Degraded collagen induces calpain-mediated apoptosis and destruction of the X-chromosome-linked inhibitor of apoptosis (xIAP) in human vascular smooth muscle cells.
OBJECTIVE
The extracellular matrix (ECM) of the atherosclerotic lesion is a crucial determinant of its stability, while its degradation by matrix metalloproteinases (MMPs) has been implied in plaque rupture. As accumulation of both MMP-derived collagen fragments and apoptotic smooth muscle cells (SMC) is observed at sites of plaque rupture, we tested the effect of polymerized and degraded type I collagen on the susceptibility of SMC to apoptosis.


METHODS
Human SMC were cultured on monomeric or polymerized collagen, and collagen gels were degraded by collagenase. Apoptosis was evaluated using antibodies to active caspases and their substrates. Calpain and caspase activity were measured using fluorogenic substrates.


RESULTS
Culture of SMC on polymerized collagen led to increased apoptosis compared to culture on monomeric collagen. In addition, we observed a distinct proteolytic degradation of the endogenous caspase inhibitor X-chromosome-linked inhibitor of apoptosis (xIAP). As MMP-1 was strongly activated in SMC on polymerized collagen, we examined the effect of degraded collagen fragments on xIAP cleavage and apoptosis. Degraded collagen induced rapid proteolytic processing of xIAP identical to that on polymerized collagen. We identified calpains as the proteolytic enzymes responsible for xIAP processing as: i) they were rapidly activated by degraded collagen; ii) recombinant calpain II processed xIAP in an identical manner, and iii) inhibition of calpains by BAPTA or calpeptin abrogated xIAP degradation in intact cells. The functional consequence of xIAP processing by calpains was a loss of its caspase-inhibitory potential. Calpain activation distinctly preceded caspase activation, and inhibition of calpains suppressed apoptosis.


CONCLUSIONS
Collagen fragments proteolytically released from the ECM by MMPs may propagate apoptosis of SMC by calpain-mediated inactivation of anti-apoptotic proteins such as xIAP. This may be a novel mechanism of SMC apoptosis in biological settings of enhanced collagen degradation such as vascular remodeling, neointima formation, and atherosclerotic plaque rupture.
von Wnuck Lipinski
Karin
K
Institute of Pathophysiology, Center of Internal Medicine, Hufelandstrasse 55, 45122 Essen, Germany.
Keul
Petra
P
Lucke
Susann
S
Heusch
Gerd
G
Wohlschlaeger
Jeremias
J
Baba
Hideo A
HA
Levkau
Bodo
B
D017209:Apoptosis; D050197:Atherosclerosis / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen / Q000494:pharmacology*; D017364:Collagenases / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism; D005782:Gels; D006801:Humans; D008856:Microscopy, Fluorescence; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D011108:Polymers; D011994:Recombinant Proteins / Q000378:metabolism; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism*
D004151:Dipeptides; D004791:Enzyme Inhibitors; D005782:Gels; D011108:Polymers; D011994:Recombinant Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; C071482:calpeptin; D004533:Egtazic Acid; D003094:Collagen; D002154:Calpain; D020169:Caspases; D017364:Collagenases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid