Chymase released from hypoxia-activated cardiac mast cells cleaves human apoA-I at Tyr192 and compromises its cardioprotective activity
2018; Elsevier BV; Volume: 59; Issue: 6 Linguagem: Inglês
10.1194/jlr.m077503
ISSN1539-7262
AutoresIlona Kareinen, Marc Baumann, Su Duy Nguyen, Katariina Maaninka, Andrey Anisimov, Minoru Tozuka, Matti Jauhiainen, Miriam Lee‐Rueckert, Petri T. Kovanen,
Tópico(s)Renin-Angiotensin System Studies
ResumoApoA-I, the main structural and functional protein of HDL particles, is cardioprotective, but also highly sensitive to proteolytic cleavage. Here, we investigated the effect of cardiac mast cell activation and ensuing chymase secretion on apoA-I degradation using isolated rat hearts in the Langendorff perfusion system. Cardiac mast cells were activated by injection of compound 48/80 into the coronary circulation or by low-flow myocardial ischemia, after which lipid-free apoA-I was injected and collected in the coronary effluent for cleavage analysis. Mast cell activation by 48/80 resulted in apoA-I cleavage at sites Tyr192 and Phe229, but hypoxic activation at Tyr192 only. In vitro, the proteolytic end-product of apoA-I with either rat or human chymase was the Tyr192-truncated fragment. This fragment, when compared with intact apoA-I, showed reduced ability to promote migration of cultured human coronary artery endothelial cells in a wound-healing assay. We propose that C-terminal truncation of apoA-I by chymase released from cardiac mast cells during ischemia impairs the ability of apoA-I to heal damaged endothelium in the ischemic myocardium. ApoA-I, the main structural and functional protein of HDL particles, is cardioprotective, but also highly sensitive to proteolytic cleavage. Here, we investigated the effect of cardiac mast cell activation and ensuing chymase secretion on apoA-I degradation using isolated rat hearts in the Langendorff perfusion system. Cardiac mast cells were activated by injection of compound 48/80 into the coronary circulation or by low-flow myocardial ischemia, after which lipid-free apoA-I was injected and collected in the coronary effluent for cleavage analysis. Mast cell activation by 48/80 resulted in apoA-I cleavage at sites Tyr192 and Phe229, but hypoxic activation at Tyr192 only. In vitro, the proteolytic end-product of apoA-I with either rat or human chymase was the Tyr192-truncated fragment. This fragment, when compared with intact apoA-I, showed reduced ability to promote migration of cultured human coronary artery endothelial cells in a wound-healing assay. We propose that C-terminal truncation of apoA-I by chymase released from cardiac mast cells during ischemia impairs the ability of apoA-I to heal damaged endothelium in the ischemic myocardium. ApoA-I, the main protein component of HDLs, is a 243 amino acid polypeptide with an apparent molecular mass of ∼28,000 Da. Circulating HDL particles contain either a single copy or multiple copies of apoA-I (1.Asztalos B.F. Tani M. Schaefer E.J. Metabolic and functional relevance of HDL subspecies.Curr. Opin. Lipidol. 2011; 22: 176-185Crossref PubMed Scopus (161) Google Scholar). Besides its role in HDL structure, apoA-I is also critical for HDL functionality (2.Lund-Katz S. Phillips M.C. High density lipoprotein structure-function and role in reverse cholesterol transport.Subcell. Biochem. 2010; 51: 183-227Crossref PubMed Scopus (178) Google Scholar). ApoA-I, both in lipid-free form and in the nascent lipid-poor form (preβ1-HDL), promotes efflux of cholesterol via the ABCA1 transporter from macrophage foam cells and so initiates the reverse cholesterol transport pathway from these cells, which is followed by facilitated hepatic uptake and ultimately excretion of the macrophage-derived cholesterol by the gut (2.Lund-Katz S. Phillips M.C. High density lipoprotein structure-function and role in reverse cholesterol transport.Subcell. Biochem. 2010; 51: 183-227Crossref PubMed Scopus (178) Google Scholar, 3.Kane J.P. Malloy M.J. Prebeta-1 HDL and coronary heart disease.Curr. Opin. Lipidol. 