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. 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. Half of all patients with chronic renal disease (CRD) die of cardiovascular causes.1Campean V. Neureiter D. Varga I. Runk F. Reiman A. Garlichs C. Achenbach S. Nonnast-Daniel B. Amann K. Atherosclerosis and vascular calcification in chronic renal failure.Kidney Blood Press Res. 2005; 28: 280-289Crossref PubMed Scopus (62) Google Scholar, 2Schiffrin E.L. Lipman M.L. Mann J.F. Chronic kidney disease: effects on the cardiovascular system.Circulation. 2007; 116: 85-97Crossref PubMed Scopus (1209) Google Scholar Patients with early stages of CRD who are not undergoing dialysis have cardiovascular disease (CVD) risk similar to that of patients with established coronary artery disease,3Foley R.N. Parfrey P.S. Sarnak M.J. Clinical epidemiology of cardiovascular disease in chronic renal disease.Am J Kidney Dis. 1998; 32: S112-S119Abstract Full Text PDF PubMed Scopus (2991) Google Scholar whereas patients with end-stage renal disease, treated by dialysis, have an approximately 30 times greater CVD risk than the general population.4de Jager D.J. Grootendorst D.C. Jager K.J. van Dijk P.C. Tomas L.M. Ansell D. Collart F. Finne P. Heaf J.G. De Meester J. Wetzels J.F. Rosendaal F.R. 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Inflammation, atherosclerosis, and coronary artery disease.N Engl J Med. 2005; 352: 1685-1695Crossref PubMed Scopus (7069) Google Scholar, 9Libby P. Aikawa M. Stabilization of atherosclerotic plaques: new mechanisms and clinical targets.Nat Med. 2002; 8: 1257-1262Crossref PubMed Scopus (499) Google Scholar Several molecular mechanisms participate in the response to injuries at the vascular wall and in the formation and progression of atherosclerotic lesions.10Businaro R. Tagliani A. Buttari B. Profumo E. Ippoliti F. Di Cristofano C. Capoano R. Salvati B. Rigano R. Cellular and molecular players in the atherosclerotic plaque progression.Ann N Y Acad Sci. 2012; 1262: 134-141Crossref PubMed Scopus (46) Google Scholar Chronic inflammation likely accelerates atherosclerosis in patients with CRD,11McIntyre C.W. Harrison L.E. Eldehni M.T. Jefferies H.J. Szeto C.C. John S.G. Sigrist M.K. Burton J.O. Hothi D. Korsheed S. Owen P.J. Lai K.B. Li P.K. Circulating endotoxemia: a novel factor in systemic inflammation and cardiovascular disease in chronic kidney disease.Clin J Am Soc Nephrol. 2011; 6: 133-141Crossref PubMed Scopus (350) Google Scholar and the combination of chronic inflammation and an imbalance in the calcium phosphate serum level in these patients exacerbates these processes.12Navarro-Gonzalez J.F. Mora-Fernandez C. Muros M. Herrera H. Garcia J. Mineral metabolism and inflammation in chronic kidney disease patients: a cross-sectional study.Clin J Am Soc Nephrol. 2009; 4: 1646-1654Crossref PubMed Scopus (75) Google Scholar, 13Chen H.Y. Chiu Y.L. Hsu S.P. Pai M.F. Lai C.F. Yang J.Y. Peng Y.S. Tsai T.J. Wu K.D. Elevated C-reactive protein level in hemodialysis patients with moderate/severe uremic pruritus: a potential mediator of high overall mortality.QJM. 2010; 103: 837-846Crossref PubMed Scopus (50) Google Scholar In addition, CRD patients receiving hemodialysis have elevated levels of circulating proinflammatory cytokines,14Lam M.F. Leung J.C. Lam C.W. Tse K.C. Lo W.K. Lui S.L. Chan T.M. Tam S. Lai K.N. Procalcitonin fails to differentiate inflammatory status or predict long-term outcomes in peritoneal dialysis-associated peritonitis.Perit Dial Int. 2008; 28: 377-384Crossref PubMed Scopus (20) Google Scholar which can initiate and perpetuate the inflammation-calcification loop. Cathepsin S plays a critical role in vascular inflammation and calcification. Monocyte-derived macrophages that mediate vascular inflammation express and secrete the cysteine protease cathepsin S.15Pan L. Li Y. Jia L. Qin Y. Qi G. Cheng J. Qi Y. Li H. Du J. Cathepsin S deficiency results in abnormal accumulation of autophagosomes in macrophages and enhances Ang II-induced cardiac inflammation.PLoS One. 2012; 7: e35315Crossref PubMed Scopus (76) Google Scholar At the basal membrane of the blood vessels, secreted cathepsin S cleaves several extracellular matrix proteins, including laminin, collagen, and, preferentially, elastin, which generate bioactive elastin peptides.16Lohoefer F. Reeps C. Lipp C. Rudelius M. Zimmermann A. Ockert S. Eckstein H.H. Pelisek J. Histopathological analysis of cellular localization of cathepsins in abdominal aortic aneurysm wall.Int J Exp Pathol. 