Activation of calpain-1 in human carotid artery atherosclerotic lesions
© Gonçalves et al; licensee BioMed Central Ltd. 2009
Received: 09 December 2008
Accepted: 18 June 2009
Published: 18 June 2009
In a previous study, we observed that oxidized low-density lipoprotein-induced death of endothelial cells was calpain-1-dependent. The purpose of the present paper was to study the possible activation of calpain in human carotid plaques, and to compare calpain activity in the plaques from symptomatic patients with those obtained from patients without symptoms.
Human atherosclerotic carotid plaques (n = 29, 12 associated with symptoms) were removed by endarterectomy. Calpain activity and apoptosis were detected by performing immunohistochemical analysis and TUNEL assay on human carotid plaque sections. An antibody specific for calpain-proteolyzed α-fodrin was used on western blots.
We found that calpain was activated in all the plaques and calpain activity colocalized with apoptotic cell death. Our observation of autoproteolytic cleavage of the 80 kDa subunit of calpain-1 provided further evidence for enzyme activity in the plaque samples. When calpain activity was quantified, we found that plaques from symptomatic patients displayed significantly lower calpain activity compared with asymptomatic plaques.
These novel results suggest that calpain-1 is commonly active in carotid artery atherosclerotic plaques, and that calpain activity is colocalized with cell death and inversely associated with symptoms.
Calpains are calcium-dependent cysteine proteases that are known to be involved in the proteolysis of a number of proteins during mitosis and cell death [1, 2]. The calpains constitute a large family of distinct isozymes that differ in structure and distribution , and three members of this family are ubiquitous – calpain-1 (μ-calpain), calpain-2 (m-calpain), and calpain-10. A study with embryonic fibroblasts from mice with genetically disrupted capn4, which codes for the regulatory subunit of both calpain-1 and -2, showed that calpains are required for activation of caspase-12 and c-Jun N-terminal kinase in ER-stress-induced apoptosis . The specific endogenous protein inhibitor calpastatin, which modulates calpain activity in vivo, is cleaved during apoptosis . The cytoskeletal protein α-fodrin is another death substrate that may be cleaved by calpains or caspases [1, 6]. Additional calpain substrates known to be involved in apoptosis are Bax , Bid , p53 , and procaspase-3, -7, -8, and -9 [10, 11]. In a previous study, we found that oxidized low-density lipoprotein (oxLDL)-induced death of human microvascular endothelial cells (HMEC-1) was accompanied by activation of calpain-1 . The calpain-1 inhibitor, PD 151746, decreased oxLDL-induced cytotoxicity, and the 80 kDa subunit of calpain-1 was autoproteolytically cleaved in oxLDL-treated HMEC-1 cells, indicating that the enzyme was activated. The Bcl-2 protein Bid was also cleaved during oxLDL-elicited cell death, and this was prevented by calpain inhibitors, but not by inhibitors of cathepsin B or caspases.
Vascular calcification is present in 80% of significant atherosclerotic lesions and in at least 90% of patients with coronary artery disease . Calcification can apparently begin at any point of plaque formation and seems to be a complex mechanism . Since vascular calcification has been shown to correlate with elevated serum calcium , and oxLDL plays a central role in atherogenesis , we hypothesized that calpains may be activated in atherosclerotic lesions. Therefore, the primary aim of the present study was to analyze atherosclerotic plaques for possible calpain activity.
Anti-calpain-1 large subunit monoclonal Ab was from Chemicon International (Temecula, CA, MAB3082), anti-α-tubulin monoclonal Ab was from Oncogene Research Products (Boston, MA, #CP06). HRP-coupled goat anti-rabbit and goat anti-mouse immunoglobulins were from Dako A/S (Glostrup, Denmark). Reagents not listed here were obtained from Sigma, unless otherwise stated in the text.
