Cathepsin L expression and regulation in human abdominal aortic aneurysm, atherosclerosis, and vascular cells
Introduction
Recent in vitro biochemical studies and animal experiments have implicated cysteine proteases in arterial remodeling. We previously demonstrated increased expression of cathepsins S and K in human atherosclerotic and aneurysmal lesions and hypothesized their potential roles in the pathogenesis of these diseases [1], [2]. In accordance with this hypothesis, mice deficient in cathepsin S or K have attenuated atherosclerosis when consuming an atherogenic diet [3], [4], and mechanistic investigations indicated their importance in extracellular matrix protein degradation via their potent elastase and collagenase activities [3].
All major cell types found in human and experimental atheromata and abdominal aortic aneurysms (AAAs), smooth muscle cells (SMC), endothelial cells (EC), and macrophages, express elastolytic and collagenolytic activity and play a role in atherogenesis and AAA formation. SMC migration and proliferation participate in the formation of atherosclerotic lesions [5]. Degradation of elastic layers, a process also mediated by cysteine proteases [6], facilitates migration of SMC from the tunica media into the intima. Further, intimal SMC, the source of interstitial collagens, a key constituent of the plaque's fibrous caps, can also produce the elastolytic and collagenolytic cathepsins S and K [1]. Together, these findings suggest that cysteine proteases participate in matrix remodeling during atherogenesis and regulation of the vulnerability of atherosclerotic plaques.
Microvascularization is a histopathology marker of abdominal aortic aneurysm (AAA) formation [7] and correlates with plaque instability and rupture [8]. Formation of microvessels involves EC migration and requires elastolytic and collagenolytic proteases [9]. Our recent investigation of the role of cysteine proteases in neovascularization [10] demonstrated that lack of cathepsin S activity reduced microvessel formation and/or extension.
Macrophages can express abundant cathepsins S, K and L. Within atherosclerotic lesions, macrophages play an active role in plaque rupture. Besides elaborating cytokines [11], [12] and matrix metalloproteinases (MMPs) [13], [14], these cells may also release cysteine proteases that mediate collagenolysis and elastolysis. Our earlier observations demonstrated increased expression of cathepsins S and K in macrophages at the shoulder regions where plaque ruptures usually occur [1]. In early atheromata, expression of cathepsins B and L localizes to apoptotic macrophages, whereas other cells appear not to contain these proteinases [15].
Most tissues and cells have basal expression of the lysosomal cathepsin L [16], [17]. However, inflammatory cytokines including interleukin-6 (IL-6), IL-1β, and interferon-γ (IFN-γ) increase cathepsin L expression in cultured human lung epithelial cells [18], muscle[19], osteosarcoma cells [20], and monocytic U937 cells [21]. Physiologically, cathepsin L resembles other lysosomal proteases with its ability to degrade endocytosed proteins in acidic environments. Given the inflammatory nature of atherosclerosis and AAA and of cathepsin L as a potent elastase (equivalent to cathepsin S [22]) and collagenase (similar to cathepsin K [23]), cathepsin L might participate in the pathogenesis of these diseases. Although, cathepsin L's expression in non-vascular cells [18], [19], [20], [21] and its ability to degrade elastin and collagen in vitro has been well studied [22], [23], its potential role in atherosclerosis or AAA formation remains unexplored. This study addresses this gap and demonstrates increased levels of cathepsin L in AAA and atherosclerotic lesions locally expressed by all major cell types found in these diseased vessels (SMC, macrophages, and EC). In vitro these cell types increase cathepsin L expression and secretion in response to pro-inflammatory mediators. Importantly, we also found systemic increase of cathepsin L levels in serum of patients with stenotic coronary arteries.
Section snippets
Immunohistology, immunoblot, and immunofluorescent colocalization
Frozen tissue samples of human carotid atheroma from endarterectomies (n = 7), normal aortas from cardiac transplant donors (n = 7), and abdominal aortic aneurysms (n = 7) were obtained according to protocols pre-approved by the Human Investigation Review Committee at the Brigham and Women's Hospital. Serial cryostat sections (6 μm) were immunostained for cathepsin L (1:150, Calbiochem) as described [1]. Protein extracts were prepared from frozen tissues and equal amounts of proteins (30 μg/sample)
Increased expression of cathepsin L in AAA and atheroma
Advanced human atherosclerotic and AAA lesions had elevated immunoreactive cathepsin L compared to non-involved arteries (Fig. 1A). In AAA, cathepsin L mainly localized in macrophage-enriched areas, but much more weakly in SMC (Fig. 1A, right two panels). In advanced atherosclerotic plaques, similar to human cathepsins S and K [1], cathepsin L protein localized in the fibrous cap, tunica media, and macrophage-rich shoulder regions (Fig. 1A, low left panel). SMC in these regions were positive
Discussion
Injury [29] or inflammation [18], [19], [20], [21] regulates the expression of cathepsin L, a potent mammalian elastase and collagenase. Inflammatory diseases such as atherosclerosis and AAA involve extensive degradation of elastin and collagen. Several protease families, including cysteine proteases [6], may participate in atherogenesis, highlighting the need to assess their relative contributions. Revealing the molecular mechanisms that trigger the pathologic processes of these common human
Acknowledgements
The authors thank Mrs. Eugenia Shvartz for technical assistance and Ms. Karen Williams for editorial assistance. This study is supported by NIH grants HL60942, HL67283 (GPS), HL67249 (GKS), HL56985 (PL) and grant from American Heart Association Grant-in-Aid (0355130Y, GPS).
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