Advanced Glycation End Products Enhance Expression of Pro-apoptotic Genes and Stimulate Fibroblast Apoptosis through Cytoplasmic and Mitochondrial Pathways

Both aging and diabetes are characterized by the formation of advanced glycation end products (AGEs). Both exhibit other similarities including deficits in wound healing that are associated with higher rates of fibroblast apoptosis. In order to investigate a potential mechanism for enhanced fibroblast apoptosis in diabetes and aged individuals, experiments were carried out to determine whether the predominant advanced glycation end product in skin, N-ϵ-(carboxymethyl) lysine (CML)-collagen, could induce fibroblast apoptosis. In vivo experiments established that CML-collagen but not unmodified collagen induced fibroblast apoptosis and that apoptosis was dependent upon caspase-3, -8, and -9 activity. In vitro experiments demonstrated that CML-collagen but not control collagen induced a time- and dose-dependent increase in fibroblast apoptosis. By use of blocking antibodies, apoptosis was shown to be mediated through receptor for AGE signaling. AGE-induced apoptosis was largely dependent on the effector caspase, caspase-3, which was activated through both cytoplasmic (caspase-8-dependent) and mitochondrial (caspase-9) pathways. CML-collagen had a global effect of enhancing mRNA levels of pro-apoptotic genes that included several classes of molecules including ligands, receptors, adaptor molecules, mitochondrial proteins, and others. However, the pattern of expression was not identical to the pattern of apoptotic genes induced by tumor necrosis factor α. Both aging and diabetes are characterized by the formation of advanced glycation end products (AGEs). Both exhibit other similarities including deficits in wound healing that are associated with higher rates of fibroblast apoptosis. In order to investigate a potential mechanism for enhanced fibroblast apoptosis in diabetes and aged individuals, experiments were carried out to determine whether the predominant advanced glycation end product in skin, N-ϵ-(carboxymethyl) lysine (CML)-collagen, could induce fibroblast apoptosis. In vivo experiments established that CML-collagen but not unmodified collagen induced fibroblast apoptosis and that apoptosis was dependent upon caspase-3, -8, and -9 activity. In vitro experiments demonstrated that CML-collagen but not control collagen induced a time- and dose-dependent increase in fibroblast apoptosis. By use of blocking antibodies, apoptosis was shown to be mediated through receptor for AGE signaling. AGE-induced apoptosis was largely dependent on the effector caspase, caspase-3, which was activated through both cytoplasmic (caspase-8-dependent) and mitochondrial (caspase-9) pathways. CML-collagen had a global effect of enhancing mRNA levels of pro-apoptotic genes that included several classes of molecules including ligands, receptors, adaptor molecules, mitochondrial proteins, and others. However, the pattern of expression was not identical to the pattern of apoptotic genes induced by tumor necrosis factor α. Advanced glycation end products (AGEs) 1The abbreviations used are: AGE, advanced glycation end product; RAGE, receptor for advanced glycation end product; CML, N-ϵ-(carboxymethyl) lysine; TNF, tumor necrosis factor; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; AFC, 7-amino-4-trifluoromethyl coumarin; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift analysis; RT, reverse transcription; NF-κB, nuclear factor κB; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.1The abbreviations used are: AGE, advanced glycation end product; RAGE, receptor for advanced glycation end product; CML, N-ϵ-(carboxymethyl) lysine; TNF, tumor necrosis factor; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; AFC, 7-amino-4-trifluoromethyl coumarin; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift analysis; RT, reverse transcription; NF-κB, nuclear factor κB; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. result from nonenzymatic reactions of carbohydrates and oxidized lipids with proteins (1.Monnier V. 