2012; 23: 367-371Crossref PubMed Scopus (49) Google Scholar, 4.Lee-Rueckert M. Blanco-Vaca F. Kovanen P.T. Escola-Gil J.C. The role of the gut in reverse cholesterol transport - Focus on the enterocyte.Prog. Lipid Res. 2013; 52: 317-328Crossref PubMed Scopus (33) Google Scholar). Importantly, lipid-poor apoA-I particles are abundant in interstitial fluids, where they can accept excess cholesterol from cholesterol-loaded cells (5.Miller N.E. Olszewski W.L. Hattori H. Miller I.P. Kujiraoka T. Oka T. Iwasaki T. Nanjee M.N. Lipoprotein remodeling generates lipid-poor apolipoprotein A-I particles in human interstitial fluid.Am. J. Physiol. Endocrinol. Metab. 2013; 304: E321-E328Crossref PubMed Scopus (25) Google Scholar). Recent data suggest that, by regulating cellular cholesterol homeostasis, HDL and apoA-I can also regulate inflammatory responses in endothelial cells and other types of cells that have been activated by proinflammatory stimuli in the arterial intima (6.Mineo C. Shaul P.W. Regulation of signal transduction by HDL.J. Lipid Res. 2013; 54: 2315-2324Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Moreover, apoA-I has a major role in binding the endotoxin (lipopolysaccharide; LPS) released from the surface membrane of Gram-negative bacteria and thereby neutralizes its toxic effects on endothelial cells (7.Van Linthout S. Spillmann F. Graiani G. Miteva K. Peng J. Van C.E. Meloni M. Tolle M. Escher F. Subasiguller A. et al.Down-regulation of endothelial TLR4 signalling after apo A-I gene transfer contributes to improved survival in an experimental model of lipopolysaccharide-induced inflammation.J. Mol. Med. (Berl.). 2011; 89: 151-160Crossref PubMed Scopus (31) Google Scholar). Mast cells are proinflammatory cells, which, upon activation and ensuing degranulation, release a variety of granule-bound neutral proteases, notably chymase, into the surrounding extracellular fluid (8.Pejler G. Ronnberg E. Waern I. Wernersson S. Mast cell proteases: multifaceted regulators of inflammatory disease.Blood. 2010; 115: 4981-4990Crossref PubMed Scopus (276) Google Scholar). In the extracellular fluid, the soluble components of the granules, such as histamine and soluble proteoglycans, disperse, whereas most of chymase remains attached to the insoluble proteoglycan fraction of the granules forming functional composite superstructures, which we have designated granule remnants (9.Lee-Rueckert M. Kovanen P.T. The mast cell as a pluripotent HDL-modifying effector in atherogenesis: from in vitro to in vivo significance.Curr. Opin. Lipidol. 2015; 26: 362-368Crossref PubMed Scopus (10) Google Scholar). Mast cells in the rat heart express chymase, which, like its human counterpart, is a chymotryptic enzyme (8.Pejler G. Ronnberg E. Waern I. Wernersson S. Mast cell proteases: multifaceted regulators of inflammatory disease.Blood. 2010; 115: 4981-4990Crossref PubMed Scopus (276) Google Scholar, 10.Urata H. Kinoshita A. Misono K.S. Bumpus F.M. Husain A. Identification of a highly specific chymase as the major angiotensin II-forming enzyme in the human heart.J. Biol. 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As a serine protease with chymotrypsin-like primary substrate specificity, chymase cleaves peptide bonds preferentially after aromatic amino acids with the order of preference Phe>Tyr>Trp (8.Pejler G. Ronnberg E. Waern I. Wernersson S. Mast cell proteases: multifaceted regulators of inflammatory disease.Blood. 2010; 115: 4981-4990Crossref PubMed Scopus (276) Google Scholar). Importantly, chymase of either rodent or human origin efficiently cleaves lipid-free and lipid-poor forms of apoA-I in vitro, so compromising the ABCA1-mediated cholesterol efflux pathway, and studies in vivo indicate that such proteolysis can also occur in specific tissue fluids in which these preferred apolipoprotein substrates are abundantly present (9.Lee-Rueckert M. Kovanen P.T. The mast cell as a pluripotent HDL-modifying effector in atherogenesis: from in vitro to in vivo significance.Curr. Opin. Lipidol. 2015; 26: 362-368Crossref PubMed Scopus (10) Google Scholar). Indeed, systemic activation of mast cells with ensuing degranulation during anaphylactic shock in the mouse proteolytically modifies circulating HDL and reduces the capacity of anaphylactic serum to act as cholesterol acceptor from cultured macrophage foam cells (13.Judström I. Jukkola H. Metso J. Jauhiainen M. Kovanen P.T. Lee-Rueckert M. Mast cell-dependent proteolytic modification of HDL particles during anaphylactic shock in the mouse reduces their ability to induce cholesterol efflux from macrophage foam cells ex vivo.Atherosclerosis. 