Superoxide-Dependent Cathepsin Activation Is Associated with Hypertensive Myocardial Remodeling and Represents a Target for Angiotensin II Type 1 Receptor Blocker Treatment
2008; Elsevier BV; Volume: 173; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2008.071126
ISSN1525-2191
AutoresXian Wu Cheng, Toyoaki Murohara, Masafumi Kuzuya, Hideo Izawa, Takeshi Sasaki, Koji Obata, Kohzo Nagata, Takao Nishizawa, Masakazu Kobayashi, Takashi Yamada, Weon Kim, Kohji Sato, Guo‐Ping Shi, Kenji Okumura, Mitsuhiro Yokota,
Tópico(s)Renin-Angiotensin System Studies
ResumoThe elastolytic activity of cathepsins in the myocardium is implicated in hypertensive heart failure (HF). Given that reactive oxygen species are also implicated in protease activation associated with cardiac remodeling, we examined the role of the reactive oxygen species-induced cathepsin activation system in cardiac remodeling during the development of hypertensive HF. Dahl salt-sensitive hypertensive rats maintained on a high-salt diet were treated with vehicle, the cathepsin inhibitor E64d, or the angiotensin receptor blocker olmesartan from 12 to 19 weeks of age. Cathepsin expression and activity were increased in the left ventricle of HF rats; olmesartan inhibited these effects, restored the balance between elastin and collagen in the left ventricle, and suppressed degradation of the elastic lamina of coronary arteries of HF rats. Furthermore, olmesartan inhibited up-regulation of NADPH oxidase subunits and activity as well as superoxide generation. These effects of olmesartan were mimicked by E64d and were accompanied by amelioration of cardiac fibrosis. Finally, olmesartan and apocynin reduced angiotensin II-induced increases in cathepsin mRNA and protein levels in cultured rat neonatal cardiac myocytes. These data suggest that cathepsins likely trigger and promote cardiac remodeling and that blocking the angiotensin II type 1 receptor attenuates cathepsin expression and activity by inhibiting the production of superoxide by NADPH oxidase, thereby attenuating cardiac remodeling and dysfunction. The elastolytic activity of cathepsins in the myocardium is implicated in hypertensive heart failure (HF). Given that reactive oxygen species are also implicated in protease activation associated with cardiac remodeling, we examined the role of the reactive oxygen species-induced cathepsin activation system in cardiac remodeling during the development of hypertensive HF. Dahl salt-sensitive hypertensive rats maintained on a high-salt diet were treated with vehicle, the cathepsin inhibitor E64d, or the angiotensin receptor blocker olmesartan from 12 to 19 weeks of age. Cathepsin expression and activity were increased in the left ventricle of HF rats; olmesartan inhibited these effects, restored the balance between elastin and collagen in the left ventricle, and suppressed degradation of the elastic lamina of coronary arteries of HF rats. Furthermore, olmesartan inhibited up-regulation of NADPH oxidase subunits and activity as well as superoxide generation. These effects of olmesartan were mimicked by E64d and were accompanied by amelioration of cardiac fibrosis. Finally, olmesartan and apocynin reduced angiotensin II-induced increases in cathepsin mRNA and protein levels in cultured rat neonatal cardiac myocytes. These data suggest that cathepsins likely trigger and promote cardiac remodeling and that blocking the angiotensin II type 1 receptor attenuates cathepsin expression and activity by inhibiting the production of superoxide by NADPH oxidase, thereby attenuating cardiac remodeling and dysfunction. The cardiac NADPH oxidase system has been implicated in the development of atherosclerotic lesions and heart failure (HF).1Griendling KK Sorescu D Ushio-Fukai M NAD(P)H oxidase: role in cardiovascular biology and disease.Circ Res. 2000; 86: 494-501Crossref PubMed Scopus (2631) Google Scholar, 2Gao L Wang W Li YL Schultz HD Liu D Cornish KG Zucker IH Superoxide mediates sympathoexcitation in heart failure: roles of angiotensin II and NAD(P)H oxidase.Circ Res. 2004; 95: 937-944Crossref PubMed Scopus (215) Google Scholar NADPH oxidase is thought to be a major source of reactive oxygen species (ROS) generated in the cardiovascular system in response to angiotensin II (Ang II).3Doerries C Grote K Hilfiker-Kleiner D Luchtefeld M Schaefer A Holland SM Sorrentino S Manes C Schieffer B Drexler H Landmesser U Critical role of the NAD(P)H oxidase subunit p47phox for left ventricular remodeling/dysfunction and survival after myocardial infarction.Circ Res. 2007; 100: 894-903Crossref PubMed Scopus (189) Google Scholar, 4Maack C Kartes T Kilter H Schafers HJ Nickenig G Bohm M Laufs U Oxygen free radical release in human failing myocardium is associated with increased activity of rac1-GTPase and represents a target for statin treatment.Circulation. 2003; 108: 1567-1574Crossref PubMed Scopus (362) Google Scholar, 5Landmesser U Cai H Dikalov S McCann L Hwang J Jo H Holland SM Harrison DG Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin II.Hypertension. 2002; 40: 511-515Crossref PubMed Scopus (495) Google Scholar Superoxide generation by NADPH oxidase has been shown to result in the activation of matrix metalloproteinases (MMPs) in vitro and in vivo as well as to contribute to the pathogenesis of atherosclerosis and cardiac fibrosis.6Grote K Flach I Luchtefeld M Akin E Holland SM Drexler H Schieffer B Mechanical stretch enhances mRNA expression and proenzyme release of matrix metalloproteinase-2 (MMP-2) via NAD(P)H oxidase-derived reactive oxygen species.Circ Res. 2003; 92: e80-e86Crossref PubMed Google Scholar, 7Luchtefeld M Grote K Grothusen C Bley S Bandlow N Selle T Struber M Haverich A Bavendiek U Drexler H Schieffer B Angiotensin II induces MMP-2 in a p47phox-dependent manner.Biochem Biophys Res Commun. 2005; 328: 183-188Crossref PubMed Scopus (107) Google Scholar, 8Yoshida J Yamamoto K Mano T Sakata Y Nishikawa N Nishio M Ohtani T Miwa T Hori M Masuyama T AT1 receptor blocker added to ACE inhibitor provides benefits at advanced stage of hypertensive diastolic heart failure.Hypertension. 2004; 43: 686-691Crossref PubMed Scopus (104) Google Scholar, 9Robbesyn F Auge N Vindis C Cantero AV Barbaras R Negre-Salvayre A Salvayre R High-density lipoproteins prevent the oxidized low-density lipoprotein-induced epidermal [corrected] growth factor receptor activation and subsequent matrix metalloproteinase-2 upregulation.Arterioscler Thromb Vasc Biol. 2005; 25: 1206-1212Crossref PubMed Scopus (62) Google Scholar However, it is unknown whether or not superoxide produced by NADPH oxidase also participates in the activation of other proteases, such as cathepsins. Cathepsins are lysosomal cysteine proteases that belong to the family of papain-like peptidases and play important roles in the degradation of the extracellular matrix (ECM).10Cheng XW Kuzuya M Nakamura K Di Q Liu Z Sasaki T Kanda S Jin H Shi GP Murohara T Yokota M Iguchi A Localization of cysteine protease, cathepsin S, to the surface of vascular smooth muscle cells by association with integrin alphanubeta3.Am J Pathol. 2006; 168: 685-694Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 11Shi GP Sukhova GK Kuzuya M Ye Q Du J Zhang Y Pan JH Lu ML Cheng XW Iguchi A Perrey S Lee AM Chapman HA Libby P Deficiency of the cysteine protease cathepsin S impairs microvessel growth.Circ Res. 2003; 92: 493-500Crossref PubMed Scopus (200) Google Scholar An increase in cathepsin gene expression or in the elastolytic activity of these proteases has been found in animals and humans with hypertensive HF or idiopathic dilated cardiomyopathy.12Cheng XW Obata K Kuzuya M Izawa H Nakamura K Asai E Nagasaka T Saka M Kimata T Noda A Nagata K Jin H Shi GP Iguchi A Murohara T Yokota M Elastolytic cathepsin induction/activation system exists in myocardium and is upregulated in hypertensive heart failure.Hypertension. 2006; 48: 979-987Crossref PubMed Scopus (85) Google Scholar, 13Sam F Siwik DA Digesting the remodeled heart: role of lysosomal cysteine proteases in heart failure.Hypertension. 2006; 48: 830-831Crossref PubMed Scopus (19) Google Scholar, 14Yan L Vatner DE Kim SJ Ge H Masurekar M Massover WH Yang G Matsui Y Sadoshima J Vatner SF Autophagy in chronically ischemic myocardium.Proc Natl Acad Sci USA. 2005; 102: 13807-13812Crossref PubMed Scopus (459) Google Scholar Cathepsins exhibit pronounced elastolytic and collagenolytic activities in vitro and in vivo.15Novinec M Grass RN Stark WJ Turk V Baici A Lenarcic B Interaction between human cathepsins K. L, and S and elastins: mechanism of elastinolysis and inhibition by macromolecular inhibitors.J Biol Chem. 2007; 282: 7893-7902Crossref PubMed Scopus (84) Google Scholar, 16Cheng XW Kuzuya M Sasaki T Arakawa K Kanda S Sumi D Koike T Maeda K Tamaya-Mori N Shi GP Saito N Iguchi A Increased expression of elastolytic cysteine proteases, cathepsins S and K, in the neointima of balloon-injured rat carotid arteries.Am J Pathol. 2004; 164: 243-251Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar On the other hand, studies of cathepsin L-deficient mice have revealed that this protease is important for the maintenance of heart structure and that its deficiency results in lysosomal impairment as well as in the consequent development of dilated cardiomyopathy.17Stypmann J Glaser K Roth W Tobin DJ Petermann I Matthias R Monnig G Haverkamp W Breithardt G Schmahl W Peters C Reinheckel T Dilated cardiomyopathy in mice deficient for the lysosomal cysteine peptidase cathepsin L.Proc Natl Acad Sci USA. 2002; 99: 6234-6239Crossref PubMed Scopus (159) Google Scholar These various observations have thus demonstrated the importance of cathepsins in cardiac remodeling. The major cardiovascular actions of Ang II have been thought to be mediated by the Ang II type 1 receptor (AT1R), and AT1R blockers (ARBs) have been widely used as antihypertensive drugs with the expectation of a cardiovascular protective effect.18Julius S Nesbitt SD Egan BM Weber MA Michelson EL Kaciroti N Black HR Grimm Jr, RH Messerli FH Oparil S Schork MA Feasibility of treating prehypertension with an angiotensin-receptor blocker.N Engl J Med. 2006; 354: 1685-1697Crossref PubMed Scopus (772) Google Scholar In this study, we explored the possibility that cathepsin activation by NADPH oxidase-derived superoxide may play an important role in hypertensive cardiac remodeling and dysfunction and might represent a target for ARB therapy in the Dahl salt-sensitive (DS) rat model. Moreover, we also evaluated the possible effects of ARB therapy on the NADPH oxidase and cathepsin activation systems in cardiac biopsy specimens from humans with hypertensive HF. Finally, we examined whether or not the Ang II signal cascade enhances cathepsin expression in cultured cardiac myocytes (CMCs) via NADPH oxidase-derived ROS. Male inbred DS rats were obtained from Eisai (Tokyo, Japan) and handled in accordance with the revised guidelines of Nagoya University Graduate School of Medicine as well as with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Weaned rats were fed laboratory chow containing 0.3% NaCl until 7 weeks of age. DS rats fed an 8% NaCl diet after 7 weeks manifest compensated concentric left ventricular (LV) hypertrophy secondary to hypertension at 12 weeks and a distinct stage of fatal LV failure with lung congestion at 19 weeks.12Cheng XW Obata K Kuzuya M Izawa H Nakamura K Asai E Nagasaka T Saka M Kimata T Noda A Nagata K Jin H Shi GP Iguchi A Murohara T Yokota M Elastolytic cathepsin induction/activation system exists in myocardium and is upregulated in hypertensive heart failure.Hypertension. 2006; 48: 979-987Crossref PubMed Scopus (85) Google Scholar, 19Nagata K Obata K Xu J Ichihara S Noda A Kimata H Kato T Izawa H Murohara T Yokota M Mineralocorticoid receptor antagonism attenuates cardiac hypertrophy and failure in low-aldosterone hypertensive rats.Hypertension. 2006; 47: 656-664Crossref PubMed Scopus (204) Google Scholar DS rats were therefore fed an 8% NaCl diet from 7 weeks of age and were randomized to an HF group, an E64d group (10 mg per kg of body mass per day, administered intraperitoneally every other day; Sigma-Aldrich, St. Louis, MO), or an olmesartan group (3 mg/kg per day in chow; Sankyo Pharmaceutical, Tokyo, Japan) from 12 to 19 weeks of age (n = 10 for each group). The doses of olmesartan (an ARB) and E64d (a synthetic cathepsin inhibitor) were determined in preliminary experiments and previous studies.20Tsuda M Iwai M Li JM Li HS Min LJ Ide A Okumura M Suzuki J Mogi M Suzuki H Horiuchi M Inhibitory effects of AT1 receptor blocker, olmesartan, and estrogen on atherosclerosis via anti-oxidative stress.Hypertension. 2005; 45: 545-551Crossref PubMed Scopus (102) Google Scholar, 21Tsubokawa T Yamaguchi-Okada M Calvert JW Solaroglu I Shimamura N Yata K Zhang JH Neurovascular and neuronal protection by E64d after focal cerebral ischemia in rats.J Neurosci Res. 2006; 84: 832-840Crossref PubMed Scopus (36) Google Scholar DS rats maintained on the 0.3% NaCl diet served as age-matched controls (control group, n = 10). At 19 weeks of age, all of the rats were euthanized by an intraperitoneal overdose of sodium pentobarbital (50 mg/kg), and the hearts were removed for biological and histological analyses.12Cheng XW Obata K Kuzuya M Izawa H Nakamura K Asai E Nagasaka T Saka M Kimata T Noda A Nagata K Jin H Shi GP Iguchi A Murohara T Yokota M Elastolytic cathepsin induction/activation system exists in myocardium and is upregulated in hypertensive heart failure.Hypertension. 2006; 48: 979-987Crossref PubMed Scopus (85) Google Scholar Arterial blood was collected from the abdominal aorta for the measurement of renin activity. Systolic blood pressure and heart rate were measured in conscious rats from 7 weeks of age, every week, using a noninvasive tail-cuff method.12Cheng XW Obata K Kuzuya M Izawa H Nakamura K Asai E Nagasaka T Saka M Kimata T Noda A Nagata K Jin H Shi GP Iguchi A Murohara T Yokota M Elastolytic cathepsin induction/activation system exists in myocardium and is upregulated in hypertensive heart failure.Hypertension. 2006; 48: 979-987Crossref PubMed Scopus (85) Google Scholar In separate experiments, 12-week-old DS rats, fed a low-salt diet from 7 weeks of age, were given vehicle, olmesartan, or E64d in the same manner as in the above experiments (n = 5 for each group), and the LV tissues for measuring targeting mRNAs and protein levels were immediately placed in liquid nitrogen and stored at −80°C. At 19 weeks of age, rats were subjected to transthoracic echocardiography. In brief, M-mode echocardiography was performed with a SONOS 7500 ultrasound system and 5- to 12-MHz ultraband transducer (Philips, Andover, MA). The peak negative myocardial velocity gradient was derived from tissue Doppler imaging as a measurement of diastolic function. After echocardiography, a 2-F high-fidelity manometer-tipped catheter (SPR-407; Millar Instruments, Houston, TX) that had been calibrated relative to atmospheric pressure was introduced through the right carotid artery into the left ventricle. Tracings of LV pressure and the electrocardiogram were digitized to determine LV end-diastolic pressure.12Cheng XW Obata K Kuzuya M Izawa H Nakamura K Asai E Nagasaka T Saka M Kimata T Noda A Nagata K Jin H Shi GP Iguchi A Murohara T Yokota M Elastolytic cathepsin induction/activation system exists in myocardium and is upregulated in hypertensive heart failure.Hypertension. 2006; 48: 979-987Crossref PubMed Scopus (85) Google Scholar Total RNA was extracted from the LV tissue and cell extracts, and the abundance of specific mRNAs was determined by reverse-transcription (RT) and real-time quantitative polymerase chain reaction (PCR) analysis as described previously.22Cheng XW Kuzuya M Nakamura K Liu Z Di Q Hasegawa J Iwata M Murohara T Yokota M Iguchi A Mechanisms of the inhibitory effect of epigallocatechin-3-gallate on cultured human vascular smooth muscle cell invasion.Arterioscler Thromb Vasc Biol. 2005; 25: 1864-1870Crossref PubMed Scopus (48) Google Scholar The sequences of primers and TaqMan probes for rats [including rat cathepsins S, K, B, D, and L; cystatin C; angiotensin-converting enzyme (ACE); AT1AR; MMP-2; and MMP-9] were described previously,10Cheng XW Kuzuya M Nakamura K Di Q Liu Z Sasaki T Kanda S Jin H Shi GP Murohara T Yokota M Iguchi A Localization of cysteine protease, cathepsin S, to the surface of vascular smooth muscle cells by association with integrin alphanubeta3.Am J Pathol. 2006; 168: 685-694Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 12Cheng XW Obata K Kuzuya M Izawa H Nakamura K Asai E Nagasaka T Saka M Kimata T Noda A Nagata K Jin H Shi GP Iguchi A Murohara T Yokota M Elastolytic cathepsin induction/activation system exists in myocardium and is upregulated in hypertensive heart failure.Hypertension. 2006; 48: 979-987Crossref PubMed Scopus (85) Google Scholar, 16Cheng XW Kuzuya M Sasaki T Arakawa K Kanda S Sumi D Koike T Maeda K Tamaya-Mori N Shi GP Saito N Iguchi A Increased expression of elastolytic cysteine proteases, cathepsins S and K, in the neointima of balloon-injured rat carotid arteries.Am J Pathol. 2004; 164: 243-251Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 19Nagata K Obata K Xu J Ichihara S Noda A Kimata H Kato T Izawa H Murohara T Yokota M Mineralocorticoid receptor antagonism attenuates cardiac hypertrophy and failure in low-aldosterone hypertensive rats.Hypertension. 2006; 47: 656-664Crossref PubMed Scopus (204) Google Scholar, 23Cheng XW Kuzuya M Sasaki T Kanda S Tamaya-Mori N Koike T Maeda K Nishitani E Iguchi A Green tea catechins inhibit neointimal hyperplasia in a rat carotid arterial injury model by TIMP-2 overexpression.Cardiovasc Res. 2004; 62: 594-602Crossref PubMed Scopus (35) Google Scholar and those for humans (including ACE, AT1R, p22phox, gp91 phox, and p47 phox) are described in Table 1. The amount of each mRNA was normalized by the corresponding amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.Table 1Sequences of Oligonucleotides Used as Forward Primers, Reverse Primers, and Detection Probes (Human)mRNAOligonucleotide sequencesGenBank locusgp91 phoxAF_229177 Forward primer (816 to 837)5′-TGCAGTCTCAGCCATATCTTC-3′ Reverse primer (878 to 899)5′-TGTGCACGCTGAAAAAGTCTTC-3′ Probe (848 to 872)5′-CACCCCTTCACCCTTACCTCTGCCC-3′P22 phoxM21186 Forward primer (121 to 140)5′-GCGCTTCACCCAGTGGTACT-3′ Reverse primer (172 to 191)5′-GGTACTCCAGCAGGCACACA-3′ Probe (142 to 164)5′-TGGTGCCTACTCCATTGTGGCGG-3′P47 phoxM32011 Forward primer (498 to 516)5′-TAGCATTGGCCACGAGCAT-3′ Reverse primer (566 to 588)5′-GGCTCATATAGCTTCTGCTTCCA-3′ Probe (523 to 548)5′-TGAGCCCAGACATTCCAAAATCGACA-3′AT1M93394 Forward primer (543 to 566)5′-TTGATCGATACCTGGCTATTGTTC-3′ Reverse primer (601 to 622)5′-GATGCAGGTGACTTTGGCTACA-3′ Probe (575 to 597)5′-AAGTCCCGCCTTCGACGCACAAT-3′ACEX16295 Forward primer (691 to 713)5′-GAGAGCCATCCTCCAGTTTTACC-3′ Reverse primer (757 to 779)5′-ACGAGTCCCCTGCATCTACATAG-3′ Probe (737 to 756)5′-CAGGCTGCCCGGCTCAATGG-3′ Open table in a new tab Transverse tissue sections (3 μm) were stained with hematoxylin and eosin (H&E) for routine histological examination; van Gieson's solution (Muto, Tokyo, Japan) was used to evaluate elastin deposition, and Picrosirius red (Sigma-Aldrich) was used to examine collagen deposition as described.12Cheng XW Obata K Kuzuya M Izawa H Nakamura K Asai E Nagasaka T Saka M Kimata T Noda A Nagata K Jin H Shi GP Iguchi A Murohara T Yokota M Elastolytic cathepsin induction/activation system exists in myocardium and is upregulated in hypertensive heart failure.Hypertension. 2006; 48: 979-987Crossref PubMed Scopus (85) Google Scholar Quantitative analyses of the cross-sectional area of cardiomyocytes and the areas of elastin and collagen deposition in the interstitial or/and perivascular regions were performed.12Cheng XW Obata K Kuzuya M Izawa H Nakamura K Asai E Nagasaka T Saka M Kimata T Noda A Nagata K Jin H Shi GP Iguchi A Murohara T Yokota M Elastolytic cathepsin induction/activation system exists in myocardium and is upregulated in hypertensive heart failure.Hypertension. 2006; 48: 979-987Crossref PubMed Scopus (85) Google Scholar For immunohistochemistry, the sections from rats and humans were stained with rabbit polyclonal antibodies to cathepsin S or K (1:200 dilutions) as described.12Cheng XW Obata K Kuzuya M Izawa H Nakamura K Asai E Nagasaka T Saka M Kimata T Noda A Nagata K Jin H Shi GP Iguchi A Murohara T Yokota M Elastolytic cathepsin induction/activation system exists in myocardium and is upregulated in hypertensive heart failure.Hypertension. 2006; 48: 979-987Crossref PubMed Scopus (85) Google Scholar Membrane and cytosolic fractions were isolated from LV tissues as previously described10Cheng XW Kuzuya M Nakamura K Di Q Liu Z Sasaki T Kanda S Jin H Shi GP Murohara T Yokota M Iguchi A Localization of cysteine protease, cathepsin S, to the surface of vascular smooth muscle cells by association with integrin alphanubeta3.Am J Pathol. 2006; 168: 685-694Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar and subjected to immunoblot analysis with antibodies to p22phox, p47phox (1:1000 dilutions; both from Santa Cruz Biotechnology, Santa Cruz, CA), or gp91phox (1:1000 dilution; BD Transduction Laboratories, Lexington, KY). Antibodies to GAPDH (Santa Cruz Biotechnology) were used to confirm equal loading of samples. Three 30-mer oligo-DNAs and 45-mer oligo-DNAs complementary to cathepsins S and K mRNA sequences were designed from the mRNA of rat cathepsins S and K. The oligo-DNAs were labeled with digoxigenin (DIG) using a DIG oligonucleotide tailing kit (Roche Diagnostics, Mannheim, Germany). In situ hybridization was performed as previously described.16Cheng XW Kuzuya M Sasaki T Arakawa K Kanda S Sumi D Koike T Maeda K Tamaya-Mori N Shi GP Saito N Iguchi A Increased expression of elastolytic cysteine proteases, cathepsins S and K, in the neointima of balloon-injured rat carotid arteries.Am J Pathol. 2004; 164: 243-251Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar In brief, the sections were immersed in 50% formamide (FA)/5× standard saline citrate (150 mmol/L NaCl and 15 mmol/L sodium citrate, pH 7.4) for 2 hours at 39°C for prehybridization. DIG-labeled oligo-DNA probes were heated for 10 minutes at 95°C to linearize the probes. For immunohistochemical detection of haptenized (DIG-labeled) antisense probes, the sections were first treated with 1% blocking reagent (DIG nucleic acid detection kit; Roche Diagnostics) in buffer containing 100 