Increased Expression and Activation of Stress-Activated Protein Kinases/c-Jun NH2-Terminal Protein Kinases in Atherosclerotic Lesions Coincide with p53
2000; Elsevier BV; Volume: 156; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)65061-4
ISSN1525-2191
AutoresBernhard Metzler, Yanhua Hu, Hermann Dietrich, Qingbo Xu,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoHyperlipidemia alters gene expression of arterial endothelial and smooth muscle cells (SMCs) and induces atherosclerotic lesions, in which cell proliferation and apoptosis co-exist. The signal transduction pathways that mediate these responses in the vessel wall in vivo have yet to be identified. Stress-activated protein kinases (SAPKs) or c-Jun NH2-terminal protein kinases (JNKs) are thought to be crucial in transmitting transmembrane signals required for cell differentiation and apoptosis in vitro. In the present study, we investigated the localization and activity of SAPK/JNK in atherosclerotic lesions of cholesterol-fed rabbits. Immunofluorescence analysis revealed abundant and heterogeneous distribution of pan-SAPK/JNK and phosphorylated SAPK/JNK, which were mainly localized in cell nuclei of the lesional cap and basal regions. Double staining of the lesions demonstrated that a portion of α-actin+ SMCs and RAM11+ macrophages contained abundant phosphorylated SAPK/JNK proteins. SAPK/JNK protein levels in protein extracts from atherosclerotic lesions were two- to threefold higher than the vessels of chow-fed rabbits. SAPK/JNK activities were elevated three- to fivefold higher than the normal vessels. Interestingly, increased SAPK/JNK in lesions was co-localized or coincided with high levels of transcription factor p53 as identified by double labeling and immunoprecipitation. Abundant pro-apoptotic protein BAX and BCL-XS were also observed. Furthermore, low-density lipoprotein (LDL) and oxidized LDL stimulated SAPK/JNK activation in cultured SMCs in a time- and dose-dependent manner. LDL also induced SAPK/JNK activation in vascular SMCs derived from LDL-receptor-deficient Watanabe rabbits, indicating a LDL-receptor-independent process. Thus, SAPK/JNK persistently hyperexpressed and activated in lesions may play a key role in mediating cell differentiation and apoptosis during the development of atherosclerosis via activation of transcription factor p53. Hyperlipidemia alters gene expression of arterial endothelial and smooth muscle cells (SMCs) and induces atherosclerotic lesions, in which cell proliferation and apoptosis co-exist. The signal transduction pathways that mediate these responses in the vessel wall in vivo have yet to be identified. Stress-activated protein kinases (SAPKs) or c-Jun NH2-terminal protein kinases (JNKs) are thought to be crucial in transmitting transmembrane signals required for cell differentiation and apoptosis in vitro. In the present study, we investigated the localization and activity of SAPK/JNK in atherosclerotic lesions of cholesterol-fed rabbits. Immunofluorescence analysis revealed abundant and heterogeneous distribution of pan-SAPK/JNK and phosphorylated SAPK/JNK, which were mainly localized in cell nuclei of the lesional cap and basal regions. Double staining of the lesions demonstrated that a portion of α-actin+ SMCs and RAM11+ macrophages contained abundant phosphorylated SAPK/JNK proteins. SAPK/JNK protein levels in protein extracts from atherosclerotic lesions were two- to threefold higher than the vessels of chow-fed rabbits. SAPK/JNK activities were elevated three- to fivefold higher than the normal vessels. Interestingly, increased SAPK/JNK in lesions was co-localized or coincided with high levels of transcription factor p53 as identified by double labeling and immunoprecipitation. Abundant pro-apoptotic protein BAX and BCL-XS were also observed. Furthermore, low-density lipoprotein (LDL) and oxidized LDL