Twenty-nine human atherosclerotic carotid plaques, from 26 patients (67 ± 8 years old, 21 males), were removed en bloc by carotid endarterectomy by one surgeon. Twelve plaques were associated with ipsilateral hemispheric symptoms in the last month and 17 were not associated to any symptoms after neurologic evaluation. Cardiovascular risk factors such as hypertension (systolic blood pressure > 140 mmHg), diabetes, coronary artery disease, tobacco use (in the past or current) and dyslipidemia were recorded, as well as the medication of these patients. Patients with atrial fibrillation, aortic valve disease, mechanic heart valves, ipsilateral carotid artery occlusion or restenosis after previous carotid endarterectomy were excluded. Informed consent was given by each patient. The study was approved by the local ethical committee. The histological characteristics of symptomatic and asymptomatic plaque samples have been published previously . In short, carotid plaques from symptomatic patients have shown lower levels of hydroxyapatite, higher levels of elastin, cholesterol esters, unesterified cholesterol, triglycerides, more cells, DNA, and soluble protein  compared to those from asymptomatic patients.
The plaques removed by endarterectomy were cleaned with isotonic NaCl containing heparin (5 U/ml), to avoid blood contamination, and thereafter the plaques were immediately snap frozen in liquid nitrogen. Two-mm-thick fragments from the stenotic region of the frozen plaques were removed for histology. Plaques were weighed, cut into pieces while still frozen, and homogenized as previously described . An aliquot was taken from each plaque for western blot analysis, and protein content was analyzed by the method of Lowry.
Immunoblotting and calpain activity
Loading buffer (final concentrations: 50 mmol/L Tris-HCl [pH 6,8], 2% SDS, 10% glycerol, 0,1% bromophenol blue, and 30 mmol/L dithiothreitol) was added to homogenized samples, and they were heated to 90°C in a heating block for 5 min. Proteins were separated under reducing conditions in SDS-polyacrylamide gels and then Western blotted onto PVDF filters. Blots were blocked with Tris-buffered saline containing 5% dry milk powder, and then incubated for 1–2 h with anti-proteolyzed 150 kDa α-fodrin pAb (diluted 1:200), anti-α-tubulin mAb (1:500), or anti-calpain-1 mAb (1:2000). The blots were subsequently incubated with a peroxidase-conjugated secondary Ab, and bound Ab was assayed by enhanced chemiluminescence detection (Santa Cruz Biotechnology, Santa Cruz, CA). To estimate the level of calpain activity, we performed densitometric analysis of Western blots with a Fluor-S MultiImager (Bio-Rad, Rockford, IL). The optical density of 150 kDa α-fodrin bands and tubulin bands was scanned, and the calculated ratio (ODα-fodrin/ODtubulin) for each plaque sample was used in statistical analysis.
Two-millimeter-thick fragments from the stenotic regions of the frozen plaques were embedded in O.C.T. compound (Tissue-Tek, Sakura), cryo-sectioned in serial 8-μm sections, and mounted on coated slides. Tissue sections for immunohistochemistry were fixed with 4% paraformaldehyde in phosphate buffer. Membranes were permeabilized in 0.5% Triton X-100. Endogenous peroxidase activity was quenched by incubating sections for 5 min in 0.9% H2O2. Thereafter sections were blocked with 10% goat serum in PBS for 30 min. Primary antibody, rabbit anti-cleaved-α-fodrin (150 kDa; ref. ), was diluted 1:200 and incubated overnight at 4°C in a humidified chamber. Sections were incubated with biotinylated secondary Ab (goat anti-rabbit, Vector Laboratories, Burlingame, CA) at a dilution of 1:200 for 60 min. Thereafter, sections were incubated with peroxidase- or alkaline phosphatase-labeled Streptavidin (for brown or blue stain, respectively; Vectastain ABC-AP kit, Vector Laboratories). In the case of double-staining, TUNEL was performed after the anti-cleaved-α-fodrin staining. Sections were developed with diaminobenzidine (Vector Laboratories), and counterstained with hematoxylin. Negative controls included substitution of the primary Ab with phosphate buffer.