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The most thoroughly studied is receptor for advanced glycation end products (RAGE). RAGE is a multi-ligand member of the immunoglobulin superfamily of cell surface receptors that binds many ligands including those of the S100/calgranulin family (22.Hofmann M. Drury S. Fu C. Qu W. Taguchi A. Lu Y. Avila C. Kambham N. Bierhaus A. Nawroth P. Neurath M. Slattery T. Beach D. Mcclary J. Nagashima M. Morser J. Stern D. Schmidt A. Cell. 1999; 97: 889-901Abstract Full Text Full Text PDF PubMed Scopus (1595) Google Scholar). Other receptors/binding proteins include macrophage scavenger receptor types I and II, oligosaccharyl transferase-48 (AGE-R1), 80K-H phosphoprotein (AGE-R2), and galectin-3 (AGE-R3). These are expressed on a wide range of cells including smooth muscle cells, monocytes, macrophages, endothelial cells, podocytes, astrocytes, microglia, and fibroblasts. Most of the biologic activities associated with AGEs have been shown to be transduced by RAGE, whereas the scavenger receptors are thought to regulate removal of AGEs (23.Stern D. Yan S. Yan S. Schmidt A. Ageing Res. Rev. 2002; 1: 1-15Crossref PubMed Scopus (233) Google Scholar, 24.Bucciarelli L. Wendt T. Rong L. Lalla E. Hofmann M. Goova M. Taguchi A. Yan S. Yan S. Stern D. Schmidt A. Cell Mol. Life Sci. 2002; 59: 1117-1128Crossref PubMed Scopus (262) Google Scholar). AGEs can modulate inflammatory events by stimulating production of reactive oxygen species, chemotaxis, activation of monocytes/macrophages, and stimulation of interleukin-1 and TNF production (25.Guzdek A. Stalinska K. Folia Histochem. Cytobiol. 1992; 30: 167-169PubMed Google Scholar, 26.Schmidt A. Yan S. Brett J. Mora R. Nowygrod R. Stern D. J. Clin. Investig. 1993; 91: 2155-2168Crossref PubMed Scopus (269) Google Scholar, 27.Vlassara H. Brownlee M. Manogue K. Dinarello C. Pasagian A. 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Caliste X. Yan S.F. Stern D.M. Schmidt A.M. Am. J. Pathol. 2001; 159: 513-525Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). One mechanism through which AGEs may affect pathologic processes is by enhanced apoptosis as supported by in vitro studies. AGEs are pro-apoptotic for cultured retinal pericytes, corneal endothelial cells, neuronal cells, and renal mesangial cells (36.Kasper M. Roehlecke C. Witt M. Fehrenbach H. Hofer A. Miyata T. Weigert C. Funk R. Schleicher E. Am. J. Respir. Cell Mol. Biol. 2000; 23: 485-491Crossref PubMed Scopus (72) Google Scholar, 37.Denis U. Lecomte M. Paget C. Ruggiero D. Wiernperger N. Lagarde M. Free Radic. Biol. Med. 2002; 33: 236-247Crossref PubMed Scopus (119) Google Scholar, 38.Kaji Y. Amano S. Usui T. Osbika T. Yamahiro K. Isbida S. Suzuki K. Tanaka S. Adamis A. Nagai R. Horiuchi S. Investig. Ophthalmol. 2003; 44: 521-528Google Scholar). The mechanisms by which AGEs lead to apoptosis are not well understood. There are reports suggesting that AGEs may enhance apoptosis indirectly through increasing oxidative stress or via induced expression of pro-apoptotic cytokines (36.Kasper M. Roehlecke C. Witt M. Fehrenbach H. Hofer A. Miyata T. Weigert C. Funk R. Schleicher E. Am. J. Respir. Cell Mol. Biol. 2000; 23: 485-491Crossref PubMed Scopus (72) Google Scholar, 38.Kaji Y. Amano S. Usui T. Osbika T. Yamahiro K. Isbida S. Suzuki K. Tanaka S. Adamis A. Nagai R. Horiuchi S. Investig. Ophthalmol. 2003; 44: 521-528Google Scholar, 39.Yamagishi S. Inagaki Y. Amano S. Okamoto T. Takeuchi M. Makita Z. Biochem. Biophys. Res. Commun. 