stimulated SAPK/JNK activation in cultured SMCs in a time- and dose-dependent manner. LDL also induced SAPK/JNK activation in vascular SMCs derived from LDL-receptor-deficient Watanabe rabbits, indicating a LDL-receptor-independent process. Thus, SAPK/JNK persistently hyperexpressed and activated in lesions may play a key role in mediating cell differentiation and apoptosis during the development of atherosclerosis via activation of transcription factor p53. Mammalian cells respond to extracellular stimuli by activating signal transduction pathways, which culminate in changes in gene expression. A critical component of signaling pathways is the activation of protein kinases that phosphorylate a host of cellular substrates, such as transcription factors controlling the induction of various genes. Stress-activated protein kinases (SAPKs) or c-Jun NH2-terminal protein kinases (JNKs) are highly activated in the cardiovascular system in response to stresses,1Force T Pombo CM Avruch JA Bonventre JV Kyriakis JM Stress-activated protein kinases in cardiovascular disease.Circ Res. 1996; 78: 947-953Crossref PubMed Scopus (187) Google Scholar, 2Zou Y Hu Y Metzler B Xu Q Signal transduction in arteriosclerosis: mechanical stress-activated MAP kinases in vascular smooth muscle cells.Int J Mol Med. 1998; 1: 827-834PubMed Google Scholar, 3Sugden PH Clerk A "Stress-responsive" mitogen-activated protein kinases (c-Jun N-terminal kinases and p38 MAP kinases) in the myocardium.Circ Res. 1998; 83: 345-352Crossref PubMed Scopus (440) Google Scholar including acute hypertension,4Xu Q Liu Y Gorospe M Udelsman R Holbrook NJ Acute hypertension activates mitogen-activated protein kinases in arterial wall.J Clin Invest. 1996; 97: 508-514Crossref PubMed Scopus (184) Google Scholar angioplasty,5Hu Y Cheng L Hochleitner BW Xu Q Activation of mitogen-activated protein kinases (ERK/JNK) and AP-1 transcription factor in rat carotid arteries after balloon injury.Arterioscler Thromb Vasc Biol. 1997; 17: 2808-2816Crossref PubMed Scopus (154) Google Scholar mechanical stress,6Reusch HP Chan G Ives HE Nemenoff RA Activation of JNK/SAPK and ERK by mechanical strain in vascular smooth muscle cells depends on extracellular matrix composition.Biochem Biophys Res Commun. 1997; 237: 239-244Crossref PubMed Scopus (87) Google Scholar, 7Hamada K Takuwa N Yokoyama K Takuwa Y Stretch activates Jun N-terminal kinase/stress-activated protein kinase in vascular smooth muscle cells through mechanisms involving autocrine ATP stimulation of purinoceptors.J Biol Chem. 1998; 273: 6334-6340Crossref PubMed Scopus (105) Google Scholar, 8Hu Y Hochleitner BW Wick G Xu Q Decline of shear stress-induced activation of extracellular signal-regulated kinases, but not stress-activated protein kinases, in in vitro propagated endothelial cells.Exp Gerontol. 1998; 33: 601-603Crossref PubMed Scopus (12) Google Scholar ischemia/reperfusion,9Yue TL Ma XL Gu JL Ruffolo Jr, RR Feuerstein GZ Carvedilol inhibits activation of stress-activated protein kinase and reduces reperfusion injury in perfused rabbit heart.Eur J Pharmacol. 1998; 345: 61-65Crossref PubMed Scopus (37) Google Scholar free radicals,10Mietus-Snyder M Glass CK Pitas RE Transcriptional activation of scavenger receptor expression in human smooth muscle cells requires AP-1/c-Jun and C/EBPbeta: both AP-1 binding and JNK activation are induced by phorbol esters and oxidative stress.Arterioscler Thromb Vasc Biol. 1998; 18: 1440-1449Crossref PubMed Scopus (43) Google Scholar heat shock,11Hu Y Metzler B Xu Q Discordant activation of stress-activated protein kinases or c-Jun NH2-terminal protein kinases in tissues of heat-stressed mice.J Biol Chem. 1997; 272: 9113-9119Crossref PubMed Scopus (60) Google Scholar and inflammatory cytokines12Modur V Zimmerman GA Prescott SM McIntyre TM Endothelial cell inflammatory responses to tumor necrosis factor alpha. 