For TUNEL staining, consecutive tissue sections were fixed with 4% paraformaldehyde in phosphate buffer and stained for apoptosis, using TUNEL In Situ Cell Death detection kit POD (Roche Applied Science, Indianapolis, USA), according to the manufacturer's instructions. Samples were viewed with an Olympus BX60 microscope and photographed.
Results were normalized to the wet weight of the plaques. We used χ2 analyses to investigate associations with dichotomous variables. Two-group comparisons were performed with the use of the Mann-Whitney non-parametric test. Spearman's rho was used for correlation analyses. Statistical analysis was performed with the use of SPSS 12.0 for Windows.
Calpain activity and apoptosis
The autoproteolytic cleavage of the 80 kDa subunit of calpain-1 and -2 is known to be associated with activation of these enzymes . To further verify the activation of calpain in atherosclerotic plaques, we used a monoclonal antibody against the 80 kDa subunit of calpain-1 on western blots, and we observed the 78 kDa autoproteolysis product of calpain-1 in all samples (Figure 1B). The detection of cleaved calpain-1 provided further evidence for active calpain in atherosclerotic plaques.
Calpain activity and plaque characteristics
Most relevant clinical characteristics of the symptomatic patients.
Symptomatic (n = 12)
current 3 and ex 5
Family history of cardiovascular disease
Peripheral arterial disease
7 (3 CCB)
Most relevant clinical characteristics of the asymptomatic patients.
Asymptomatic (n = 17)
current 6 and ex 2
Family history of cardiovascular disease
Peripheral arterial disease
11 (5 CCB)
Our present data demonstrate unequivocally that calpain was activated in atherosclerotic plaques and that calpain activity was co-localized with cell death. Interestingly, in a previous study on these carotid plaques, those associated with symptoms had 70% lower amounts of calcium (hydroxyapatite) . This is in accordance with other studies suggesting that calcium could make plaques more stable , limiting the spread of inflammation . A calcified nodule within or close to the plaque cap can protrude and lead to rupture . However, if the calcified areas coalesce, the interfaces between rigid and distensible areas as well as the mechanical stress decrease . Therefore, depending on their topography in the lesion, calcified areas can function as a protective "shell".
The presence of bone proteins as well as bone and cartilage formation in calcified vascular lesions has suggested that osteogenic mechanisms may play a role in vascular calcification . Interestingly, calpain-2 has been shown to regulate matrix mineralization in a rat growth plate chondrocyte culture model , suggesting that calpains could be involved in vascular calcification. Furthermore, it has been suggested that apoptotic bodies derived from vascular smooth muscle cells may act as nucleating structures for calcium crystal formation and thus initiate vascular calcification . A recent paper showed that vascular smooth muscle cell apoptosis in transgenic mice induced features of plaque vulnerability in atherosclerosis . The fact that calpain regulates oxLDL-induced apoptosis [12, 28], and possibly other types of vascular cell death, combined with the above-mentioned findings, suggests that this enzyme may be a central regulator of vascular calcification, and play an important role in the development of vulnerable plaques.
Our results suggest that calpain-1 is commonly active in carotid artery atherosclerotic plaques, and that calpain activity is colocalized with cell death and inversely associated with symptoms.
oxidized low-density lipoprotein.
This research was supported by funding from foundations at Malmö University Hospital, the Alfred Österlund's Foundation, the Zoèga's Foundation, the Royal Physiographic Society in Lund, and the Swedish Heart and Lung Foundation.
