effect in the setting of inflammation is warranted.

Cost-effectiveness of the polypill versus risk assessment for prevention of cardiovascular disease

Objective There is an international trend towards recommending medication to prevent cardiovascular disease (CVD) in individuals at increasingly lower cardiovascular risk. We assessed the cost-effectiveness of a population approach with a polypill including a statin (simvastatin 20 mg) and three antihypertensive agents (amlodipine 2.5 mg, losartan 25 mg and hydrochlorothiazide 12.5 mg) and periodic risk assessment with different risk thresholds. Methods We developed a microsimulation model for lifetime predictions of CVD events, diabetes, and death in 259 146 asymptomatic UK Biobank participants aged 40–69 years. We assessed incremental costs and quality-adjusted life-years (QALYs) for polypill scenarios with the same combination of agents and doses but differing for starting age, and periodic risk assessment with 10-year CVD risk thresholds of 10% and 20%. Results Restrictive risk assessment, in which statins and antihypertensives were prescribed when risk exceeded 20%, was the optimal strategy gaining 123 QALYs (95% credible interval (CI) −173 to 387) per 10 000 individuals at an extra cost of £1.45 million (95% CI 0.89 to 1.94) as compared with current practice. Although less restrictive risk assessment and polypill scenarios prevented more CVD events and attained larger survival gains, these benefits were offset by the additional costs and disutility of daily medication use. Lowering the risk threshold for prescription of statins to 10% was economically unattractive, costing £40 000 per QALY gained. Starting the polypill from age 60 onwards became the most cost-effective scenario when annual drug prices were reduced below £240. All polypill scenarios would save costs at prices below £50. Conclusions Periodic risk assessment using lower risk thresholds is unlikely to be cost-effective. The polypill would become cost-effective if drug prices were reduced.

Clinical ScienceAssociations between metabolic dysregulation and circulating biomarkers of fibrosis: the Cardiovascular Health Study

AIM Fibrosis is one postulated pathway by which diabetes produces cardiac and other systemic complications. Our aim was to determine which metabolic parameters are associated with circulating fibrosis-related biomarkers transforming growth factor-β (TGF-β) and procollagen type III N-terminal propeptide (PIIINP). METHODS We used linear regression to determine the cross-sectional associations of diverse metabolic parameters, including fasting glucose, fasting insulin, body mass index, fatty acid binding protein 4, and non-esterified fatty acids, with circulating levels of TGF-β (n = 1559) and PIIINP (n = 3024) among community-living older adults in the Cardiovascular Health Study. RESULTS Among the main metabolic parameters we examined, only fasting glucose was associated with TGF-β (P = 0.03). In contrast, multiple metabolic parameters were associated with PIIINP, including fasting insulin, body mass index, and non-esterified fatty acids (P<0.001, P<0.001, P=0.001, respectively). These associations remained statistically significant after mutual adjustment, except the association between BMI and PIIINP. CONCLUSIONS Isolated hyperglycemia is associated with higher serum concentrations of TGF-β, while a broader phenotype of insulin resistance is associated with higher serum PIIINP. Whether simultaneous pharmacologic targeting of these two metabolic phenotypes can synergistically reduce the risk of cardiac and other manifestations of fibrosis remains to be determined.

Associations between metabolic dysregulation and circulating biomarkers of fibrosis: the Cardiovascular Health Study

AIM Fibrosis is one postulated pathway by which diabetes produces cardiac and other systemic complications. Our aim was to determine which metabolic parameters are associated with circulating fibrosis-related biomarkers transforming growth factor-β (TGF-β) and procollagen type III N-terminal propeptide (PIIINP). METHODS We used linear regression to determine the cross-sectional associations of diverse metabolic parameters, including fasting glucose, fasting insulin, body mass index, fatty acid binding protein 4, and non-esterified fatty acids, with circulating levels of TGF-β (n = 1559) and PIIINP (n = 3024) among community-living older adults in the Cardiovascular Health Study. RESULTS Among the main metabolic parameters we examined, only fasting glucose was associated with TGF-β (P = 0.03). In contrast, multiple metabolic parameters were associated with PIIINP, including fasting insulin, body mass index, and non-esterified fatty acids (P<0.001, P<0.001, P=0.001, respectively). These associations remained statistically significant after mutual adjustment, except the association between BMI and PIIINP. CONCLUSIONS Isolated hyperglycemia is associated with higher serum concentrations of TGF-β, while a broader phenotype of insulin resistance is associated with higher serum PIIINP. Whether simultaneous pharmacologic targeting of these two metabolic phenotypes can synergistically reduce the risk of cardiac and other manifestations of fibrosis remains to be determined.