2012; 93: 252-258Crossref PubMed Scopus (28) Google Scholar, 17Samouillan V. Dandurand J. Nasarre L. Badimon L. Lacabanne C. Llorente-Cortes V. Lipid loading of human vascular smooth muscle cells induces changes in tropoelastin protein levels and physical structure.Biophys J. 2012; 103: 532-540Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Elastin-derived peptides and fragments, also known as matrikines, can incite inflammation. Elastin peptides stimulate macrophage chemotaxis18Simpson C.L. Lindley S. Eisenberg C. Basalyga D.M. Starcher B.C. Simionescu D.T. Vyavahare N.R. Toward cell therapy for vascular calcification: osteoclast-mediated demineralization of Pathol. 2007; PubMed Scopus Google Scholar and vascular inflammation and J. G.K. P. cysteine in PubMed Scopus Google Scholar In addition, cathepsin S with of elastin in atherosclerotic P.A. J.Y. D. inhibition of cathepsin S atherosclerotic lesions in 2010; PubMed Scopus Google Scholar We that cathepsin S deficiency to reduced activity and decreased vascular inflammation and calcification in the of hypercholesterolemic mice with in cathepsin in and aortic E. Aikawa M. P. G. Y. D. R. Arterial and aortic calcification by cathepsin S deficiency in chronic renal 2009; PubMed Scopus Google Scholar a of the the that treatment with a selective cathepsin S inhibitor attenuates inflammation and atherosclerotic lesion formation in the of hypercholesterolemic mice with CRD. mice = from The were fed a high-fat high-cholesterol diet for At of mice were to with the diet = or to 5/6 nephrectomy = CRD mice were treated with 6.6 or 60 mg/kg of a potent and selective cathepsin S inhibitor = admixed with the high-fat high-cholesterol diet for an The on all the blood cells were by in and were for in with human and to differentiate macrophages. cells were from and in with We an established to chronic renal by the of kidney E. Aikawa M. P. G. Y. D. R. Arterial and aortic calcification by cathepsin S deficiency in chronic renal 2009; PubMed Scopus Google Scholar to E. Aikawa M. P. G. Y. D. R. Arterial and aortic calcification by cathepsin S deficiency in chronic renal 2009; PubMed Scopus Google Scholar, S. J.F. E. K. Chronic renal accelerates atherogenesis in apolipoprotein Am Soc Nephrol. 2003; PubMed Scopus Google Scholar the and of the of the kidney was mice received of molecular imaging a and a calcium for and for in fluorescence of was a fluorescence as E. M. Aikawa M. R. with inflammation in atherosclerosis by molecular imaging in 2007; 116: PubMed Scopus Google Scholar, E. M. D. Aikawa M. R. molecular imaging and osteogenic in early aortic 2007; PubMed Scopus Google Scholar the heart with to and were and were an imaging were and were for of the and mice = received and blood were at in The were on and at to of levels in plasma was by was in which were in with protease were and were to was with primary and with The membrane was by analysis Blood was the and was in a serum was at levels of osteogenic including protein or osteocalcin, secreted or osteopontin, and osteoprotegerin or factor were by were in and were and with and for general phosphatase activity was on (alkaline phosphatase was to analysis for macrophages cathepsin S cathepsin and growth differentiation factor-15 was the were and an or were for of was cathepsin S and or muscle were with to were an cells were with siRNA cathepsin S or control siRNA for the the was from human and murine macrophages was an and was by and was in the The from were and and and and and The levels of the were to levels for human and to levels for murine activity was by the in fluorescence by of a a the of which is in the The of and The at were as human and cathepsins and were for cathepsin which from was and was was to the in and the was with was accumulation