Characterizing Cathepsin Activity and Macrophage Subtypes in Excised Human Carotid Plaques

Abstract

T he formation and progression of atherosclerotic plaques is characterized by accumulation of oxidized lipids and inflammatory cells covered by a fibrous cap of smooth muscle cells and collagen-rich matrix.1 Most severe clinical events result from plaque rupture, which exposes prothrombotic material in the plaque to the blood and causes intraluminal thrombus formation. Plaques vulnerable to rupture are characterized by a thin fibrous cap, a large lipid/necrotic core, positive vascular remodeling, and substantial infiltration of inflammatory cells.2 In particular, macrophages have emerged as a key cellular element of atherosclerotic plaque pathogenesis and pose a significant risk for causing plaque rupture.3 Recently, studies have observed heterogeneous subsets of macrophages, such as M1 and M2, in human atherosclerotic lesions. 4 Progressive necrosis of foam cells causes secretion of several factors that sustain the inflammatory processes and produce tissue-degrading proteases, such as matrix metalloproteinases and elastases.5 Plaque destabilization is attributed mostly to extracellular matrix (ECM) degradation (collagenolysis elastinolysis) by multiple ECM degrading enzymes, such as matrix metalloproteinases, serine hydrolyses, and cysteine proteases. Cysteine proteases, including cathepsins B, L, and S, display profound ECM catabolic activity in vitro and were found to be overexpressed in activated macrophages and advanced atherosclerotic lesions. 6,7 Cathepsin B, L, and S play key roles in macrophage function, with their mRNA expression and activity increasing significantly in the M2 phenotype.8 Additionally, M1 macrophages show elevated cathepsin S and L mRNA levels. Moreover, both mRNA and protein levels of cathepsin B, L, and S are overexpressed in murine atherosclerotic plaques. Similarly, in advanced human atherosclerotic plaques, cathepsin B and S were found overexpressed within macrophages, Background and Purpose-Atherosclerosis is a leading cause of mortality worldwide, contributing to both strokes and heart attacks. Macrophages are key players in atherogenesis, promoting vascular inflammation and arterial remodeling through cysteine cathepsin proteases. We used a cathepsin-targeted activity-based probe in human carotid plaque to assess its diagnostic potential and evaluate macrophage subtypes ex vivo. Methods-Carotid plaque specimens surgically removed during endarterectomy from 62 patients (age range, 38% female, 28% symptomatic) were graded pathologically as either stable (Grade 1) or unstable (Grade 2 or 3). A cathepsin activitybased probe was used to quantify individual cathepsins in plaque tissue and macrophage subtypes. Results-Cathepsin B and S activities were increased in unstable carotid plaques. They were quantified using the probe to biochemically investigate individual cathepsins (Cathepsin B and S: 0.97

Cysteine proteases in atherosclerosis

“…These included members of the cysteine proteases such as cathepsins B and Z among other tissue remodeling factors such as matrix metalloproteinase . Likewise, we recently reported on altered gene expression pattern and activity of cysteine cathepsins in macrophages from unstable lesions . This molecular information implies that signals emanating from the plaque microenvironment will provide more accurate predictions relating to patient outcome compared with standard assessment techniques .…”

“…Furthermore, the covalent binding of the enzyme to the small molecule enables downstream analysis, such as by SDS/PAGE. We recently applied this method to human carotid endarterectomy samples to show that cathepsin activity may be used as a surrogate marker to distinguish symptomatic patients . In addition, labeling cathepsin activity with qABP has been successfully implemented in preclinical animal models of atherosclerosis .…”

Atherosclerosis Treatment with Stimuli‐Responsive Nanoagents: Recent Advances and Future Perspectives

“…As mentioned before, cathepsin B is a protease secreted by macrophages that plays a major role in ECM degradation together with MMPs. In unstable plaques, cathepsin B activity is significantly higher than in stable plaques . Therefore, it can serve as both a stimulus and a target for drug delivery in atherosclerosis management.…”

Engineered nanomaterial-induced lysosomal membrane permeabilization and anti-cathepsin agents

“…Cathepsin B has been associated with ischemic cell death resulting from LMP in cerebral ischemia/reperfusion injured rats, an effect that is attenuated by CA074-me, a cathepsin B inhibitor that protects against lysosomal rupture (Xu et al 2016). Cathepsin B and S activities are also elevated in unstable carotid plaques, which contribute to the inflammatory development of atherosclerosis (Abd-Elrahman et al 2016), and cathepsin B activity is increased in patients with active arthritis (Däbritz et al 2016). Cathepsin B was also identified as a key regulator of lysosomal biogenesis (Qi et al 2016).…”