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.4, for 1 hour, and then were incubated with alkaline phosphatase-conjugated sheep anti-DIG IgG (1:500) dissolved in 1% blocking buffer for 1 hour. After being washed three times, the sections were visualized with the color substrate NBT/BCIP (DIG nucleic acid detection kit; Roche Diagnostics) according to the manufacturer's instructions. Control experiments were used to confirm the specificity of the cathepsin S or K mRNA signals. Sense probes were hybridized with some sections as a negative control. The cathepsins S and K antisenses were described previously.16Cheng XW Kuzuya M Sasaki T Arakawa K Kanda S Sumi D Koike T Maeda K Tamaya-Mori N Shi GP Saito N Iguchi A Increased expression of elastolytic cysteine proteases, cathepsins S and K, in the neointima of balloon-injured rat carotid arteries.Am J Pathol. 2004; 164: 243-251Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar Specific myocardial NADPH oxidase activity was measured in total homogenates of the fresh LV tissue with the use of a lucigenin-based enhanced chemiluminescence assay as described previously.24Li Y Zhu H Kuppusamy P Roubaud V Zweier JL Trush MA Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems.J Biol Chem. 1998; 273: 2015-2023Crossref PubMed Scopus (481) Google Scholar A low lucigenin concentration (5 μmol/L) was used to minimize artifactual O2− production attributable to redox cycling. In brief, 1 mg of homogenate protein diluted in 1 ml of lysis buffer (in mmol/L, Tris-HCl, 20; NaCl, 150; EDTA, 1; EGTA, 1; and 1% Triton X-100, pH 7.5) was transferred to an assay tube, and NADPH and dark-adapted lucigenin were added to final concentrations of 100 and 5 μmol/L, respectively, immediately before the measurement of chemiluminescence. The chemiluminescence signal was sampled every minute for 12 minutes with a tube luminometer (20/20; Turner Designs, Sunnyvale, CA), and the respective background counts were subtracted from the experimental values. Dihydroethidium staining for superoxide generation was performed as described.25Tao L Gao E Jiao X Yuan Y Li S Christopher TA Lopez BL Koch W Chan L Goldstein BJ Ma XL Adiponectin cardioprotection after myocardial ischemia/reperfusion involves the reduction of oxidative/nitrative stress.Circulation. 2007; 115: 1408-1416Crossref PubMed Scopus (403) Google Scholar After treatment with acetone for 10 minutes, cryostat myocardial sections (5 μm) were incubated for 30 minutes at 37°C with 5 μmol/L dihydroethidium in the presence or absence of an NADPH oxidase inhibitor diphenyliodonium (10 μmol/L; Sigma-Aldrich), a polyethylene glycol-SOD (500 U/ml; Sigma-Aldrich), and a native SOD (500 U/ml; Sigma-Aldrich), and then examined with a laser-scanning confocal microscope (WinROOF version 5.0; WinROOF, Tokyo, Japan). The levels of SOD and catalase were determined by assay kits obtained from Cayman (Ann Arbor, MI). Total SOD activity was assessed by the disappearance of superoxide detected by a water-soluble tetrazolium salt.26Peskin AV Winterbourn CC A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1).Clin Chim Acta. 2000; 293: 157-166Crossref PubMed Scopus (401) Google Scholar Catalase activity was determined as the conversion of methanol to formaldehyde in the presence of H2O2 using the method of Johansson and Borg.27Johansson LH Borg LA A spectrophotometric method for determination of catalase activity in small tissue samples.Anal Biochem. 