was in human cells by cells were from human with of cells a were with of RO5444101 for and were in with protease were and were to was with primary and The membrane was by analysis are as of and were an inhibitor with high to cathepsin S not cathepsins the of cathepsin S with high was an in and cathepsins and or to of to The cellular activity of the was in of in human Cathepsin S cleaves the a of the of cathepsin S in cells results in the accumulation of an of the by analysis The of RO5444101 was to We also the effects of in mice and that significantly induced accumulation in in and in levels and the response in of cathepsin S of in chronic inflammatory and the of RO5444101 and in mice and an inhibitor to the of cathepsin S in the of chronic inflammatory including and of the Cathepsin S or effects of RO5444101 on cathepsin S from and human The high and cathepsins in also high cathepsin S in in a new of the Cathepsin S in a new In effects of RO5444101 on cathepsin S from and human The high and cathepsins in also high cathepsin S in levels of secreted and osteoprotegerin factor in mice with CRD 5/6 nephrectomy of CRD mice with the cathepsin S inhibitor RO5444101 these We in plasma levels of (P < P < 60 (P < P < 60 and osteoprotegerin (P < P = 60 with CRD results that RO5444101 at 60 mg/kg reduced of these proinflammatory and to with Cathepsin S of and in with CRD with < the treatment and the CRD < the treatment and the CRD < the treatment and the CRD < the treatment and the CRD < the treatment and the CRD < the treatment and the CRD CRD 6.6 < the treatment and the CRD < the treatment and the CRD < the treatment and the CRD CRD 60 < the treatment and the CRD < the treatment and the CRD < the treatment and the CRD were from mice at the of the treatment and plasma levels of the osteogenic were by chronic renal P < the treatment and the CRD P < the treatment and the CRD in a new Blood were from mice at the of the treatment and plasma levels of the osteogenic were by chronic renal fluorescence in overall cathepsin activity with a in imaging that is by including cathepsins and S. with CRD in the with control an that cathepsin S inhibitor treatment limited In addition, fluorescence imaging to cathepsin activity in the and We a significant reduction in in mice treated with RO5444101 (P < 60 mg/kg CRD and analysis that cathepsin S inhibitor treatment accumulation of cathepsin S in the atherosclerotic and the molecular imaging and analysis results that cathepsin S inhibitor treatment reduced activity and cathepsin S protein levels in the of CRD mice. Cathepsin S is the potent elastin in the of aortic We that RO5444101 treatment significantly reduced the of elastin as by van (P = and results that mice treated with a high of cathepsin S inhibitor had reduced of cathepsin S and limited elastin that the inhibition of cathepsin S elastin analysis for cathepsin and muscle in from CRD mice the cell responsible for cathepsin S expression and We that cathepsin S in macrophages that cathepsin S expression was also in smooth muscle CRD mice treated with the cathepsin S inhibitor RO5444101 had decreased plaque in the of the aortic (P = and The were to the atherosclerotic lesions to develop in mice on a high-fat high-cholesterol diet for the of CRD and cathepsin S inhibition for the effects of the cathepsin inhibitor RO5444101 on established atherosclerotic not on the initiation of atherosclerosis. We significant in the lesion size and macrophage accumulation in CRD mice treated with a high of the treatment reduced the of CRD mice to levels similar to of with CRD. results that can the development of atherosclerosis in CRD. the mechanisms of plaque size macrophage accumulation and RO5444101 treatment decreased macrophage accumulation and in and as by for (P < for 6.6 and P < for 60 and and macrophage P < for 6.6 and 60 and results that cathepsin S inhibition can the inflammatory and and atherosclerotic lesion formation in CRD. the cell of cathepsin S of cathepsin S and macrophages or smooth muscle that cathepsin S is in and macrophages CRD mice treated with 60 mg/kg of RO5444101 not have significantly reduced osteogenic in the and and