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Geriatr Gerontol Int 2016; 16: 522–527
ORIGINAL ARTICLE: BIOLOGY
Advanced glycation end-products accelerate the cardiac aging process through the receptor for advanced glycation end-products/transforming growth factor-β-Smad signaling pathway in cardiac fibroblasts Min Fang,1* Junhong Wang,1* Shiling Li1 and Yan Guo1,2 1
Department of Gerontlogy, the First Affiliated Hospital of Nanjing Medical University, Nanjing, and 2Department of Cardioangiology, Shengze Hospital of Jiangsu Province, Suzhou, China
Aims: The current study was carried out to evaluate the effect of advanced glycation end-products (AGE) on cardiac aging and to explore its underlying mechanisms. Methods: Neonatal rat cardiac fibroblasts were cultured and divided into four groups: control; AGE; AGE + receptor for AGE antibody and AGE + SB431542 (transforming growth factor-β [TGF-β]/Smad signaling pathway inhibitor, 10 μmol/L) group. After being cultured for 48 h, the cells were harvested and the senescence-associated betagalactosidase expression was analyzed. Then the level of p16, TGF-β, Smad/p-smad and matrix metalloproteinases-2 was evaluated by western blot. Results: Significantly increased senescence-associated beta-galactosidase activity as well as p16 level was observed in the AGE group. Furthermore, AGE also significantly increased the TGF-β1, p-smad2/3 and metalloproteinases-2 expression in cardiac fibroblasts (all P < 0.01). Meanwhile, either pretreatment with receptor for AGE-Ab or SB431542 significantly inhibited the upregulated cardiac senescence (beta-galactosidase activity and P16) and fibrosis-associated (TGF-β1, p-smad2/3 and metalloproteinases-2) markers induced by AGE. Conclusions: Taken together, all these results suggested that AGE are an important factor for cardiac aging and fibrosis, whereas the receptor for AGE and TGF-β/Smad signaling pathway might be involved in the AGE-induced cardiac aging process. Geriatr Gerontol Int 2016; 16: 522–527. Keywords: advanced glycation end-products, cardiac aging, receptor for advanced glycation end-products, transforming growth factor-β-Smad pathway.
Introduction Advanced glycation end-products (AGE) are stable end-products that are formed by proteins, nucleic acids or lipids through non-enzymatic glycation, also known as the Maillard reaction, including glycoxidation or lipoxidation products, such as N-epsiloncarboxymethyllysine, malondialdehyde- lysine and pentosidine.1 Advanced glycation can directly change protein function, turnover and trafficking though intra-
Accepted for publication 24 February 2015. Correspondence: Professor Yan Guo MD PhD, Department of Gerontology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China. Email: [email protected] *These two authors contributed equally to this work.
522 |
doi: 10.1111/ggi.12499
cellular post-translational modification of proteins.2 Several mechanisms have been regarded by which AGE contribute to development of pathological conditions and trigger negative effects: modification of proteins and injury of cell function by intracellular glycation of proteins; altering tissue structure and function, such as compliance of myocardium and vascular smooth muscle; forming cross-link by extracellular matrix proteins; and binding of specific cell surface receptors, receptor for AGE (RAGE) to activate intracellular signaling pathways.3–7 Cardiac aging, an independent risk factor for cardiovascular diseases, is associated with structural remodeling and functional decline in the heart.8,9 Cardiac aging leads to an elevated prevalence of cardiac hypertrophy, diastolic dysfunction and compromised myocardial performance.8 During the aging process, AGE are a major cause of cardiac and vascular dysfunction,10 and plasma © 2015 Japan . . Geriatrics Society
AGE and cardiac aging
AGE level is reported to be associated with diastolic dysfunction of aging humans.11 However, the underlying mechanisms of AGE-induced cardiac aging remain largely unknown. As cardiac fibroblasts are an important player in the process of cardiac fibrosis and aging, the purpose of the present study was to investigate the effects of AGE on cardiac aging in cardiac fibroblasts (CF), and the underlying mechanisms were studied by using anti-RAGE antibody and transforming growth factor-β (TGF-β)-Smad inhibitor in order to identify the involved signaling pathways.12
Materials and methods Materials and reagents AGE were purchased from Calbiochem (Temecula, CA, USA), which was prepared by reacting bovine serum albumin with glycoaldehyde under sterile conditions as described previously.13 A neutralizing anti-RAGE antibody was purchased from R&D systems (Minneapolis, MN, USA). Antibodies against phospho-smad2/3 and smad2/3 were from Cell Signaling Technology (Beverly, MA, USA). SB431542 was from Gene Operation (Ann Arbor, MI, USA). Rabbit anti-TGF-β1 and matrix metalloproteinases-2 (MMP-2) antibodies were from Epitomics (Burlingame, CA, USA). P16 was from Santa Cruz Biotechnology (Dallas, TX, USA). Senescence-βGalactosidase Staining kit was from Beyotime Institute of Biotechnology (Shanghai, China). Fetal bovine serum as well as Dulbecco’s modified Eagle’s medium were from Grand Island Biological Company (Gibco, Grand Island, NY, USA). The protein quantification assay kit was purchased from Bio-Rad (Hercules, CA, USA).