2002; 296: 877-882Crossref PubMed Scopus (201) Google Scholar). Interestingly, enhanced apoptosis of these cells is also associated with diabetic complications such as retinopathy, neuropathy, nephropathy, and accelerated vasculopathy (38.Kaji Y. Amano S. Usui T. Osbika T. Yamahiro K. Isbida S. Suzuki K. Tanaka S. Adamis A. Nagai R. Horiuchi S. Investig. Ophthalmol. 2003; 44: 521-528Google Scholar, 39.Yamagishi S. Inagaki Y. Amano S. Okamoto T. Takeuchi M. Makita Z. Biochem. Biophys. Res. Commun. 2002; 296: 877-882Crossref PubMed Scopus (201) Google Scholar, 40.Huang D. Wang J. Kivisakk P. Rollins B. Ransohoff R. J. Exp. Med. 2001; 193: 713-726Crossref PubMed Scopus (508) Google Scholar). Enhanced apoptosis has been linked to many of the detrimental effects of aging, which is also associated with AGE accumulation (41.Higami Y. Shimokawa I. Cell Tissue Res. 2000; 301: 125-132Crossref PubMed Scopus (153) Google Scholar). Because fibroblasts play important roles in the maintenance and healing of dermal connective tissue, the accumulation of AGEs in skin may have a detrimental effect, in part, through promoting fibroblast apoptosis. To address this issue, we investigated the apoptotic effect of AGEs on fibroblasts and the mechanisms through which AGEs induce apoptosis in these cells. CML-collagen—CML-collagen was prepared by chemical modification of acid-soluble bovine skin collagen (Sigma), as described previously (21.Santana R. Xu L. Chase H. Amar S. Graves D. Trackman P. Diabetes. 2003; 52: 1502-1510Crossref PubMed Scopus (197) Google Scholar, 42.Kislinger T. Fu C. Huber B. Qu W. Taguchi A. Du Yan S. Hofmann M. Yan S. Pischesrieder M. Stern D. Schmidt A. J. Biol. Chem. 1999; 274: 31740-31749Abstract Full Text Full Text PDF PubMed Scopus (790) Google Scholar). Briefly, 50 mg of collagen was dissolved in 25 ml of 1 mm HCl freshly made in sterile water and incubated at 37 °C with occasional mixing. Sterile PBS (25 ml) was added, followed by sodium cyanoborohydride (1.42 g) and glyoxylic acid (0.715 g). Control collagen was prepared at the same time, except that no glyoxylic acid was added. All samples were then incubated at 37 °C for 24 h. AGE collagen and control collagen were then exhaustively dialyzed against distilled water. In dose-response experiments where higher concentrations of CML-collagen were used, AGE and control collagen was dialyzed against PBS. Both CML-collagen and control collagen were soluble at the concentrations stored and tested. In total, 3–8% of lysine residues in CML-collagen were converted to CML, as determined by the trinitrobenzenesulfonic acid assay (43.Habeeb A. Anal. Biochem. 1966; 14: 328-336Crossref PubMed Scopus (1917) Google Scholar). The percentage of modification of collagen that we have generated is 10-fold less than the amount used in a recent report to assess CML binding and activation of NF-κB (42.Kislinger T. Fu C. Huber B. Qu W. Taguchi A. Du Yan S. Hofmann M. Yan S. Pischesrieder M. Stern D. Schmidt A. J. Biol. Chem. 1999; 274: 31740-31749Abstract Full Text Full Text PDF PubMed Scopus (790) Google Scholar) and only a small amount higher than that reported for the skin of aged or diabetic individuals (31.Dyer D. Dunn J. Thorpe S. Bailie K. Lyons T. McCance D. Baynes J. J. Clin. Investig. 1993; 91: 2463-2469Crossref PubMed Scopus (639) Google Scholar). CML-collagen was highly reactive on Western blots with anti-CML monoclonal antibody 6D12 (Wako, Richmond, VA), whereas control collagen was not reactive. The amount of endotoxin contamination was measured by Pyrochrome Limulus Amebocyte Lysate assay (Associates of Cape Cod, Inc., Woods Hole, MA) and found to be low (0.094 ± 0.01 ng/mg lipopolysaccharide in control collagen and 0.087 ± 0.01 ng/mg lipopolysaccharide in CML-collagen). Animals—CD1 mice were purchased from Charles River Laboratories, (Waltham, MA). These are outbred mice that were selected because they do not exhibit strain-associated