Familial hypercholesterolemia (FH) is increasingly associated with inflammation, a phenotype that persists despite treatment with lipid lowering therapies. The alternative C3 complement system (C3), as a key inflammatory mediator, seems to be involved in the atherosclerotic process; however, the relationship between C3 and lipids during plaque progression remains unknown. The aim of the study was to investigate by a systems biology approach the role of C3 in relation to lipoprotein levels during atherosclerosis (AT) progression and to gain a better understanding on the effects of C3 products on the phenotype and function of human lipid-loaded vascular smooth muscle cells (VSMCs). By mass spectrometry and differential proteomics, we found the extracellular matrix (ECM) of human aortas to be enriched in active components of the C3 complement system, with a significantly different proteomic signature in AT segments. Thus, C3 products were more abundant in AT-ECM than in macroscopically normal segments. Furthermore, circulating C3 levels were significantly elevated in FH patients with subclinical coronary AT, evidenced by computed tomographic angiography. However, no correlation was identified between circulating C3 levels and the increase in plaque burden, indicating a local regulation of the C3 in AT arteries. In cell culture studies of human VSMCs, we evidenced the expression of C3, C3aR (anaphylatoxin receptor) and the integrin α_Mβ₂ receptor for C3b/iC3b (RT-PCR and Western blot). C3mRNA was up-regulated in lipid-loaded human VSMCs, and C3 protein significantly increased in cell culture supernatants, indicating that the C3 products in the AT-ECM have a local vessel-wall niche. Interestingly, C3a and iC3b (C3 active fragments) have functional effects on VSMCs, significantly reversing the inhibition of VSMC migration induced by aggregated LDL and stimulating cell spreading, organization of F-actin stress fibers and attachment during the adhesion of lipid-loaded human VSMCs. This study, by using a systems biology approach, identified molecular processes involving the C3 complement system in vascular remodeling and in the progression of advanced human atherosclerotic lesions.

Acute decompensated heart failure (ADHF) is a life-threatening clinical syndrome involving multi-organ function deterioration. ADHF results from multifaceted, dysregulated pathways that remain poorly understood. Better characterization of proteins associated with heart failure decompensation is needed to gain understanding of the disease pathophysiology and support a more accurate disease phenotyping. In this study, we used an untargeted mass spectrometry (MS) proteomic approach to identify the differential urine protein signature in ADHF patients and examine its pathophysiological link to disease evolution. Urine samples were collected at hospital admission and compared with a group of healthy subjects by two-dimensional electrophoresis coupled to MALDI-TOF/TOF mass spectrometry. A differential pattern of 26 proteins (>1.5-fold change, p < 0.005), mostly of hepatic origin, was identified. The top four biological pathways (p < 0.0001; in silico analysis) were associated to the differential ADHF proteome including retinol metabolism and transport, immune response/inflammation, extracellular matrix organization, and platelet degranulation. Transthyretin (TTR) was the protein most widely represented among them. Quantitative analysis by ELISA of TTR and its binding protein, retinol-binding protein 4 (RBP4), validated the proteomic results. ROC analysis evidenced that combining RBP4 and TTR urine levels highly discriminated ADHF patients with renal dysfunction (AUC: 0.826, p < 0.001) and significantly predicted poor disease evolution over 18-month follow-up. In conclusion, the MS proteomic approach enabled identification of a specific urine protein signature in ADHF at hospitalization, highlighting changes in hepatic proteins such as TTR and RBP4.