Cell culture Animal handling and use complied with the Guide for the Care and Use of Laboratory Animals published by the USA National Institutes of Health (NIH Publication No.85-23, revised 1996), and were approved by the Animal Care and Use Committee of Nanjing Medical University. Cardiac fibroblasts were isolated from neonatal Sprague–Dawley rats (1- to 3-day-old) as described previously.14 Hearts were carefully extracted, and then isolated ventricles were incubated in collagenase II solutions for further digestion. The homogenized tissue was allowed to adhere to the 100-mm dish in culture media (Dulbecco’s modified Eagle’s medium) containing 10% fetal bovine serum for 2 h at 37°C. CF were separated from cardiomyocytes by differential adhesion to the dish during this process. Cells were washed twice with phosphate-buffered saline, trypsinized and passaged twice. Cells at passage 3 were used for the present study, and were divided into four groups: control; AGE (200 μg/mL); RAGE-Ab (2 μg/ mL) + AGE (200 μg/mL; A + RAGE-Ab); SB431542 © 2015 Japan Geriatrics Society
(10 μmol/L) + AGE (200 μg/mL; A + SB) and cultured for 48 h. Nε-carboxy-methyl-lysine (CML) was the major form of AGE in vivo. In order to address the “gap” difference in the concentrations between the present culture condition (200 μg/mL of bovine-derived AGE) and the reported concentrations of AGE in humans, the CML concentration was measured by an enzyme-linked immunosorbent assay kit (Uscn Life Science, Wuhan, China). Bovine-derived AGE was characterized by higher CML concentration compared with the unmodified bovine serum albumin (2.97 nmol/mg protein in bovinederived AGE vs 0.15 nmol/mg protein in unmodified bovine serum albumin). We therefore examined the effects of 200 μg/mL bovine-derived AGE (the CML concentration in 200 μg/mL AGE was 0.59 nmol/mL (121.31 mg/L) on cardiac fibroblasts, whose CML concentrations are representative of those found in the plasma of aged diabetic patients.15
Western blot analysis For western blot, the protein samples were extracted from whole cells according to the procedure, and total protein (30 μg) was fractionated on sodium dodecylsulfatepolyacrylamide gel, and transferred to polyvinylidenefluoride (PVDF) membranes as described previously.9 After transfer, the membranes were blocked with 5% non-fat milk, and incubated overnight with primary antibodies against TGF-β, p-smad2/3, smad2/3, MMP-2, p16 and glyceraldehyde 3-phosphate dehydrogenase. Then the membranes were washed with tris buffered saline tween-20 (TBST), and incubated with anti-rabbit HRP-conjugated secondary antibodies for two hours at 4°C, washed with TBST, and visualized by ECL reagent (Life Technology Company, Rockford, lL, USA), then detected by Odyssey Infrared Imaging System (LI-COR Inc, Lincoln, Nebraska, USA). Protein of interest signal was normalized versus glyceraldehyde 3-phosphate dehydrogenase.
Beta-galactosidase activity Beta-galactosidase (SA-β-Gal) activity is detected by Senescence-β-Galactosidase assay chemically at pH 6.0 wash according to the instructions (Senescence-βGalactosidase Staining kit; Beyotime Institute of Biotechnology, Wuhan, China). Briefly, the cells were grown in six-well plates, interfered for 72 h, fixed with 5% fixative solution for 15 min at room temperature; washed three times with phosphate-buffered saline; and incubated in staining solution containing X-gal at 37°C overnight. Cells were photographed by ordinary optical microscope at ×10 magnification, and the proportions of SA-β-Gal positive cells were shown as a percentage of the total number of cells quantified in each dish. The data were expressed as the mean of triplicates ± SD. | 523
M Fang et al.