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The atherosclerotic lesion is defined by arterial intimal cell proliferation, lipid accumulation, and connective tissue deposition. The major cellular components of the human atherosclerotic plaques are smooth muscle cells (SMCs), which dominate the fibrous cap, and macrophages, which are the most abundant cell type in the lipid-rich core region.24Xu QB Oberhuber G Gruschwitz M Wick G Immunology of atherosclerosis: cellular composition and major histocompatibility complex class II antigen expression in aortic intima, fatty streaks, and atherosclerotic plaques in young and aged human specimens.Clin Immunol Immunopathol. 1990; 56: 344-359Crossref PubMed Scopus (161) Google Scholar, 25Ross R The pathogenesis of atherosclerosis: a perspective for the 1990s.Nature. 1993; 362: 801-809Crossref PubMed Scopus (9959) Google Scholar Although the pathogenic mechanism of atherosclerosis is not fully understood, a high concentration of circulating cholesterol or low-density lipoproteins (LDL) is believed to be a major risk factor. LDL delivers cholesterol to vascular SMCs and macrophages, stimulates cell proliferation, and induces gene expression of platelet-derived growth factor (PDGF), PDGF receptors, c-fos, and egr-125Ross R The pathogenesis of atherosclerosis: a perspective for the 1990s.Nature. 1993; 362: 801-809Crossref PubMed Scopus (9959) Google Scholar, 26Sachinidis A Ko Y Wieczorek A Weisser B Locher R Vetter W Vetter H Lipoproteins induce expression of the early growth response gene-1 in vascular smooth muscle cells from rat.Biochem Biophys Res Commun. 1993; 192: 794-799Crossref PubMed Scopus (31) Google Scholar in the arterial wall. LDL or oxidized LDL stimulation can also evoke cell apoptosis,27Kinscherf R Claus R Wagner M Gehrke C Kamencic H Hou D Nauen O Schmiedt W Kovacs G Pill J Metz J Deigner HP Apoptosis caused by oxidized LDL is manganese superoxide dismutase and p53 dependent.FASEB J. 1998; 12: 461-467Crossref PubMed Scopus (127) Google Scholar, 28Harada-Shiba M Kinoshita M Kamido H Shimokado K Oxidized low density lipoprotein induces apoptosis in cultured human umbilical vein endothelial cells by common and unique mechanisms.J Biol Chem. 1998; 273: 9681-9687Crossref PubMed Scopus (232) Google Scholar, 29Li D Yang B Mehta JL Ox-LDL induces apoptosis in human coronary artery endothelial cells: role of PKC, PTK, bcl-2, and Fas.Am J Physiol. 1998; 275: H568-H576PubMed Google Scholar which has been observed in atherosclerotic lesions.30Geng YJ Libby P Evidence for apoptosis in advanced human atheroma: colocalization with interleukin-1 beta-converting enzyme.Am J Pathol. 1995; 147: 251-266PubMed Google Scholar, 31Bennett MR Evan GI Schwartz SM Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques.J Clin Invest. 1995; 95: 2266-2274Crossref PubMed Scopus (610) Google Scholar, 32Isner JM Kearney M Bortman S Passeri J Apoptosis in human atherosclerosis and restenosis.Circulation. 1995; 91: 2703-2711Crossref PubMed Scopus (625) Google Scholar, 33Han DK Haudenschild CC Hong MK Tinkle BT Leon MB Liau G Evidence for apoptosis in human atherogenesis and in a rat vascular injury model.Am J Pathol. 1995; 147: 267-277PubMed Google Scholar In addition, p53 has been identified to be highly expressed in atherosclerotic lesions and to be involved in oxidized LDL-induced apoptosis.27Kinscherf R Claus R Wagner M Gehrke C Kamencic H Hou D Nauen O Schmiedt W Kovacs G Pill J Metz J Deigner HP Apoptosis caused by oxidized LDL is manganese superoxide dismutase and p53 dependent.FASEB J. 1998; 12: 461-467Crossref PubMed Scopus (127) Google Scholar However, the precise signal transduction pathways that link hypercholesterolemia or LDL stimulation to quantitative changes in gene expression and, to apoptosis in the pathogenesis of atherosclerosis, remain to be elucidated. In the present