2010; 208: 148-154Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The initial indication for a role of chymase in myocardial pathology derives from the observation that chymase is the major angiotensin II-forming enzyme in the human heart (10.Urata H. Kinoshita A. Misono K.S. Bumpus F.M. Husain A. Identification of a highly specific chymase as the major angiotensin II-forming enzyme in the human heart.J. Biol. Chem. 1990; 265: 22348-22357Abstract Full Text PDF PubMed Google Scholar). Cardiac chymase has also been shown to induce adverse cardiac remodeling after estrogen depletion in experimental rat models (11.Li J. Jubair S. Janicki J.S. Estrogen inhibits mast cell chymase release to prevent pressure overload-induced adverse cardiac remodeling.Hypertension. 2015; 65: 328-334Crossref PubMed Scopus (36) Google Scholar, 14.Wang H. da Silva J. Alencar A. Zapata-Sudo G. Lin M.R. Sun X. Ahmad S. Ferrario C.M. Groban L. Mast cell inhibition attenuates cardiac remodeling and diastolic dysfunction in middle-aged, ovariectomized Fischer 344 × Brown Norway rats.J. Cardiovasc. Pharmacol. 2016; 68: 49-57Crossref PubMed Scopus (18) Google Scholar). Moreover, other reports demonstrate that cardiac chymase contributes to the development of the adverse effects of hypertensive heart failure in rats (15.Shiota N. Rysa J. Kovanen P.T. Ruskoaho H. Kokkonen J.O. Lindstedt K.A. A role for cardiac mast cells in the pathogenesis of hypertensive heart disease.J. Hypertens. 2003; 21: 1935-1944Crossref PubMed Scopus (118) Google Scholar) and that it promotes acute ischemia/reperfusion injury (IRI) in pigs (16.Oyamada S. Bianchi C. Takai S. Chu L.M. Sellke F.W. Chymase inhibition reduces infarction and matrix metalloproteinase-9 activation and attenuates inflammation and fibrosis after acute myocardial ischemia/reperfusion.J. Pharmacol. Exp. Ther. 2011; 339: 143-151Crossref PubMed Scopus (75) Google Scholar). Importantly, in the experimental pig model, inhibition of cardiac mast cell-derived chymase resulted in decreased infarction size after IRI, thus making chymase alone responsible for at least some of the harmful effects of IRI (16.Oyamada S. Bianchi C. Takai S. Chu L.M. Sellke F.W. Chymase inhibition reduces infarction and matrix metalloproteinase-9 activation and attenuates inflammation and fibrosis after acute myocardial ischemia/reperfusion.J. Pharmacol. Exp. Ther. 2011; 339: 143-151Crossref PubMed Scopus (75) Google Scholar). Similarly, complete degranulation of cardiac mast cells shortly before IRI in rats has been shown to attenuate inflammation and to result in improved cardiac function due to a decrease in the release of cytotoxic mediators from such granule-deficient cardiac mast cells during IRI (17.Jaggi A.S. Singh M. Sharma A. Singh D. Singh N. Cardioprotective effects of mast cell modulators in ischemia-reperfusion-induced injury in rats.Methods Find. Exp. Clin. Pharmacol. 2007; 29: 593-600Crossref PubMed Scopus (33) Google Scholar). Both human plasma HDL and reconstituted HDL (rHDL) have been found to exert cardioprotective effects in experimental IRI in rats (18.Calabresi L. Rossoni G. Gomaraschi M. Sisto F. Berti F. Franceschini G. High-density lipoproteins protect isolated rat hearts from ischemia-reperfusion injury by reducing cardiac tumor necrosis factor-alpha content and enhancing prostaglandin release.Circ. Res. 2003; 92: 330-337Crossref PubMed Scopus (140) Google Scholar, 19.Rossoni G. Gomaraschi M. Berti F. Sirtori C.R. Franceschini G. Calabresi L. Synthetic high-density lipoproteins exert cardioprotective effects in myocardial ischemia/reperfusion injury.J. Pharmacol. Exp. Ther. 2004; 308: 79-84Crossref PubMed Scopus (49) Google Scholar). Thus, it was shown that administration of HDL before ischemia in an isolated rat heart model reduces cardiac TNF-α content and increases prostaglandin release, resulting in improved postischemic functional recovery. Importantly, this