Author + information
- Aeron M. Small, MD, MTR,
- Daniel H. Kiss, MD,
- Saif Anwaruddin, MD,
- Jay Giri, MD,
- Yuchi Han, MD,
- Lei Zhao, MD, PhD,
- Lisa Salvador, PhD,
- Mary Ellen Cvijic, PhD,
- Zhuyin Li, PhD,
- Julio A. Chirinos, MD, PhD,
- Scott M. Damrauer, MD and
- Daniel J. Rader, MD∗ (, )@PennMedicine
- ↵∗University of Pennsylvania, 11-125 Smilow Center for Translational Research, 3400 Civic Center Blvd, Philadelphia, Pennsylvania 19104
Calcific aortic stenosis (CAS) is the end manifestation of calcific aortic valve disease (CAVD). There are no medical therapies to reverse or slow the progression of CAVD (1). Circulating biomarkers may provide insights into CAVD pathogenesis. Biomarkers identified in association with CAVD include osteopontin, osteoprotegerin, fibroblast growth factor (FGF-23), B-type natriuretic peptide (BNP), low-density lipoprotein cholesterol, and lipoprotein(a) [Lp(a)] (2). Lp(a) has been established as a causal risk factor for CAVD through Mendelian randomization (3).
We performed a targeted discovery experiment to identify novel biomarkers of CAS using a multiplexed assay of 48 candidate proteins related to cardiovascular disease. We leveraged a large biobank at the University of Pennsylvania (PennMedicine Biobank) and used text mining of the electronic health record to identify CAS cases and non-CAS control subjects, followed by electronic health record review for confirmation of case status. We then assayed 48 proteins among 711 subjects with CAS and 802 age- and sex-matched controls (cases: 78 ± 10 years of age, 84% Caucasian, 6% Black; control subjects: 74 ± 9 years of age, 85% Caucasian, 6% Black).
In a multiple linear regression model adjusted for age, sex, ethnicity, and common clinical conditions (hypertension, coronary artery disease, heart failure, diabetes, peripheral vascular disease, and renal disease), 17 proteins were significantly (false discovery rate <0.05) associated with CAS. These included BNP, which had increased plasma levels in CAS cases relative to control subjects. Osteopontin, osteoprotegerin, and FGF-23 were significantly increased in cases in unadjusted regression, but were not significant after adjusting for demographic and clinical covariates. Of the novel protein associations, soluble FMS-like tyrosine kinase (sFLT)-1 was the biomarker with the greatest significant (p = 3.1E-07) difference between CAS cases (7.9 log[pg/ml] ± 2.8) and controls (7.1 log[pg/ml] ± 3.1). sFLT-1 levels varied by disease severity (p = 7.7E-13), with mean levels of 7.2, 7.4, and 8.5 log[pg/ml] in subjects with mild (n = 182), moderate (n = 145), and severe (n = 384) aortic stenosis.
We subsequently validated the association between sFLT-1 and CAS in an independent sample of 222 CAS cases with 385 matched control subjects. sFLT-1 levels were significantly higher in CAS cases (p = 7.3E-07). In inverse-variance weighted meta-analysis of the discovery and validation adjusted regression models, sFLT-1 achieved a combined β of 0.59 (p = 1.0E-06) (Figure 1).
We performed sensitivity analyses to account for hemodynamic parameters that potentially impact plasma levels of sFLT-1. In a subset of the discovery cohort with available hemodynamic data (CAS = 596, control subjects = 131), we performed multiple linear regression against aortic valve mean gradient. sFLT-1 maintained a strong association to mean gradient after adjustment for left ventricular ejection fraction, age, sex, and ethnicity (β = 0.02; p = 5.3E-04). sFLT-1 also maintained a significant association with CAS after adjusting for BNP (β = 0.59; p = 4.1E-04), a biomarker with a strong association with heart failure.
Lp(a) is well studied in CAS and is an established genetic risk factor for the disease. We replicated the association between CAS and Lp(a) in the discovery cohort, with higher plasma levels of Lp(a) in cases (p = 3.0E-04). After adjusting for Lp(a) in multivariable regression, the association between sFLT-1 and CAS remained significant (β = 0.71; p = 8.0E-06).
Our results represent one of the first applications of multiplexed protein biomarker discovery in CAS. We identified and validated in an independent sample a novel association of higher sFLT-1 levels in CAS, which was independent of heart failure and Lp(a). sFLT-1 is a soluble splice variant of the transmembrane protein FLT-1 (or VEGFR-1), and is proposed to have an antiangiogenic function by either sequestration of soluble VEGF or by heterodimerization with transmembrane FLT-1 and inactivation of its downstream effects. Angiogenesis is common in late-stage CAS, and although normal aortic valves are avascular, CAS histological samples frequently demonstrate neovessel formation within the valve intima (4). Additional studies are warranted to distinguish whether sFLT-1 plays a causal role in the pathobiology of CAS or is a useful biomarker of the disease.
Please note: Dr. Anwaruddin has been a consultant and speaker for Edwards Lifesciences and Medtronic. Dr. Giri has received institutional research funding from St. Jude Medical and Recor Medical; and has served on an advisory board for AstraZeneca. Drs. Zhao, Cvijic, and Li are employees of and hold stock in Bristol-Myers Squibb. Dr. Salvador is an employee of Bristol-Myers Squibb. Dr. Chirinos has received institutional research grants from the National Institutes of Health, American College of Radiology Network, Fukuda-Denshi, BMS, and Microsoft; and consulting honoraria from BMS, OPKO Healthcare, Fukuda-Denshi, Microsoft, Ironwood, Sanifit, Pfizer, Vital Labs, Bayer, Merck, and Akros Pharma. Dr. Damrauer has received research funding from CytoVas, LLC. Drs. Small and Rader received funding from the Doris Duke Charitable Foundation that supported in part this work. Dr. Rader serves on scientific advisory boards for Alnylam, Novartis, and Pfizer. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. The authors would like to acknowledge the following individuals, without whom this work would not have been possible: Giovanni Ferrari, Robert Wilensky, Marie Guerraty, Aditya Munshi, Wanda Anselmo, Samuel Montgomery, Melissa Yarde, Jie Pan, and Michael Basso.
- 2019 American College of Cardiology Foundation
- Small A.,
- Kiss D.,
- Giri J.,
- et al.
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