study, we examined SAPK/JNK expression, localization, and activation in atherosclerotic lesions of cholesterol-fed rabbits and provide the first evidence of SAPK/JNK overexpression and activation in lesions. Furthermore, we demonstrate that LDL and oxidized LDL stimulate SAPK/JNK activation in cultured SMCs independent of classic LDL receptors. New Zealand White male rabbits weighing between 1,800 and 2,200 g were obtained from Charles River (Kissleg, Germany). All animals were selected for serum cholesterol levels under 100 mg/dl, individually housed in wire-bottomed cages at 22°C with a relative humidity of 55%. All received water ad libitum and were fed either a normal standard chow diet (T775; Tagger & Co., Graz, Austria) or a cholesterol-enriched diet (0.2% w/w) for 16 weeks, as described previously.34Xu Q Dietrich H Steiner HJ Gown AM Mikuz G Kaufmann SHE Wick G Induction of arteriosclerosis in normocholesterolemic rabbits by immunization with heat shock protein 65.Arterioscler Thromb. 1992; 12: 789-799Crossref PubMed Scopus (349) Google Scholar, 35Xu Q Kleindienst R Waitz W Dietrich H Wick G Increased expression of heat shock protein 65 coincides with a population of infiltrating T lymphocytes in atherosclerotic lesions of rabbits specifically responding to heat shock protein 65.J Clin Invest. 1993; 91: 2693-2702Crossref PubMed Scopus (181) Google Scholar Animals were sacrificed by heart puncture under ketamine (25 mg/kg) and xylazine (5 to 10 mg/kg) anesthesia. Serum was collected for cholesterol assays and LDL isolation. The aortas were carefully removed intact from the aortic arch to the iliac bifurcation, immediately put into cold phosphate-buffered saline (PBS) (4°C) and prepared for histological analysis, tissue culture, and protein extractions. The total surface area of aortas covered by lesions was evaluated. For conventional histology, tissue fragments were fixed in 4% buffered (pH 7.2) formaldehyde, embedded in paraffin, and sectioned for hematoxylin and eosin (H&E) staining. Serum cholesterol values were measured every 2 or 4 weeks using an enzymatic procedure (Sigma Chemical Co., St. Louis, MO). The procedure used for immunohistochemical staining was similar to that described elsewhere.34Xu Q Dietrich H Steiner HJ Gown AM Mikuz G Kaufmann SHE Wick G Induction of arteriosclerosis in normocholesterolemic rabbits by immunization with heat shock protein 65.Arterioscler Thromb. 1992; 12: 789-799Crossref PubMed Scopus (349) Google Scholar, 35Xu Q Kleindienst R Waitz W Dietrich H Wick G Increased expression of heat shock protein 65 coincides with a population of infiltrating T lymphocytes in atherosclerotic lesions of rabbits specifically responding to heat shock protein 65.J Clin Invest. 1993; 91: 2693-2702Crossref PubMed Scopus (181) Google Scholar Briefly, serial 4-μm-thick frozen sections were overlaid with mouse monoclonal antibodies against α-actin (Sigma), macrophages (RAM11; DAKO, Copenhagen, Denmark), or T cells (L11/135; ATCC, Rockville, MD) and incubated with rabbit anti-mouse immunoglobulin (Ig) conjugated with peroxidase (DAKO) and developed for 20 minutes at room temperature using a substrate solution. For immunofluorescence staining, a monoclonal antibody against SAPK/JNK1 (Transduction Lab., Lexington, KY) was added to the sections. After three washes with PBS, the sections were incubated with a rabbit anti-mouse Ig-tetramethylrhodamine B isothiocyanate (TRITC) conjugate (DAKO) for 30 minutes, or sections were incubated with a monoclonal antibody against phosphorylated SAPK/JNK conjugated with fluorescein isothiocyanate (FITC) (p-JNK; Santa Cruz Biochem., Santa Cruz, CA), mounted in glycerol/PBS, and examined in an epiillumination immunofluorescence microscope equipped with appropriate filter combinations for the three wavelength method (Leitz, Wetzlar, Germany). For immunofluorescence double staining, frozen