effect was attenuated when either type of HDL was administered during the reperfusion phase, revealing that in order to exhibit cardioprotective properties, HDL has to be infused before but not during reperfusion to prevent attenuation of their functionality. Such partial loss of the ability of HDL to preserve myocardial function when injected into the coronary circulation in the postischemic phase was explained by an emerging resistance of the myocardium to the cardioprotective effects of HDL (18.Calabresi L. Rossoni G. Gomaraschi M. Sisto F. Berti F. Franceschini G. High-density lipoproteins protect isolated rat hearts from ischemia-reperfusion injury by reducing cardiac tumor necrosis factor-alpha content and enhancing prostaglandin release.Circ. Res. 2003; 92: 330-337Crossref PubMed Scopus (140) Google Scholar, 19.Rossoni G. Gomaraschi M. Berti F. Sirtori C.R. Franceschini G. Calabresi L. Synthetic high-density lipoproteins exert cardioprotective effects in myocardial ischemia/reperfusion injury.J. Pharmacol. Exp. Ther. 2004; 308: 79-84Crossref PubMed Scopus (49) Google Scholar). Another explanation is that the loss of cardioprotection may actually have resulted from HDL becoming dysfunctional already during cardiac ischemia, i.e., during the time period preceding the onset of reperfusion. Since IRI in hearts isolated from rats (17.Jaggi A.S. Singh M. Sharma A. Singh D. Singh N. Cardioprotective effects of mast cell modulators in ischemia-reperfusion-induced injury in rats.Methods Find. Exp. Clin. Pharmacol. 2007; 29: 593-600Crossref PubMed Scopus (33) Google Scholar, 20.Gilles S. Zahler S. Welsch U. Sommerhoff C.P. Becker B.F. Release of TNF-alpha during myocardial reperfusion depends on oxidative stress and is prevented by mast cell stabilizers.Cardiovasc. Res. 2003; 60: 608-616Crossref PubMed Scopus (105) Google Scholar), dogs (21.Frangogiannis N.G. Lindsey M.L. Michael L.H. Youker K.A. Bressler R.B. Mendoza L.H. Spengler R.N. Smith C.W. Entman M.L. Resident cardiac mast cells degranulate and release preformed TNF-alpha, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion.Circulation. 1998; 98: 699-710Crossref PubMed Scopus (414) Google Scholar), guinea pigs, and mice (22.Mackins C.J. Kano S. Seyedi N. Schafer U. Reid A.C. Machida T. Silver R.B. Levi R. Cardiac mast cell-derived renin promotes local angiotensin formation, norepinephrine release, and arrhythmias in ischemia/reperfusion.J. Clin. Invest. 2006; 116: 1063-1070Crossref PubMed Scopus (173) Google Scholar) induces cardiac mast cell activation with ensuing chymase release, these findings actually suggest a pathological scenario in which chymase could promote local proteolysis of apoA-I. Given that mast cell chymase can degrade apoA-I in interstitial fluids, e.g., in aortic intimal fluid (23.Lindstedt L. Lee M. Castro G.R. Fruchart J-C. Kovanen P.T. Chymase in exocytosed rat mast cell granules effectively proteolyzes apolipoprotein AI-containing lipoproteins, so reducing the cholesterol efflux-inducing ability of serum and aortic intimal fluid.J. Clin. Invest. 1996; 97: 2174-2182Crossref PubMed Scopus (69) Google Scholar), here, we studied the ability of chymase exocytosed by hypoxia-activated rat cardiac mast cells to proteolyze apoA-I when it is infused into the coronary circulation using the Langendorff isolated heart perfusion system. The results demonstrate that cardiac mast cell activation in the ischemic isolated rat heart is associated with the production of a C-terminally truncated apoA-I fragment, which we could identify as a proteolytic product generated solely by mast cell chymase. Of note, such proteolytic cleavage reduced the ability of apoA-I to promote migration of cultured human endothelial cells, when assessed using a wound healing assay. The results suggest that activated mast cells in the ischemic heart may compromise the ability of apoA-I to maintain endothelial integrity in the microvascular coronary arterial tree. Heparin Leo Orifarm (5,000 IU/ml) and isoflurane were purchased from Orion Pharmaceuticals (Espoo, Finland). Purified human lipid-free apoA-I has been kindly provided by Dr. Peter Lerch (Swiss Red Cross, Bern, Switzerland). Poorly lipidated