- Vanags DM, Pörn-Ares MI, Coppola S, Burgess DH, Orrenius S: Protease involvement in fodrin cleavage and phosphatidylserine exposure in apoptosis. J Biol Chem. 1996, 271: 31075-31085. 10.1074/jbc.271.49.31075.View ArticlePubMedGoogle Scholar
- Lankiewicz S, Luetjens CM, Truc Bui N, Krohn AJ, Poppe M, Cole GM, Saido TC, Prehn JH: Activation of calpain I converts excitotoxic neuron death into a caspase-independent cell death. J Biol Chem. 2000, 275: 17064-17071. 10.1074/jbc.275.22.17064.View ArticlePubMedGoogle Scholar
- Saido TC, Sorimachi H, Suzuki K: Calpain: new perspectives in molecular diversity and physiological-pathological involvement. FASEB J. 1994, 8: 814-822.PubMedGoogle Scholar
- Tan Y, Dourdin N, Wu C, De Veyra T, Elce JS, Greer PA: Ubiquitous calpains promote caspase-12 and JNK activation during endoplasmic reticulum stress-induced apoptosis. J Biol Chem. 2006, 281: 16016-16024. 10.1074/jbc.M601299200.View ArticlePubMedGoogle Scholar
- Pörn-Ares MI, Samali A, Orrenius S: Cleavage of the calpain inhibitor, calpastatin, during apoptosis. Cell Death Differ. 1998, 5: 1028-1033. 10.1038/sj.cdd.4400424.View ArticlePubMedGoogle Scholar
- Martin SJ, O'Brien GA, Nishioka WK, McGahon AJ, Mahboubi A, Saido TC, Green DR: Proteolysis of fodrin (non-erythroid spectrin) during apoptosis. J Biol Chem. 1995, 270: 6425-8642. 10.1074/jbc.270.12.6425.View ArticlePubMedGoogle Scholar
- Gao G, Dou QP: N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome C release and apoptotic cell death. J Cell Biochem. 2000, 80: 53-72. 10.1002/1097-4644(20010101)80:1<53::AID-JCB60>3.0.CO;2-E.View ArticlePubMedGoogle Scholar
- Mandic A, Viktorsson K, Strandberg L, Heiden T, Hansson J, Linder S, Shoshan MC: Calpain-mediated Bid cleavage and calpain-independent Bak modulation: two separate pathways in cisplatin-induced apoptosis. Mol Cell Biol. 2002, 22: 3003-3013. 10.1128/MCB.22.9.3003-3013.2002.View ArticlePubMedPubMed CentralGoogle Scholar
- Kubbutat MHG, Vousden KH: Proteolytic cleavage of human p53 by calpain: a potential regulator of protein stability. Mol Cell Biol. 1997, 17: 460-468.View ArticlePubMedPubMed CentralGoogle Scholar
- McGinnis KM, Gnegy ME, Park YH, Mukerjee N, Wang KKW: Procaspase-3 and poly(ADP)ribose polymerase (PARP) are calpain substrates. Biochem Biophys Res Commun. 1999, 263: 94-99. 10.1006/bbrc.1999.1315.View ArticlePubMedGoogle Scholar
- Chua BT, Guo K, Li P: Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases. J Biol Chem. 2000, 275: 5131-5135. 10.1074/jbc.275.7.5131.View ArticlePubMedGoogle Scholar
- Pörn-Ares MI, Saido T, Andersson T, Ares MPS: Oxidized low-density lipoprotein induces calpain-dependent cell death and ubiquitination of caspase-3 in HMEC-1 endothelial cells. Biochem J. 2003, 374: 403-411. 10.1042/BJ20021955.View ArticlePubMedPubMed CentralGoogle Scholar
- Parhami F, Boström K, Watson K, Demer LL: Role of molecular regulation in vascular calcification. J Atheroscler Thromb. 1996, 3 (2): 90-94.View ArticlePubMedGoogle Scholar
- Abedin M, Tintut Y, Demer LL: Vascular calcification: mechanisms and clinical ramifications. Arterioscler Thromb Vasc Biol. 2004, 24: 1161-1170. 10.1161/01.ATV.0000133194.94939.42.View ArticlePubMedGoogle Scholar
- Raggi P, Boulay A, Chasan-Taber S, Amin N, Dillon M, Burke SK, Chertow GM: Cardiac calcification in adult hemodialysis patients. J Am Coll Cardiol. 2002, 39: 695-701. 10.1016/S0735-1097(01)01781-8.View ArticlePubMedGoogle Scholar