sections were incubated with monoclonal antibodies against α-actin Cy3 conjugate (Sigma) or RAM11 macrophages. After three washes with PBS, the sections were incubated with a rabbit anti-mouse Ig-TRITC conjugate (DAKO) for 30 minutes, rinsed, blocked with normal mouse serum (1:5), and stained with a mouse monoclonal antibody against phosphorylated-SAPK/JNK-FITC conjugate (p-JNK). For p53 and JNK1 double staining, a mouse monoclonal anti-p53 antibody and rabbit anti-JNK1 antibodies (Santa Cruz Biotech. Inc.) were used and visualized with swine anti-mouse Ig-FITC and goat anti-rabbit Ig-TRITC. For visualization of nuclei, sections were counterstained with the DNA stain Hoechst 33258 (1 μg/ml PBS; Lambda Probes, Graz, Austria) for 1 minute. The procedure used for protein extracts was similar to that described previously36Metzler B Hu Y Sturm G Wick G Xu Q Induction of mitogen-activated protein kinase phosphatase-1 by arachidonic acid in vascular smooth muscle cells.J Biol Chem. 1998; 273: 33320-33326Crossref PubMed Scopus (56) Google Scholar with a slight modification. Briefly, the atherosclerotic-lesioned intima and media were dissected on ice from the remaining adventitia with tweezers and scissors. Tissues were homogenized in buffer A (20 mmol Hepes, pH 7.4, 50 mmol/L β-glycerophosphate, 2 mmol/L EGTA, 1 mmol/L dithiothreitol, 1 mmol/L Na3VO4, 1% Triton X-100, 10% glycerol, 1 μg/ml leupeptin, 100 μmol/L phenylmethyl sulfonyl fluoride (PMSF), and 1 μg/ml aprotinin), and proteins were extracted. Proteins (50 μg/lane) were separated by electrophoresis through a 10. SDS-polyacrylamide gel and transferred to an immobilon-p transfer membrane. The membranes were processed with monoclonal antibodies against SAPK/JNK1, actin, p53, BAX, and BCL-XS/L (Santa Cruz Biotech. Inc.), respectively, and specific antigen-antibody complexes were detected with the ECL Western Blot Detection Kit (Amersham Co., Little Chalfont, UK). One-half milliliter of the supernatant containing 0.5 mg proteins was incubated with 10 μl of rabbit anti-SAPK/JNK1 antibodies and 40 μl of protein A-agarose suspension (Santa Cruz Biochem.). The immunocomplexes were precipitated by centrifugation and washed 2 times with buffer A, buffer B (500 mmol/L LiCl, 100 mmol/L Tris, 1 mmol/L dithiothreitol, 0.1% Triton X-100, pH 7.6), and buffer C (20 mmol/L Mops, 2 mmol/L EGTA, 10 mmol/L MgCl2, 1 mmol/L dithiothreitol, 0.1% Triton X-100, pH 7.2), respectively. The activities of SAPK/JNK in the immunocomplexes were measured as described previously.11Hu Y Metzler B Xu Q Discordant activation of stress-activated protein kinases or c-Jun NH2-terminal protein kinases in tissues of heat-stressed mice.J Biol Chem. 1997; 272: 9113-9119Crossref PubMed Scopus (60) Google Scholar, 16Kyriakis JM Banerjee P Nikolakaki E Dai T Rubie EA Ahmad MF Avruch J Woodgett JD The stress-activated protein kinase subfamily of c-Jun kinases.Nature. 1994; 369: 156-160Crossref PubMed Scopus (2411) Google Scholar Briefly, immunocomplexes were incubated with 35 μl of buffer C supplemented with glutathione S-transferase (GST)-c-Jun (5 μg), γ-32P-ATP (Amersham, Little Chalfont, UK), and MgCl2 (50 mmol/L) for 20 minutes at 37°C with vortexing every 5 minutes. The GST-c-Jun fusion protein (plasmid provided by Dr. Woodgett, Toronto, Canada) was produced in Escherichia coli and isolated using glutathione Sepharose 4B RediPack Columns (Pharmacia Biotech Inc., Piscataway, NJ) according to the manufacturer's protocol. To stop the reaction, 15 μl of 4× Laemmli buffer was added and the mixture was boiled for 5 minutes. Proteins in the kinase reaction were resolved by SDS-polyacrylamide gel electrophoresis (12% gel) and subjected to autoradiography. For immunoprecipitation of SAPK/JNK1 and p53 Western blot analysis, 1 ml of the supernatant containing 2 mg of protein was incubated with 50 μl of rabbit anti-SAPK/JNK1 