apoA-I-containing rHDL were prepared by the cholate dialysis method as previously described (24.Jauhiainen M. Dolphin P.J. Human plasma lecithin-cholesterol acyltransferase. An elucidation of the catalytic mechanism.J. Biol. Chem. 1986; 261: 7032-7043Abstract Full Text PDF PubMed Google Scholar) and used within a week. The final rHDL preparation (human apoA-I:egg yolk phosphatidyl choline:cholesterol, 1:30:12.5, mol/mol/mol ratio) exhibited preβ-mobility in agarose gel electrophoresis. The polyclonal anti-human apoA-I antibody R261 was produced in New Zealand white rabbits using purified lipid-free apoA-I as antigen. Compound 48/80 (48/80), phthaldialdehyde (OPTA), histamine dihydrochloride, N-benzoyl-L-tyrosine ethyl ester (BTEE), and soybean trypsin inhibitor (SBTI) were purchased from Sigma Aldrich. Recombinant human chymase (specific activity 80 BTEE units/μ;g) expressed in the baculovirus-insect cell system was kindly provided by Dr. Hidenori Kasai (Teijin Ltd. Co., Shizuoka, Japan). The preparation was diluted in a buffer containing 5 mM Tris-HCl, 150 mM NaCl, and 1 mM EDTA, pH 7.4 (TNE buffer) before use. The enzyme preparation was fully inhibited by adding SBTI at a final concentration of 100 μ;g/ml. The mouse monoclonal antibody 16-4 Mab was used to detect the C-terminally truncated human apoA-I at the cleavage site Phe225, as previously described (25.Usami Y. Matsuda K. Sugano M. Ishimine N. Kurihara Y. Sumida T. Yamauchi K. Tozuka M. Detection of chymase-digested C-terminally truncated apolipoprotein A-I in normal human serum.J. Immunol. Methods. 2011; 369: 51-58Crossref PubMed Scopus (17) Google Scholar). Goat anti-rabbit Ig antibody P0448 and goat-anti-mouse Ig antibody P0447 were purchased from Dako. Pierce ECL Western Blotting Substrate was from Thermo Scientific. Experiments were conducted in conformity with the Finnish regulations and the Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Animal experiments and the protocols were approved by The National Animal Experiment Board of Finland (ELLA; license no. ESAVI-2010-05448/Ym-23). Experiments were performed using female Wistar rats (200–250 g) purchased from Harlan Laboratories (Venray, The Netherlands). Rats were housed (three per cage), allowed to access regular rodent diet (Teklad Global, Harlan Laboratories) and water ad libitum, and maintained in an automatic 12/12 h dark–light cycle. Rats were kept in the animal facility at least for 1 week before experimentation. Rat peritoneal mast cells were obtained from euthanized rats by peritoneal lavage with PBS medium containing 0.5 mg/ml BSA, 1 mg/ml glucose, and 0.05 mg/ml heparin. Peritoneal cells present in the recovered PBS medium were sedimented by centrifugation at 200 g for 10 min, then resuspended in RPMI-medium, and cultured at 37°C in humidified CO2 for 2 h to allow attachment of the peritoneal macrophages. After incubation, the medium was collected and centrifuged at 200 g for 5 min to sediment the nonadherent cells (mast cells). To obtain a maximum yield of the cytoplasmic secretory mast cell granules, the sedimented mast cells were resuspended in 0.3 M sucrose and lysed by subjecting them to 6 freeze-thaw cycles. Nonlysed mast cells and cellular debris were removed by centrifugation at 300 g, and the supernatant containing membrane-covered mast cell granules was collected. The supernatant was then centrifuged at 20,000 g for 30 min to sediment the granules, after which the granules were resuspended in distilled water to deprive them of membranes and thereby to convert them into "granule remnants" in which the heparin-bound chymase is exposed to the surrounding fluid. Finally, the granule remnants were resedimented to obtain a preparation of washed granule remnants. Chymase activity in mast cell granules was measured as described previously (26.Woodbury R.G. Everitt M.T. Neurath H. Mast cell proteases.Methods Enzymol. 