Complement regulation in murine and human hypercholesterolemia and role in the control of macrophage and smooth muscle cell proliferation.
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 (approximately 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 gamma chain, C1s and C3c alpha 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.
Laboratory 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.
Francisco
F
Verdeguer
Claudia
C
Castro
Markus
M
Kubicek
Davinia
D
Pla
Marian
M
Vila-Caballer
Angela
A
Vinué
Fernando
F
Civeira
Miguel
M
Pocoví
Juan José
JJ
Calvete
Vicente
V
Andrés
D000595:Amino Acid Sequence; D000818:Animals; D001057:Apolipoproteins E / Q000502:physiology; D049109:Cell Proliferation; D003176:Complement C3 / Q000502:physiology; D015926:Complement C3a / Q000502:physiology; D003165:Complement System Proteins / Q000502:physiology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology; D006801:Humans; D006937:Hypercholesterolemia / Q000209:etiology*; D008264:Macrophages / Q000166:cytology*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000502:physiology*
D001057:Apolipoproteins E; D003176:Complement C3; D015926:Complement C3a; D003165:Complement System Proteins; D048049:Extracellular Signal-Regulated MAP Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go