as by the calcium treatment significantly phosphatase activity (P < and and expression (P < and in the of aortic with the these results demonstrated that mice with CRD have osteogenic which were reduced with cathepsin S The in that cathepsin inhibition by RO5444101 reduced macrophage accumulation and expression of a for macrophage in the of CRD mice and The was a in from reduced macrophages or also decreased macrophage the that cathepsin S inhibition expression by macrophage in cell RO5444101 reduced expression induced by in human primary macrophages from blood and cells The RO5444101 to cathepsin S as siRNA of the also reduced by RO5444101 or cathepsin S by siRNA reduced expression of monocyte chemotactic a proinflammatory than of all patients with CRD die of cardiovascular causes.1Campean V. Neureiter D. Varga I. Runk F. Reiman A. Garlichs C. Achenbach S. Nonnast-Daniel B. Amann K. Atherosclerosis and vascular calcification in chronic renal failure.Kidney Blood Press Res. 2005; 28: 280-289Crossref PubMed Scopus (62) Google Scholar, 2Schiffrin E.L. Lipman M.L. Mann J.F. Chronic kidney disease: effects on the cardiovascular system.Circulation. 2007; 116: 85-97Crossref PubMed Scopus (1209) Google Scholar Despite effects in CRD M.J. Levey A.S. Schoolwerth A.C. Coresh J. Culleton B. Hamm L.L. McCullough P.A. Kasiske B.L. Kelepouris E. Klag M.J. Parfrey P. Pfeffer M. Raij L. Spinosa D.J. Wilson P.W. Kidney disease as a risk factor for development of cardiovascular disease: a statement from the American Heart Association councils on kidney in cardiovascular disease, high blood pressure research, clinical cardiology, and epidemiology and prevention.Hypertension. 2003; 42: 1050-1065Crossref PubMed Scopus (957) Google Scholar, B.C. Jardine A.G. Schmieder R.E. Holdaas H. Bannister K. Beutler J. Chae D.W. Chevaile A. Cobbe S.M. Gronhagen-Riska C. De Lima J.J. Lins R. Mayer G. McMahon A.W. Parving H.H. Remuzzi G. Samuelsson O. Sonkodi S. Sci D. Suleymanlar G. Tsakiris D. Tesar V. Todorov V. Wiecek A. Wuthrich R.P. Gottlow M. Johnsson E. Zannad F. Rosuvastatin and cardiovascular events in patients undergoing hemodialysis.N Engl J Med. 2009; 360: 1395-1407Crossref PubMed Scopus (1622) Google Scholar need new that cardiovascular risk in CRD The that and selective inhibition of cathepsin S inflammation and calcification in mice with a novel for CVD risk in We that of the selective cathepsin S inhibitor which not the activity of several key of in of atherosclerosis in CRD including reduction of atherosclerotic plaque reduction of macrophage accumulation and in atherosclerotic as by expression of and the proinflammatory reduction of cathepsin activity and the of elastin in the atherosclerotic reduction of osteogenic activity and of vascular and reduction of plasma levels of circulating osteogenic The results of RO5444101 are in results that to cathepsin S activity vascular inflammation and in cathepsin E. Aikawa M. P. G. Y. D. R. Arterial and aortic calcification by cathepsin S deficiency in chronic renal 2009; PubMed Scopus Google Scholar In addition, the results that of elastin by selective inhibition of cathepsin S activity inflammation and and cardiovascular in patients with CRD. Cathepsin cysteine as cathepsin S key in several including aneurysm Y. J. Y. L. H. Li H. C. Du J. of cathepsin S attenuates II-induced abdominal aortic aneurysm formation in apolipoprotein Res. 2012; PubMed Scopus Google Scholar G.K. Y. Y. M. S. M.L. Y. M.T. P. of cathepsin S atherosclerosis in LDL 2003; PubMed Scopus Google Scholar and vascular E. Aikawa M. P. G. Y. D. R. Arterial and aortic calcification by cathepsin S deficiency in chronic renal 2009; PubMed Scopus Google Scholar Cathepsin S is of the potent cysteine by macrophages in In the basal membrane of the blood cathepsin S as a potent and promoting inflammation and E. Aikawa M. P. G. Y. D. R. Arterial and aortic calcification by cathepsin S deficiency in chronic renal 2009; PubMed Scopus Google Scholar, G.K. Y. Y. M. S. M.L. Y. M.T. P. of cathepsin S atherosclerosis in LDL 2003; PubMed Scopus Google Scholar thus a for We that mice cathepsin S with induced CRD significantly reduced