1981; 80: 588-609Crossref PubMed Scopus (75) Google Scholar). In this assay, the BTEE units are calculated as Δabs/Δtime × 1,000 in 1 µl of mast cell lysate, and 1 BTEE unit corresponds to a 0.001 increase in absorbance per minute, when measured at 256 nm. ApoA-I (1 mg/ml) was incubated at 37°C for the indicated periods of time in TNE buffer containing 40 BTEE units/ml either recombinant human chymase or rat chymase (present in granule remnants; see above). To stop proteolysis, each incubation vial was placed on ice. To inhibit the activity of recombinant human chymase, SBTI was added to the respective incubation vials. To remove rat granule remnant-bound chymase from the incubation mixtures, the vials were centrifuged at 10,000 g for 5 min at +4°C to remove the granule remnants. The apoA-I–containing supernatants were collected for later analysis or used in the wound-healing assay. All samples were stored at −80°C until analysis. The ex vivo experiments were performed using the isolated heart perfusion according to Langendorff. For this purpose, rats were anesthetized using inhalation anesthesia (Univentor U-400, AgnTho's, Lidingö, Sweden) in a chamber saturated with 4% isoflurane, and anesthesia was maintained with 2.5% isoflurane with a rostral mask. Rats received i.v. injection of heparin (5,000 IU/kg) into the lateral tail vein. Anesthesia was then deepened by adjusting the vaporizer to 4% isoflurane, and once the pedal reflex was nonresponsive, ∼4 ml of blood was collected from the vena cava caudalis in the terminally anesthetized animals. Hearts were then rapidly excised, mounted on the Langendorff apparatus, cannulated over the aortic valve, and perfused with Krebs-Henseleit buffer (125 mM NaCl, 4.7 mM KCl, 20 mM NaHCO3, 0.43 mM NaH2PO4, 1.0 mM MgCl2, 1.3 mM CaCl2, and 9.1 mM D-glucose; pH 7.4). The perfusion buffer was continuously purged with carbogen (95% O2/5% CO2) and maintained at 37°C. Hearts were equilibrated for 30 min at a flow rate of 6.6 ml/min before the mast cell activation-inducing treatments. To study the effect of mast cell activation on the intactness of apoA-I perfused through the isolated rat heart, 1 mg of apoA-I in 100 µl of buffer was administered into the perfusion line near the inlet to the heart at a slow flow of 0.6 ml/min under the following conditions: after the 30 min equilibration period, immediately after a bolus injection of the mast cell degranulating 48/80 (300 µg in 500 µl of buffer) (Fig. 1A), or after low-flow ischemia induced by reducing the flow of the perfusion buffer to 0.6 ml/min for 20 min (Fig. 1B). Every compound was administered as a bolus injection into the perfusion line within a period of 1 min (for clarity, these short time intervals were not included in the time line shown in Fig. 1). After each apoA-I injection, fractions of the coronary effluent were collected at 0.6 ml/min flow for 5 min (1 fraction per min). When apoA-I was perfused into the system, a single protein peak eluted within 5 min after the injection. The fractions corresponding to the highest protein concentrations were taken for analysis of apoA-I proteolysis. In other experiments, 1 mg of apoA-I-containing rHDL (see Materials) was perfused through the rat hearts in the Langendorff system using the above described flow settings, and the intactness of apoA-I was evaluated in the fractions collected within 5 min after the injection, which contained the highest protein concentrations. Hearts were sectioned transversely and submerged into tissue section medium Tissue-Tek O.C.T. (Sakura Finetek Europe). Hearts were then rapidly frozen in liquid nitrogen and stored at −80°C until sectioned into 5 µm slices. The frozen sections were stained in 0.1% Toluidine blue, and the number of mast cells and the extent of mast cell activation were determined (magnification ×40). Mast cell activation was defined as the presence of several extracellular granules extruded into the immediate vicinity of the parent cell and was determined as described previously (27.Laine P. Kaartinen M. Penttilä A. Panula P. Paavonen T. Kovanen P.T. Association between myocardial infarction and the mast cells in the adventitia of the infarct-related coronary artery.Circulation. 