Selective Cathepsin S Inhibition Attenuates Atherosclerosis in Apolipoprotein E–Deficient Mice with Chronic Renal Disease

Abstract

Chronic renal disease (CRD) accelerates the development of atherosclerosis. The potent protease cathepsin S cleaves elastin and generates bioactive elastin peptides, thus promoting vascular inflammation and calcification. We hypothesized that selective cathepsin S inhibition attenuates atherogenesis in hypercholesterolemic mice with CRD. CRD was induced by 5/6 nephrectomy in high-fat high-cholesterol fed apolipoprotein E-deficient mice. CRD mice received a diet admixed with 6.6 or 60 mg/kg of the potent and selective cathepsin S inhibitor RO5444101 or a control diet. CRD mice had significantly higher plasma levels of osteopontin, osteocalcin, and osteoprotegerin (204%, 148%, and 55%, respectively; P < 0.05), which were inhibited by RO5444101 (60%, 40%, and 36%, respectively; P < 0.05). Near-infrared fluorescence molecular imaging revealed a significant reduction in cathepsin activity in treated mice. RO5444101 decreased osteogenic activity. Histologic assessment in atherosclerotic plaque demonstrated that RO5444101 reduced immunoreactive cathepsin S (P < 0.05), elastin degradation (P = 0.01), plaque size (P = 0.01), macrophage accumulation (P < 0.01), growth differentiation factor-15 (P = 0.0001), and calcification (alkaline phosphatase activity, P < 0.01; osteocalcin, P < 0.05). Furthermore, cathepsin S inhibitor or siRNA significantly decreased expression of growth differentiation factor-15 and monocyte chemotactic protein-1 in a murine macrophage cell line and human primary macrophages. Systemic inhibition of cathepsin S attenuates the progression of atherosclerotic lesions in 5/6 nephrectomized mice, serving as a potential treatment for atherosclerosis in patients with CRD.

Dual-Modality Activity-Based Probes as Molecular Imaging Agents for Vascular Inflammation

“…The intensity of the labeling pattern of the arterial wall architecture in fresh frozen tissue samples of both the ligated and the unligated carotid arteries of the diseased hyperlipidemic and diabetic FVB mice, compared with the control animal sections, was somewhat surprising. When these sections were costained with elastin, the signal for the probe and elastin colocalized, consistent with previous reports that cathepsin S and elastin colocalize, particularly in regions of elastin breaks or remodeling (28). The labeling of these architectural elements in the unligated carotid artery of the diseased animal was particularly interesting, as these sections did not contain plaques.…”

“…Cellular studies using in situ and ex vivo optical methods were also performed and confirm that probes are highly specific and that they can provide an accurate readout of levels of these proteases that correlates with disease severity. In concurrence with previous studies (27,28), probe labeling also colocalized with elastin in carotid samples. We also demonstrate the use of the probes for detecting macrophage-derived cathepsins in human carotid endarterectomy specimen by topical application of the probe to the tissue samples.…”

“…These proteases have been shown to play important roles in extracellular matrix remodeling and are implicated in the development and progression of atherosclerosis (26). For example, cathepsin S has been shown to colocalize with a major protein of the extracellular matrix, elastin, in the arterial medium of atheromas (27), especially in regions of elastin breaks (28). Several cysteine cathepsin-specific probes have been developed for both optical and PET imaging of tumor progression and lung inflammation (29)(30)(31).…”

A novel approach for reliable detection of cathepsin S activities in mouse antigen presenting cells

“…CTSS dysregulation-associated autoimmune diseases are, i.e. atherosclerosis (Figueiredo et al, 2015), multiple sclerosis (Haves-Zburof et al, 2011;Fissolo et al, 2008), psoriasis (Schonefuss et al, 2010) and the Sjögren's syndrome (Hamm-Alvarez et al, 2014). Additionally, enhanced CTSS secretion into the intestinal lumen is reported to promote pain induction during inflammatory bowel disease (Cattaruzza et al, 2011).…”

“…Furthermore, CTSS dysregulation is also involved in cancer progression (Sobotic et al, 2015). This involvement of CTSS in the pathogenesis of various diseases makes this protease an intensely studied drug target (Figueiredo et al, 2015;Kohl et al, 2015;Vazquez et al, 2015;Jadhav et al, 2014). In order to evaluate the precise role of CTSS function in this context, not only protein concentrations of CTSS should be detected but rather its catalytic activity as protein amount and enzymatic activity do not necessarily need to correlate.…”