Cytokeratin 8 in association with sdLDL and ELISA development

Abstract

Background:Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality worldwide. Cytokeratins (CKs) which may also be expressed in vascular smooth muscle cells (SMCs) are generally considered to be markers for the differentiation of epithelial cells. Small, dense, low-density lipoprotein (sdLDL) particles, also termed LDL-IV, independently predict risk of CVD.Aims:The aims of this study were to develop an analytical method, apart from ultracentrifugation capable of isolating sdLDL in order to study any associated proteins.Materials and Methods:Using modified gradient gel electrophoresis (GGE), de-identified sdLDL-enriched plasma was used to physically elute and isolate sdLDL particles. To validate the finding, additional plasma from 77 normal and 48 higher risk subjects were used to measure sdLDL particles and CK8. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting method were used to identify the characteristics of proteins associated with sdLDL. An enzyme-linked immunosorbent assay (ELISA) method was developed and validated for the measurement of CK8 in plasma.Results:The validation of the CK8 ELISA method showed good analytical performance. The isolated sdLDL particles were verified with nondenaturing GGE with the apolipoprotein B component confirmed by Western immunoblotting. Confirmed by SDS-PAGE and Western immunoblotting, CK8 was associated with sdLDL. Two-tailed statistical analysis showed that CK8 and sdLDL particles were significantly higher in the high-risk CVD group compared to control group (P < 0.01 and P < 0.01, respectively).Conclusion:This study reports a novel association between CK8 and sdLDL in individuals with CVD who have a predominance of sdLDL.

Keratin 8 is a potential self-antigen in the coronary artery disease immunopeptidome: A translational approach

“…The lack of increase in PD-1 mRNA expression by Keratin 8-stimulated PBMCs from CAD patients suggests an aberration of normal tolerance pathways to the self-antigen. Aberrant self-reactive immune responses to Keratin 8 in the context of CAD needs further investigation but its potential involvement is supported by the report that Keratin 8 binds to small dense, low-density lipoprotein (sdLDL) [70]. Analysis of over 11,000 subjects in the Atherosclerosis Risk in Communitites (ARIC) study showed that sdLDL concentration predicted risk for coronary heart disease [71].…”