1999; 99: 361-369Crossref PubMed Scopus (294) Google Scholar). To establish the degree of myocardial mast cell activation, histamine was determined in all effluent samples collected during the Langendorff experimental protocols, as shown in Fig. 1. In short, the samples were precipitated with 50% TCA and centrifuged, and the precipitate was dissolved in 1 N NaOH and incubated with OPTA, which was used as the fluorescent reagent (28.Shore P.A. Burkhaltera A. Cohn Jr, V.H. A method for the fluorometric assay of histamine in tissues.J. Pharmacol. Exp. Ther. 1959; 127: 182-186PubMed Google Scholar). After addition of 2 N H2SO4, the absorbance was measured in a spectrophotometer at excitation/emission 355/450 nm. Commercial histamine dihydrochloride was used as standard. ApoA-I proteolysis was evaluated by analyzing the collected effluent samples (1 µg protein) on NuPAGE Novex 4-12% Bis-Tris gradient gels (Life Technologies) under reducing conditions. The gels were stained with Instant Blue stain (Expedeon), and the protein bands were documented by scanning the gels. For Western blotting analysis, the samples were first run on 15% SDS-PAGE under reducing conditions. Proteins were electrotransferred onto nitrocellulose membrane (Hybond-C Extra, Amersham). After blocking in 5% defatted milk, the membranes were incubated overnight at 4°C with either rabbit anti-human apoA-I polyclonal antibody (R261, diluted 1:2,000) or mouse anti-human C-terminally truncated apoA-I monoclonal antibody (16-4 Mab, diluted 1:1,000). The membranes were then incubated at room temperature for 1 h with either goat-anti-rabbit IgG antibody (diluted 1:2,000) or with goat-anti-mouse IgM antibody (diluted 1:5,000). Immune complexes were visualized with HRP-conjugated ECL. Ponceau S staining was used to confirm even protein transfer to nitrocellulose and equivalent protein mass loading. The degradation products of apoA-I generated either in the Langendorff perfusion system (coronary effluent) or by incubating apoA-I with human or rat chymase in vitro (total incubation mixture) were identified by MALDI-TOF MS. MALDI-TOF and TOF/TOF analyses were carried out with an UltraFleXtreme 2000 Hz laser instrument (Bruker Daltonics, Bremen, Germany) equipped with a SmartBeamTM Nd/YAG laser (355 nm), operated in positive and linear modes. Typically, a mass spectrum was acquired by accumulating spectra of 2,000 and up to 20,000 laser shots. External calibration was performed for molecular mass assignments using a protein calibration standard (ProtCal I, Bruker Daltonics, Leipzig, Germany). A few picograms of each sample were mixed with a protein matrix solution [sinapinic acid in 30:70 (vol/vol) acetonitrile:trifluoroacetic acid 0.1% in water] in a ratio varying between 1:1 and 1:5. The samples were then applied to a stainless-steel sample plate (Bruker Daltonic) and placed into the instrument. A standard method optimized to a mass range of 5–50 kDa was used in the linear and positive mode according to the manufacturer's instructions. Human coronary artery endothelial cells (HCAECs; PromoCell) were cultured in T-75 flasks in Endothelial Cell Basal Medium MV (Basal Medium; catalog no. C-22220, PromoCell) supplemented with 5% FCS, 0.4% endothelial cell growth supplement, 10 ng/ml epidermal growth factor, 90 µg/ml heparin, 1 μ;g/ml hydrocortisone (all as components of supplement pack, catalog no. C-39220, PromoCell), 100 U/ml penicillin/streptomycin solution, and 50 ng/ml amphotericin B to yield Complete Medium according to the instructions of the manufacturer. Confluent HCAECs were washed with 15 ml of PBS, trypsinized, and replated in Complete Medium. Experiments were performed with cells at their fifth to eighth passage. The wound-healing assay was carried out using ibidi™ culture inserts (Ibidi, Planegg, Germany). Approximately 15,000 cells were seeded in each chamber of the insert and cultured for 1 day to reach 90–100% confluency. The cells were then washed with prewarmed HBSS and the insert was removed, after which the cells were incubated in Basal Medium only or in the presence of 1% FCS (PromoCell), apoA-I, or rat chymase-treated apoA-I (25 or 50 µg/ml each) for 10 h. In additional experiments, HCAECs were incubated with untreated or chymase-treated rHDL particles (50 µg/ml) for 10 h and their endothelial healing effect was evaluated using the above described conditions. Images were captured at 30 min intervals us
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