elastin and calcification in the and aortic E. Aikawa M. P. G. Y. D. R. Arterial and aortic calcification by cathepsin S deficiency in chronic renal 2009; PubMed Scopus Google Scholar In the expression of cathepsins or matrix not We thus that cathepsin S accelerates cardiovascular calcification an The a with on cathepsin mice, selective cathepsin S inhibition by of novel RO5444101 reduced elastin in hypercholesterolemic mice with CRD. RO5444101 also reduced osteogenic activity in atherosclerotic of cathepsin S activity or similar to a role for cathepsin S inhibition in the of elastin and vascular calcification. The also the role of cathepsin S in atherosclerosis in mice with CRD and that selective cathepsin S inhibition significantly atherosclerotic plaque likely from reduced as decreased plaque accumulation of macrophages. the initiation and progression of atherosclerotic plaque and E. M. Aikawa M. R. with inflammation in atherosclerosis by molecular imaging in 2007; 116: PubMed Scopus Google Scholar, M. P. The atherosclerotic and Pathol. PubMed Scopus Google Scholar We that of elastin E. Aikawa M. P. G. Y. D. R. Arterial and aortic calcification by cathepsin S deficiency in chronic renal 2009; PubMed Scopus Google Scholar Elastin-derived proinflammatory including macrophage C.L. Lindley S. Eisenberg C. Basalyga D.M. Starcher B.C. Simionescu D.T. Vyavahare N.R. Toward cell therapy for vascular calcification: osteoclast-mediated demineralization of Pathol. 2007; PubMed Scopus Google Scholar to vascular J. G.K. P. cysteine in PubMed Scopus Google Scholar et G.K. Y. Y. M. S. M.L. Y. M.T. P. of cathepsin S atherosclerosis in LDL 2003; PubMed Scopus Google Scholar that cathepsin S reduced atherosclerotic plaque macrophage and proinflammatory in hypercholesterolemic mice. The similar in hypercholesterolemic apolipoprotein E–deficient mice with CRD that the of atherosclerosis and not the effects of cathepsin S is a of the growth factor and was as a novel with CVD in the general M. S. Wu Y. Li H. Y. F. Y. for the of cardiovascular events in patients with to coronary artery a Heart J. 2012; PubMed Scopus Google Scholar and in patients with J.J. Tsai Wu L. L. P. O. L. S. A. H. M. P. and mortality in end-stage renal Dial 2012; PubMed Scopus Google Scholar protein in and cathepsin S inhibition significantly reduced accumulation in aortic in primary macrophages and macrophage cell demonstrated that cathepsin S inhibitor or siRNA reduced macrophage with the in a known of macrophage S.M. A.G. M. M. S. K. a novel macrophage is a of the Acad Sci S A. PubMed Scopus Google Scholar to the proinflammatory role of cathepsin S. also mortality risk in patients with J.J. Tsai Wu L. L. P. O. L. S. A. H. M. P. and mortality in end-stage renal Dial 2012; PubMed Scopus Google Scholar atherosclerotic lesion development and CRD. macrophage in a and growth factor Jager B. I. M. A. V. C. van T.J. J. R. differentiation factor deficiency atherosclerosis by macrophage Exp Med. 2011; PubMed Scopus Google Scholar We thus the effects of cathepsin S inhibition on that cathepsin S inhibition macrophage expression of selective cathepsin S inhibition macrophage in atherosclerotic lesions and vascular inflammation and plaque as cathepsin inflammation. to the effects of cathepsin S inhibition from in In to effects on macrophages and inhibitor the levels and the response in and inhibition in that cathepsin S in cell or as reduced response of cells and of cells and J. E. H. A. F. E. V. P. cathepsin not cathepsin in Acad Sci S A. 2003; PubMed Scopus (76) Google Scholar, C. A. M.J. P. Cathepsin S by cells in 2005; PubMed Scopus Google Scholar mechanisms cathepsin S as an for than atherosclerosis, as and In the significantly elevated risk of cardiovascular and mortality in patients with CRD statin treatment for new for a that demonstrated the of cathepsin S on cardiovascular inflammation and calcification by selective cathepsin S inhibition as a novel that the progression of atherosclerotic lesions and cardiovascular in patients with CRD. that of molecular mechanisms and assessment of the cathepsin S inhibitor in patients new the of CVD in CRD We and for