antibodies and 100 μl of protein A-agarose suspension. The immunocomplexes were precipitated as described for the kinase assay and resolved in SDS-polyacrylamide gel electrophoresis (15% gel). Human and rabbit vascular SMCs were cultivated from arteries using the procedure described by Ross and Kariya37Ross R Kariya B Smooth muscle cells in culture.in: Bohr DF Somlyo AP Sparks HV Handbook of Physiology: Circulation, Vascular Smooth Muscle. American Phyiological Society, Bethesda1980: 69-91Google Scholar with a slight modification.38Xu Q Hu Y Kleindienst R Wick G Nitric oxide induces heat-shock protein 70 expression in vascular smooth muscle cells via activation of heat shock factor 1.J Clin Invest. 1997; 100: 1089-1097Crossref PubMed Scopus (127) Google Scholar In short, specimens of human carotid arteries were obtained from the Department of Vascular Surgery, University Hospital of Innsbruck. Intima and inner layer of media were dissected from the arteries and cut into pieces, digested with collagenase and elastase, and cultured in Dulbecco's modified essential medium (DMEM; Life Technologies, Inc., Grand Island, NY). Thoracic aortas of chow- and cholesterol-fed rabbits were removed, and lesioned intima and normal media were carefully dissected from the vessel, cut into pieces (∼1 mm3), and implanted onto a gelatin-coated (0.02%) plastic bottle (Becton Dickinson, Oxnard, CA). The bottle was incubated up-side-down at 37°C in a humidified atmosphere of 95% air/5% CO2 for 3 hours, and then medium supplemented with 20% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) was slowly added. Cells were passaged by treatment with 0.05% trypsin/0.02. ethylenediaminetetraacetic acid (EDTA) solution. Experiments were conducted on SMCs that had just achieved confluence. Similarly, aortic SMCs from Watanabe rabbits (La Roche, Basel, Switzerland) were cultivated and used for SAPK/JNK kinase assays. EDTA plasma was pooled from normolipemic, fasting (12 to 14 hours. male and female donors, aged 20 to 30 years. Lipoproteins were prepared by differential centrifugation using solid KBr to adjust the density, as described previously.39Jürgens G Xu Q Huber LA Böck G Howanietz H Wick G Traill KN Promotion of lymphocyte growth by high density lipoproteins (HDL): physiological significance of the HDL binding site.J Biol Chem. 1989; 264: 8549-8556PubMed Google Scholar, 40Xu Q Jürgens G Huber LA Böck G Wolf H Wick G Lipid utilization by human lymphocytes is correlated with high-density-lipoprotein binding site activity.Biochem J. 1992; 285: 105-112PubMed Google Scholar LDL were obtained in fraction between 1.020 to 1.050 g/ml. Concentrations of LDL were determined gravimetrically by aliquot weight after drying, and quantities of lipoproteins were expressed as total weights.39Jürgens G Xu Q Huber LA Böck G Howanietz H Wick G Traill KN Promotion of lymphocyte growth by high density lipoproteins (HDL): physiological significance of the HDL binding site.J Biol Chem. 1989; 264: 8549-8556PubMed Google Scholar, 40Xu Q Jürgens G Huber LA Böck G Wolf H Wick G Lipid utilization by human lymphocytes is correlated with high-density-lipoprotein binding site activity.Biochem J. 1992; 285: 105-112PubMed Google Scholar LDL oxidation was performed by incubation of LDL (1 mg/ml PBS) with 10 μmol/L CuCl2 at 37°C for 18 hours.41Amberger A Maczek C Jürgens G Michaelis D Schett G Xu Q Wick G Co-expression of ICAM-1, VCAM-1, ELAM-1 and Hsp60 in human arterial venous endothelial cells in response to cytokines and oxidized low density lipoproteins.Cell Stress Chaperones. 1997; 2: 94-103Crossref PubMed Scopus (167) Google Scholar The extent of oxidation was assessed by measurement of thiobarbituric acid reactive substances (TBARS) (9.8 ± 1.3 nmol/mg).42Buege JA August SD Microsomal lipid peroxidation.Methods Enzymol. 