responses, as has been reported in some cases where a particular strain of mouse has been studied (45.Belyavskyi M. Levy G. Leibowitz J. Adv. Exp. Med. Biol. 1998; 440: 619-625Crossref PubMed Scopus (3) Google Scholar, 46.Shi Z. Wakil A. Rockey D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10663-10668Crossref PubMed Scopus (283) Google Scholar). All procedures involving mice were approved by the Boston University Medical Center Institutional Animal Care and Use Committee. Mice were anesthetized with injection of ketamine (80 mg/kg) and xylazine (10 mg/kg) in sterile PBS. CML-collagen or unmodified collagen was injected into the loose connective tissue adjacent to calvarial bone at a point on the midline of the skull located between the ears. Injection at this anatomic site can be reproducibly achieved. For each data point, there were six mice (n = 6). We undertook preliminary experiments to identify a dose for CML-collagen that gave a moderate number of apoptotic cells. On this basis, 100 μg of CML-collagen or an equal amount of unmodified collagen was injected. Mice were euthanized 24 h after injection. In addition to CML-collagen or unmodified collagen alone, some animals were treated by intraperitoneal injection of caspase-3, -8, or -9 inhibitor (1 mg/kg) 1 h before CML-collagen injection and locally (25 μg) at the time of CML-collagen injection. The caspase-3 inhibitor Z-DEVD-fmk, the caspase-8 inhibitor Z-IETD-fmk, and the caspase-9 inhibitor Z-LEHD-fmk were purchased from R&D Systems (Minneapolis, MN). Control mice received CML-collagen or unmodified collagen containing 2% Me2SO (Sigma-Aldrich). Preparation of Histologic Sections—Animals were euthanized by decapitation, and their heads were fixed for 72 h in cold 4% paraformaldehyde and decalcified by incubation with cold Immunocal (Decal Corp., Congers, NY) for ∼12 days with solution changed daily. Paraffin-embedded sagittal sections were prepared at a thickness of 5–6 μm. TUNEL Assay and Quantitative Histologic Analysis—Apoptotic cells were detected by an in situ TUNEL assay by means of a TACS 2 TdT-Blue Label kit purchased from Trevigen (Gaithersburg, MD), following the manufacturer's instructions. Sections were counterstained with nuclear fast red. The number of fibroblastic apoptotic cells was determined in six specimens per group by counting the number of TUNEL-positive cells that had the characteristic microscopic appearance of fibroblasts. Counts and measurements were confirmed by reanalysis of all the specimens by an independent examiner. The intra- and inter-examiner variations were <10%. Student's t test was used to determine significant differences between the experimental and control groups at the p < 0.05 level. Caspase Activity—Caspase-3, -8, and -9 activities from in vitro and in vivo experiments were assayed with fluorometric kits purchased from R&D Systems. Briefly, after sacrifice at the indicated time points, murine scalps were immediately dissected from the calvaria and frozen in liquid nitrogen. Frozen tissues were pulverized, and lysates were prepared using cell lysis buffer provided by R&D Systems. After centrifugation, total protein was quantitated using a BCA protein assay kit (Pierce). Caspase-3 activity was detected by using the specific caspase-3 fluorogenic substrate, DEVD peptide conjugated to 7-amino-4-trifluoromethyl coumarin (AFC). Caspase-8 activity was detected by using the specific caspase-8 fluorogenic substrate, IETD-AFC. Caspase-9 activity was detected by using the specific caspase-9 fluorogenic substrate, LEHD-AFC. Measurements were made on a fluorescent microplate reader using filters for excitation (400 nm) and detection of emitted light (505 nm). In some assays, recombinant caspase-3 enzyme (R&D Systems) was used as a positive control. Buffers without cell lysate and cell lysate without substrate were used