Copyright © 2007, European Society of Cardiology
Complement regulation in murine and human hypercholesterolemia and role in the control of macrophage and smooth muscle cell proliferation
aLaboratory of Vascular Biology, Department of Molecular and Cellular Pathology and Therapy, Instituto de Biomedicina de Valencia (IBV–CSIC), Spanish Council for Scientific Research, 46010 Valencia, Spain
bClinical Institute for Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, A-1090 Vienna, Austria
cRed Temática de Investigación Cooperativa en Enfermedades Cardiovasculares, Instituto de Salud Carlos III, Spain
dDepartments of Medicine and Biochemistry and Molecular and Cellular Biology, Universidad de Zaragoza, Zaragoza, Spain
eLaboratory of Structural Proteinomics, Department of Genomics and Proteomics, Instituto de Biomedicina de Valencia (IBV–CSIC), Spanish Council for Scientific Research, 46010 Valencia, Spain
*Corresponding author. Instituto de Biomedicina de Valencia, Jaime Roig 11, 46010 Valencia, Spain. Tel.: +34 963391752; fax: +34 963391751. vandres{at}ibv.csic.es
Objective Mounting evidence suggests that activation of complement, an important constituent of innate immunity, contributes to atherosclerosis. Here we investigated the expression of complement components (CCs) in the setting of experimental and clinical hypercholesterolemia, a major risk factor for atherosclerosis, their effects on vascular smooth muscle cell (VSMC) and macrophage proliferation, and the underlying molecular mechanisms.
Methods For this study we analyzed the mRNA and protein expression of several CCs in plasma and aorta of hypercholesterolemic atherosclerosis-prone apolipoprotein E-null mice (apoE-KO) and in plasma of normocholesterolemic subjects and familial hypercholesterolemia (FH) patients. We also carried out in vitro molecular studies to assess the role of CCs on the control of macrophage and VSMC proliferation.
Results Fat-fed apoE-KO mice experiencing severe hypercholesterolemia (
400 mg/dL), but not fat-fed wild-type controls with plasma cholesterol level<110 mg/dL, displayed in aortic tissue upregulation of several CC mRNAs, including C3, C4, C1s, and C1q. In apoE-KO mice, induction of C3 mRNA was already apparent two days after fat feeding when hypercholesterolemia was manifested yet atherosclerotic lesions were absent or incipient. Rapid C3 and C4 protein upregulation was also observed in the plasma of fat-fed apoE-KO mice, and FH patients exhibited higher plasmatic C3a, C4 
chain, C1s and C3c 
chain protein levels than normocholesterolemic subjects. In vitro, C3 and C3a, but not C3a-desArg, C4 and C1q, promoted macrophage and VSMC proliferation through Gi protein-dependent activation of extracellular signal-regulated kinase 1/2 (ERK1/2). We also found that C3-enriched FH plasma evoked a stronger mitogenic response in macrophages than normocholesterolemic plasma, and treatment with anti-C3 antibodies eliminated this difference.
Conclusions Both experimental and clinical hypercholesterolemia coincides with a concerted activation of several CCs. However, only C3 and C3a elicited a mitogenic response in cultured VSMCs and macrophages through Gi protein-dependent ERK1/2 activation. Thus, excess of C3/C3a in hypercholesterolemic apoE-KO mice and FH patients may contribute to atheroma growth by promoting neointimal cell proliferation.
KEYWORDS Hypercholesterolemia; Complement activation; Vascular smooth muscle cell; Macrophage; Proliferation; ERK
1 Present address: Laboratory of Gene Expression Development and Disease, Department of Developmental Biology, Institut Pasteur, 75724 Paris, France.
2 These authors contributed equally to this work.
3 Present address: Instituto de Medicina y Biología Experimental de Cuyo (IMBECU), CONICET, Facultad de Ciencia Médicas UNCuyo, Mendoza, Argentina.