1978; 52: 302-310Crossref PubMed Scopus (10696) Google Scholar The Mann-Whitney U test was used for comparison between two groups. P < 0.05 was considered statistically significant. Animals in the control group (chow diet) had blood cholesterol levels <100 mg/dl, whereas blood cholesterol levels in rabbits receiving the 0.2% cholesterol diet were significantly elevated and reached 500 mg/dl at 4 weeks, and ∼700 mg/dl at 16 weeks. Rabbit aortas from both groups were examined morphologically and immunohistologically 16 weeks after chow or cholesterol feeding. Fifty percent to 80% of aortic intima was covered by atherosclerotic lesions, including fatty streaks and plaques in cholesterol-fed rabbits. Aortic lesions of rabbits fed with a cholesterol-enriched diet were characterized by cell proliferation, foam cell accumulation, and lipid deposition in the intima. Atherosclerotic lesions displayed great variations in cell type and distribution. The overlying endothelium was intact to variable degrees, and α-actin-positive SMCs appeared in various stages of lesions, most frequently in advanced lesions (Figure 1) Cells expressing the macrophage antigen identified by the RAM11 antibody were observed in all atherosclerotic lesions. T lymphocytes identified by monoclonal antibody L11/135 were also found in lesions (Figure 1) SAPK/JNK kinases are activated by dual phosphorylation of tyrosine and threonine residues in response to stress stimuli or mitogens.13Davis RJ The mitogen-activated protein kinase signal transduction pathway.J Biol Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 14Seger R Krebs EG The MAPK signaling cascade.FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3192) Google Scholar, 15Cano E Mahadevan LC Parallel signal processing among mammalian MAPKs.Trends Biochem Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (997) Google Scholar After activation, they translocate from cytoplasm into the nucleus in cultured cells. We performed an immunofluorescent analysis of atherosclerotic lesions using the antibody recognizing the pan-SAPK/JNK and phosphorylated SAPK/JNK, respectively. Intimal endothelium, media, and adventitia of normal aortas showed very weak SAPK/JNK staining (Figure 2, a and d), while the lesion-covered areas in intima from rabbits receiving a cholesterol-rich diet showed increased immunostaining intensity (Figure 2, c, e, and f). Nonspecific reactivity was minimal in the negative controls stained with a normal mouse Ig (Figure 2b). The pattern of pan-SAPL/JNK and phosphorylated SAPK/JNK staining in the lesioned aortas is shown in Figure 2, c, e, and f. Heterogeneity of pan-SAPK/JNK and phosphorylated SAPK/JNK staining became more evident in atherosclerotic plaques. Sites of increased pan-SAPK/JNK and phosphorylated SAPK/JNK were mainly within the cap and base regions of the atherosclerotic plaque (Figure 2, c, e, and f). Fatty streaks displayed elevated SAPK/JNK content in subendothelial regions. Next, we performed immunofluorescence double staining using the antibody against phosphorylated-SAPK/JNK to identify the cells responsible for the elevated SAPK/JNK expression. Figure 3, a–d, shows data representing double immunostaining with antibodies against phosphorylated-SAPK/JNK (a and c; green), α-actin (b; red), and RAM11 macrophages (d; red). Nonspecific staining was minimal in the corresponding negative controls stained with a normal mouse Ig. Typical double-positive cells are indicated by arrows, demonstrating a portion of SMCs and macrophages in lesions overexpressing SAPKs/JNKs. Because of the increased intensity of immunostaining for lesions, it would be interesting to determine SAPK/JNK1 protein levels of atherosclerotic lesions. Protein extracts from tissues of normal intima/media, lesioned intima, and media were analyzed by Western blot analysis. Abundant SAPK/JNK1 proteins in atherosclerotic
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