- Steinberg D: Low-density lipoprotein oxidation and its pathobiological significance. J Biol Chem. 1997, 272: 20963-20966. 10.1074/jbc.272.34.20963.View ArticlePubMedGoogle Scholar
- Gonçalves I, Lindholm MW, Pedro LM, Dias N, Fernandes e Fernandes J, Fredrikson GN, Nilsson J, Moses J, Ares MPS: Elastin and calcium rather than collagen or lipid content are associated with echogenicity of human carotid plaques. Stroke. 2004, 35: 2795-2800. 10.1161/01.STR.0000147038.12073.59.View ArticlePubMedGoogle Scholar
- Gonçalves I, Moses J, Pedro LM, Dias N, Fernandes e Fernandes J, Nilsson J, Ares MP: Echolucency of carotid plaques correlates with plaque cellularity. Eur J Vasc Endovasc Surg. 2003, 26: 32-38. 10.1053/ejvs.2002.1907.View ArticlePubMedGoogle Scholar
- Gonçalves I, Moses J, Dias N, Pedro LM, Fernandes e Fernandes J, Nilsson J, Ares MPS: Changes related to age and cerebrovascular symptoms in the extracellular matrix of human carotid plaques. Stroke. 2003, 34: 616-622. 10.1161/01.STR.0000058157.69113.F6.View ArticlePubMedGoogle Scholar
- Saido TC, Yokota M, Nagao S, Yamaura I, Tani E, Tsuchiya T, Suzuki K, Kawashima S: Spatial resolution of fodrin proteolysis in postischemic brain. J Biol Chem. 1993, 268: 25239-25243.PubMedGoogle Scholar
- Cheng G, Shan J, Xu G, Huang J, Ma J, Ying S, Zhu L: Apoptosis induced by simvastatin in rat vascular smooth muscle cell through Ca2+-calpain and caspase-3 dependent pathway. Pharmacol Res. 2003, 48: 571-578. 10.1016/S1043-6618(03)00245-7.View ArticlePubMedGoogle Scholar
- Hunt JL, Fairman R, Mitchell ME, Carpenter JP, Golden M, Khalapyan T, Wolfe M, Neschis D, Milner R, Scoll B, Cusack A, Mohler ER: Bone formation in carotid plaques: a clinicopathological study. Stroke. 2002, 5: 1214-1219. 10.1161/01.STR.0000013741.41309.67.View ArticleGoogle Scholar
- Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM: Lessons from sudden coronary death. Arterioscl Thromb Vasc Biol. 2000, 20: 1262-1275.View ArticlePubMedGoogle Scholar
- Giachelli CM: Vascular calcification mechanisms. J Am Soc Nephrol. 2004, 15: 2959-2964. 10.1097/01.ASN.0000145894.57533.C4.View ArticlePubMedGoogle Scholar
- Yasuda T, Shimizu K, Nakagawa Y, Yamamoto S, Niibayashi H, Yamamuro T: m-Calpain in rat growth plate chondrocyte cultures: Its involvement in the matrix mineralization process. Dev Biol. 1995, 170: 159-168. 10.1006/dbio.1995.1204.View ArticlePubMedGoogle Scholar
- Proudfoot D, Skepper JN, Hegyi L, Bennett MR, Shanahan CM, Weissberg PL: Apoptosis regulates human vascular calcification in vitro. Circ Res. 2000, 87: 1055-1062.View ArticlePubMedGoogle Scholar
- Clarke MCH, Figg N, Maguire JJ, Davenport AP, Goddard M, Littlewood TD, Bennett MR: Apoptosis of vascular smooth muscle cells induces features of plaque vulnerability in atherosclerosis. Nat Med. 2006, 12: 1075-1080. 10.1038/nm1459.View ArticlePubMedGoogle Scholar
- Vindis C, Elbaz M, Escargueil-Blanc I, Auge N, Heniquez A, Thiers JC, Negre-Salvayre A, Salvayre R: Two distinct calcium-dependent mitochondrial pathways are involved in oxidized LDL-induced apoptosis. Arterioscl Thromb Vasc Biol. 2005, 25: 639-645. 10.1161/01.ATV.0000154359.60886.33.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/9/26/prepub
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