Objective-Lysosomal proteinases have been implicated in a number of pathologies associated with extracellular matrix breakdown. Therefore, we investigated the possibility that the lysosomal proteinase cathepsin S may be involved in atherosclerotic plaque destabilization. Methods and Results-Atherosclerotic plaques in the brachiocephalic arteries of fat-fed apolipoprotein E/cathepsin S double knockout mice had 73% fewer acute plaque ruptures (Pϭ0.026) and were 46% smaller (Pϭ0.025) than those in age-, strain-, and sex-matched apolipoprotein E single knockout controls. When the incidence of acute plaque rupture was normalized for plaque size, the reduction in the double knockouts was 72% (Pϭ0.039). The number of buried fibrous layers, indicative of an unstable plaque phenotype, was reduced by 67% in the double knockouts (Pϭ0.008). The cysteine proteinase inhibitor, egg white cystatin, was biotinylated and used as an active-site-directed probe for cathepsins. Biotinylated cystatin selectively detected cathepsin S in extracts of human carotid atherosclerotic plaque. Active cathepsin S was detectable in extracts of human atherosclerotic plaque but not in nondiseased carotid arteries. Active cathepsins were especially prominent in macrophages in the shoulder regions of plaques, areas considered to be vulnerable to rupture. Cathepsin S protein colocalized with regions of elastin degradation in human coronary plaques. Conclusion-These data provide direct evidence that an endogenous proteinase, cathepsin S, plays an important role in atherosclerotic plaque destabilization and rupture. Key Words: atherosclerosis Ⅲ plaque Ⅲ pathology Ⅲ cathepsin T he highly thrombogenic gruel in the core of an atherosclerotic plaque is luminally covered by a fibrous cap, weakening of which leads to plaque rupture and thrombus formation. Macrophages and T cells accumulate at sites of plaque disruption. 1 Degradation of collagen by macrophagederived matrix metalloproteinases has been reported, 2 but less is known of other classes of proteinase, which may be released by activated macrophages.The lysosomal cathepsins have been implicated in the development and progression of atherosclerosis. Increased levels of cathepsins F, K, and S are present in atherosclerotic lesions, 3,4 whereas levels of the major extracellular inhibitor of cysteine proteinases, cystatin C, are decreased. 5 In humans, an association has been shown between a genetically determined decrease in cystatin C levels and the severity of coronary artery disease. 6 Cathepsin S/low-density lipoprotein (LDL) receptor double knockout mice have impaired atherogenesis when compared with LDL receptor single knockout controls. 7 Grading of atherosclerosis in the aortic arch showed that there was a delay in lesion progression in the double knockouts. For example, 12 weeks of feeding atherogenic diet to the double knockouts resulted in an average lesion severity similar to that seen in single knockout controls after just 8 weeks of feeding, and 26 weeks of atherogenic diet feeding in the ...

The effects of chloride channel blockers on pressure‐induced constriction, K+‐induced force, and whole‐cell calcium channel currents were tested in rat cerebral arteries using isobaric and isometric myography, and patch clamp. Under isobaric conditions at 75 mmHg, NPPB), a chloride channel blocker, reversibly depressed the myogenic constriction with an IC50 of 32.8 ± 0.52 μm (mean ± s.e.m., n= 5). Blockers of Ca2+‐activated chloride channels, flufenamic acid (100 μm) and 9‐AC; 1 mm), and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel blocker, glibenclamide (100 μm), were without effect in this tissue (n= 3). Under isobaric conditions at 20 mmHg, 37 °C, raising [K+]o to 45 mm induced a constriction which was unaffected by 100 μm NPPB (n= 4). In contrast, at 75 mmHg and 18‐21 °C, 100 μm NPPB completely and reversibly blocked a 45 mm K+‐induced constriction (n= 3). Under isometric conditions, NPPB reversibly depressed a 45 mm K+‐induced force with an IC50 of 10.0 ± 0.76 μm (mean ± s.e.m., n= 5). IAA‐94), another chloride channel blocker, depressed the K+‐induced force with an IC50 of 17.0 ± 1.2 μm (mean ± s.e.m., n= 4). Using whole‐cell patch clamp, 100 μm NPPB or 200 μm IAA‐94 blocked calcium channel currents carried by 10 mm Ba2+ by 79.1 ± 1.7 and 39.8 ± 7.0 %, respectively (mean ± s.e.m., n= 6). In summary, chloride channel blockers depress calcium channel currents in rat cerebral arteries, which could contribute to a reduction in myogenic contraction.

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Streptomycin has been demonstrated to inhibit mechanosensitive conductances in a wide variety of cell types, including muscle. The action of streptomycin on rat cerebral arteries that exhibit pressure‐induced myogenic response was investigated. Pressure‐induced tone, measured using isobaric myography, in isolated pressurized cerebral arteries was reversibly and concentration‐dependently inhibited by streptomycin with an IC50 of 2.6 mM. Isometric K+‐induced force, measured using isometric myography, is supported by voltage‐gated Ca2+ entry. Streptomycin reversibly and concentration‐dependently inhibited isometric force with an IC50 of 1.71 mM. Voltage‐gated macroscopic inward Ca2+ channel currents were recorded from freshly isolated rat basilar myocytes. These were reversibly and concentration‐dependently inhibited by streptomycin with an IC50 of 1.79 and 0.47 mM when 10 and 1.8 mM CaCl2, respectively, was used as the charge carrier. These data suggest that streptomycin inhibits myogenic tone and K+‐induced isometric force largely by blockade of L‐type, dihydropyridine‐sensitive Ca2+ channels. In conclusion, streptomycin is not useful in the investigation of stretch‐activated channels which may underlie the myogenic response of rat small cerebral arteries.

The time-course of endothelial regrowth and functional recovery following polytetrafluoroethylene filament-induced endothelial denudation in vivo was studied in the left common carotid artery of the mouse. This technique does not result in any intimal hyperplasia, enabling the investigation of endothelial function without any confounding effect of intimal thickening. Endothelial coverage was assessed histologically, and functional recovery was assessed as restoration of receptor-mediated, endothelium-dependent relaxation to acetylcholine in vitro. Re-endothelialization of the carotid artery was complete within 8 days of denudation. However, relaxations to acetylcholine, which are mediated by endothelium-derived nitric oxide, were only partially restored 10 days after the procedure. At this time point, arterial responses to either phenylephrine, the receptor-independent endothelium-dependent dilator cyclopiazonic acid, or the nitric oxide donor diethylamine NONOate, were not significantly different to controls. At 25 days after denudation, acetylcholine-evoked responses remained significantly depressed compared to controls but at 90 days full recovery was observed. These data indicate that following mechanical denudation of the mouse carotid artery, although endothelial re-growth is complete within 8 days, recovery of endothelial cell function – assessed as the ability of the regenerated endothelium to mediate acetylcholine-stimulated relaxation – remains impaired for a prolonged period.

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