Cellular mechanisms by which proinsulin C-peptide prevents insulin-induced neointima formation in human saphenous vein.
OBJECTIVE
Endothelial cells (ECs) and smooth muscle cells (SMCs) play key roles in the development of intimal hyperplasia in saphenous vein (SV) bypass grafts. In diabetic patients, insulin administration controls hyperglycaemia but cardiovascular complications remain. Insulin is synthesised as a pro-peptide, from which C-peptide is cleaved and released into the circulation with insulin; exogenous insulin lacks C-peptide. Here we investigate modulation of human SV neointima formation and SV-EC and SV-SMC function by insulin and C-peptide.


METHODS
Effects of insulin and C-peptide on neointima formation (organ cultures), EC and SMC proliferation (cell counting), EC migration (scratch wound), SMC migration (Boyden chamber) and signalling (immunoblotting) were examined. A real-time RT-PCR array identified insulin-responsive genes, and results were confirmed by real-time RT-PCR. Targeted gene silencing (siRNA) was used to assess functional relevance.


RESULTS
Insulin (100 nmol/l) augmented SV neointimal thickening (70% increase, 14 days), SMC proliferation (55% increase, 7 days) and migration (150% increase, 6 h); effects were abrogated by 10 nmol/l C-peptide. C-peptide did not affect insulin-induced Akt or extracellular signal-regulated kinase signalling (15 min), but array data and gene silencing implicated sterol regulatory element binding transcription factor 1 (SREBF1). Insulin (1-100 nmol/l) did not modify EC proliferation or migration, whereas 10 nmol/l C-peptide stimulated EC proliferation by 40% (5 days).


CONCLUSIONS
Our data support a causative role for insulin in human SV neointima formation with a novel counter-regulatory effect of proinsulin C-peptide. Thus, C-peptide can limit the detrimental effects of insulin on SMC function. Co-supplementing insulin therapy with C-peptide could improve therapy in insulin-treated patients.
Mughal
R S
RS
Division of Cardiovascular and Neuronal Remodelling, University of Leeds, Worsley Building, Leeds LS2 9JT, UK.
Scragg
J L
JL
Lister
P
P
Warburton
P
P
Riches
K
K
O'Regan
D J
DJ
Ball
S G
SG
Turner
N A
NA
Porter
K E
KE
D000704:Analysis of Variance; D015153:Blotting, Western; D002096:C-Peptide / Q000378:metabolism*; D002452:Cell Count; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D006965:Hyperplasia / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012501:Saphenous Vein / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D015398:Signal Transduction / Q000187:drug effects; D017539:Tunica Intima / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*
D002096:C-Peptide; D007328:Insulin; D034741:RNA, Small Interfering; D051057:Proto-Oncogene Proteins c-akt
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