as negative controls. The same caspase-3, -8, and-9 inhibitors were used in both in vivo and in vitro studies. In addition, the pan-caspase (general caspase) inhibitor Z-VAD-fmk was purchased from R&D Systems for in vitro assays. For each group, there were six specimens (n = 6). Cell Culture—Primary human adult dermal fibroblasts were purchased from Cambrex (Walkersville, MD). Cells were propagated and maintained in Dulbecco's modified Eagle's medium (Cambrex) supplemented with 10% fetal bovine serum, gentamicin (100 μg/ml), and amphotericin B (100 ng/ml) at 37 °C in a humidified atmosphere of 5% CO2. Experiments with CML-collagen were performed in culture medium supplemented with 0.5% fetal bovine serum. Assays were performed when the cultures reached 75–85% confluence. In most experiments, 200 μg/ml CML-collagen or unmodified control collage was used, which is equivalent to 2 μm. Apoptosis of fibroblasts was determined by an ELISA technique measuring histone-associated DNA fragments (Roche Applied Science), following the manufacturer's instructions. In these studies, 20,000 fibroblasts/cm2 were treated with CML-collagen (200 μg/ml) or unmodified collagen (200 μg/ml) for 24 h. Apoptosis was determined by ELISA, and the cell numbers were assessed in corresponding wells to normalize apoptosis measurements. For caspase activity measurements, the same technique was used as described above for in vivo studies. In some cases, cells were treated with caspase-3, caspase-8, caspase-9, caspase-8 + caspase-9, or pancaspase inhibitors (50 μm) at the time of CML-collagen stimulation. The dose of each inhibitor was selected based on our previous results (47.Alikhani M. Alikhani Z. He H. Liu R. Popek I. Graves D. J. Biol. Chem. 2003; 278: 52901-52908Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Control cells were incubated in assay medium supplemented with vehicle alone (2% Me2SO) in the culture media. Inhibition of RAGE—Antibodies to the extracellular domain of RAGE have been shown to inhibit binding of CML modified proteins to RAGE and have been used to assess the impact of RAGE activation of cellular events (42.Kislinger T. Fu C. Huber B. Qu W. Taguchi A. Du Yan S. Hofmann M. Yan S. Pischesrieder M. Stern D. Schmidt A. J. Biol. Chem. 1999; 274: 31740-31749Abstract Full Text Full Text PDF PubMed Scopus (790) Google Scholar, 48.Oldfield M. Bach L. Forbes J. Nikolic-Paterson D. McRober A. Thallas V. Atkins R. Osicka T. Jerums G. Cooper M. J. Clin. Investig. 2001; 108: 1853-1863Crossref PubMed Scopus (392) Google Scholar). To study the role of RAGE, fibroblast cultures were incubated with CML-collagen or unmodified collagen (200 μg/ml) in the presence or absence of anti-RAGE polyclonal antibodies specific for the extracellular domain of RAGE (10 μg/μl) or non-immune serum (10 μg/μl) for 24 h (Biocompare, San Francisco, CA). As controls, cells were incubated with 10 μg/ml RAGE antiserum or non-immune serum without AGE stimulation. The extent of apoptosis was determined by ELISA. Statistical difference between samples was determined by one-way analysis of variance followed by Tukey's multiple comparison tests. EMSA—Fibroblast cultures were incubated in assay medium for 1 h with 200 μg/ml CML-collagen or unmodified collagen in the presence or absence of a specific NF-κB inhibitor, SN50 (100 μg/ml) (Biomol, Plymouth Meeting, PA). Nuclear proteins were extracted using protein extraction kit (Pierce) following the manufacturer's instructions. Concentrations of nuclear proteins were measured by using BCA protein assay kit (Pierce). Interaction between NF-κB in the protein extract and DNA probe was investigated using EMSA kit from Panomics (Redwood City, CA) following the manufacturer's instruction. Microarray—Fibroblast cell cultures were exposed to CML-collagen (200 μg/ml) or TNF-α (20 ng/ml) for 6 h. TNF-α was purchased