1988; 174: 331-336Crossref PubMed Scopus (788) Google Scholar The Ang II concentration in the LV myocardium was measured by radioimmunoassay from the LV homogenates collected in chilled tubes containing a mixture of 25 mmol/L EDTA (Sigma-Aldrich), 0.44 mmol/L 1,20-orthophenanthrolene monohydrate, 1 mmol/L Na+ parachloromercuribenzoate, and 3 μmol/L WFML (rat renin inhibitor: acetyl-His-Pro-Phe-Val-Statine-Leu-Phe), and the total amount of Ang II was corrected by LV weight. Details of patients' information for human myocardial biopsy are provided in Supplementary Table 1 (see http://ajp.amjpathol.org). Human LV endomyocardial biopsy specimens were obtained as described previously.12Cheng XW Obata K Kuzuya M Izawa H Nakamura K Asai E Nagasaka T Saka M Kimata T Noda A Nagata K Jin H Shi GP Iguchi A Murohara T Yokota M Elastolytic cathepsin induction/activation system exists in myocardium and is upregulated in hypertensive heart failure.Hypertension. 2006; 48: 979-987Crossref PubMed Scopus (85) Google Scholar The patients who had cardiomyopathy such as ischemic, valvular, diabetic, alcoholic, inflammatory, systemic, hypertrophic, idiopathic dilated cardiomyopathy were excluded by established clinical, hemodynamic, echocardiographic, and cardiac catheterization criteria. Hypertensive HF patients were treated with (n = 6) or without olmesartan (n = 4). Endomyocardial biopsy samples were performed for real-time PCR analysis and immunohistochemistry. Baseline characteristics of the humans for this study are summarized in Supplementary Table 1 at http://ajp.amjpathol.org). Normal LV myocardial samples (n = 7) were obtained from donor hearts not matched for transplantation. For all normal hearts there was no history of cardiac diseases. Informed consent was obtained from all of the patients for use of the myocardial specimens in the present study. All of the patients provided informed consent for use of the myocardial specimens in the present study. CMCs were isolated from the LV myocardium of 1-day-old Wistar rats and were cultured in a mixture (50:50, v/v) of Dulbecco's modified Eagle's medium and Ham's F-12 (DMEM/F-12; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and antibiotics as previously described.12Cheng XW Obata K Kuzuya M Izawa H Nakamura K Asai E Nagasaka T Saka M Kimata T Noda A Nagata K Jin H Shi GP Iguchi A Murohara T Yokota M Elastolytic cathepsin induction/activation system exists in myocardium and is upregulated in hypertensive heart failure.Hypertension. 2006; 48: 979-987Crossref PubMed Scopus (85) Google Scholar CMCs (5 × 104/well) were cultured in 12-well plates in serum-free DMEM/F-1 for 24 hours. After pretreatment with or without olmesartan (1 μmol/L), apocynin (APO, 100 μmol/L), xanthine oxidase (XO) inhibitor allopurinol (ALL, 10 μmol/L), or PEG-SOD (500 U/ml) for 30 minutes, CMCs were cultured in the presence or absence of Ang II (1 μmol/L) for 24 hours, and total RNA was extracted for quantitative real-time PCR assay. CMCs were also stimulated with H2O2 and XO/xanthine (4 μg/ml and 0.5 mmol/L) for examination of examined targeting gene expression. CMCs were cultured in 24-well plates until confluent. After pretreatment with or without APO (100 μmol/L) and PEG-SOD (500 U/ml), CMCs were cultured in the presence or absence of Ang II (1 μmol/L) in serum-free medium containing BODIPY fluorescein-conjugated DQ elastin from bovine neck ligament (300 μg/ml; Molecular Probes, Eugene, OR) as described previously.28Liu J Sukhova GK Yang JT Sun J Ma L Ren A Xu WH Fu H Dolganov GM Hu C Libby P Shi GP Cathepsin L expression and regulation in human abdominal aortic aneurysm, atherosclerosis, and vascular cells.Atherosclerosis. 2006; 184: 302-311Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar After 24 hours of incubation, the cultured medium was analyzed for degraded elastin by Fluoroskan Ascent CF (excitation/emission: 485/530; Labsystems, Helsinki, Finland). The elastolytic activity in the extracts of rat LV tissues was also studied. Data were presented as relative un
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