from R&D Systems. Control cells were exposed to either unmodified collagen (200 μg/ml) or vehicle alone (PBS). RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA). Human Apoptosis GEArray Q series kit that includes 96 key genes involved in apoptosis was purchased from SuperArray, Inc. (Bethesda, MD). Total RNA was used as the template for 32P-labeled cDNA probe synthesis following the manufacturer's instructions. After overnight hybridization with labeled probe, the GEArray membranes were exposed using a PhosphorImager. The resulting digital image was converted to a raw data file using Scanalyze software (www.microarray.org/software.html). GEArray Analyzer software (SuperArray, Inc.) was used for data analysis. The microarray was performed twice with similar results, and the mean values of the two experiments are presented for each gene. The microarray for TNF-α stimulation was performed simultaneously with the AGE stimulation array. RT-PCR—Five μg of total RNA was used to produce cDNA using a First Strand cDNA Synthesis Kit (SuperArray, Inc.). PCR was performed on the synthesized cDNA using the SingleGene PCR Kit purchased from SuperArray, Inc. following the manufacturer's protocol. Specific primers were used for each gene, and GAPDH was used as internal control. This kit has the advantage that GAPDH and the gene of interest are amplified in the same tube and for the same number of cycles. Based on a pilot study, 24 and 28 cycles were chosen to compare the gene expression. After electrophoresis on a 2% agarose gel containing 0.5 μg/ml ethidium bromide, bands were visualized on a UV-box. The density of each band was measured and compared using Quantity One software (Bio-Rad). The optical density of each band was normalized by the value of the GAPDH in the same lane. Two separate RT-PCRs were performed with similar results. Student's t test was used to determine significant differences between the experimental and control groups. In Vivo Induction of Apoptosis by CML-collagen—To study the effect of AGEs on fibroblast apoptosis in vivo, 100 μg of CML-collagen or control collagen was injected in the scalp of CD1 mice. Histologic sections were examined for fibroblast apoptosis by their characteristic appearance in the TUNEL assay (Fig. 1). Fibroblast apoptosis could be detected in the CML-collagen-treated group. In comparison, there was virtually no induction of fibroblast apoptosis in mice treated with unmodified collagen compared with the zero time point (Fig. 1A). Quantitative analysis indicated that CML-collagen stimulated a 4-fold increase in apoptotic cells compared with unmodified collagen (Fig. 1B) (p < 0.05). When expressed as the percentage of TUNEL-positive fibroblasts, the levels were 0.85 ± 0.05 in the CML-collagen-inoculated group and 0.21 ± 0.01 (p < 0.05) for control collagen-treated group. In Vitro Induction of Apoptosis by CML-collagen—In vitro studies were carried out to measure the direct effect of AGEs on fibroblast apoptosis under well-defined conditions. Apoptosis of human adult primary skin fibroblasts was measured by the relative amount of histone-associated DNA fragments in the cytoplasm as determined by ELISA. A time course experiment determined that AGE-induced apoptosis was not detected before 6 h (Fig. 2A). At the 6 h time point, a 1.7-fold increase was noted, whereas at 24 h, a 3-fold increase in apoptosis was detected. The increase at 6 and 24 h was statistically significant (p < 0.05). CML-collagen induced a dose-dependent increase in fibroblast apoptosis, whereas control collagen did not (Fig. 2B). At 200 μg/ml, CML-collagen compared with unmodified collagen induced a 3-fold increase in apoptosis, which was statistically significant (p < 0.05). Saturating levels were reached at 400 μg/ml CML-collagen with a 5-fold increase in apoptosis (p < 0.05). In subsequent studies,