Statistical analysis All data are shown as mean ± SD. Statistical analyses were carried out with GraphPad Prism 5.0 software (Graphpad Software, Inc., San Diego, CA, USA). Oneway ANOVA was used to compare the difference in those groups. Statistical significance was determined at a P value of
Geriatr Gerontol Int 2016; 16: 522–527
ORIGINAL ARTICLE: BIOLOGY
Advanced glycation end-products accelerate the cardiac aging process through the receptor for advanced glycation end-products/transforming growth factor-β-Smad signaling pathway in cardiac fibroblasts Min Fang,1* Junhong Wang,1* Shiling Li1 and Yan Guo1,2 1
Department of Gerontlogy, the First Affiliated Hospital of Nanjing Medical University, Nanjing, and 2Department of Cardioangiology, Shengze Hospital of Jiangsu Province, Suzhou, China
Aims: The current study was carried out to evaluate the effect of advanced glycation end-products (AGE) on cardiac aging and to explore its underlying mechanisms. Methods: Neonatal rat cardiac fibroblasts were cultured and divided into four groups: control; AGE; AGE + receptor for AGE antibody and AGE + SB431542 (transforming growth factor-β [TGF-β]/Smad signaling pathway inhibitor, 10 μmol/L) group. After being cultured for 48 h, the cells were harvested and the senescence-associated betagalactosidase expression was analyzed. Then the level of p16, TGF-β, Smad/p-smad and matrix metalloproteinases-2 was evaluated by western blot. Results: Significantly increased senescence-associated beta-galactosidase activity as well as p16 level was observed in the AGE group. Furthermore, AGE also significantly increased the TGF-β1, p-smad2/3 and metalloproteinases-2 expression in cardiac fibroblasts (all P < 0.01). Meanwhile, either pretreatment with receptor for AGE-Ab or SB431542 significantly inhibited the upregulated cardiac senescence (beta-galactosidase activity and P16) and fibrosis-associated (TGF-β1, p-smad2/3 and metalloproteinases-2) markers induced by AGE. Conclusions: Taken together, all these results suggested that AGE are an important factor for cardiac aging and fibrosis, whereas the receptor for AGE and TGF-β/Smad signaling pathway might be involved in the AGE-induced cardiac aging process. Geriatr Gerontol Int 2016; 16: 522–527. Keywords: advanced glycation end-products, cardiac aging, receptor for advanced glycation end-products, transforming growth factor-β-Smad pathway.
Introduction Advanced glycation end-products (AGE) are stable end-products that are formed by proteins, nucleic acids or lipids through non-enzymatic glycation, also known as the Maillard reaction, including glycoxidation or lipoxidation products, such as N-epsiloncarboxymethyllysine, malondialdehyde- lysine and pentosidine.1 Advanced glycation can directly change protein function, turnover and trafficking though intra-
Accepted for publication 24 February 2015. Correspondence: Professor Yan Guo MD PhD, Department of Gerontology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China. Email: [email protected] *These two authors contributed equally to this work.
522 |
doi: 10.1111/ggi.12499
cellular post-translational modification of proteins.2 Several mechanisms have been regarded by which AGE contribute to development of pathological conditions and trigger negative effects: modification of proteins and injury of cell function by intracellular glycation of proteins; altering tissue structure and function, such as compliance of myocardium and vascular smooth muscle; forming cross-link by extracellular matrix proteins; and binding of specific cell surface receptors, receptor for AGE (RAGE) to activate intracellular signaling pathways.3–7 Cardiac aging, an independent risk factor for cardiovascular diseases, is associated with structural remodeling and functional decline in the heart.8,9 Cardiac aging leads to an elevated prevalence of cardiac hypertrophy, diastolic dysfunction and compromised myocardial performance.8 During the aging process, AGE are a major cause of cardiac and vascular dysfunction,10 and plasma © 2015 Japan . . Geriatrics Society
AGE and cardiac aging
AGE level is reported to be associated with diastolic dysfunction of aging humans.11 However, the underlying mechanisms of AGE-induced cardiac aging remain largely unknown. As cardiac fibroblasts are an important player in the process of cardiac fibrosis and aging, the purpose of the present study was to investigate the effects of AGE on cardiac aging in cardiac fibroblasts (CF), and the underlying mechanisms were studied by using anti-RAGE antibody and transforming growth factor-β (TGF-β)-Smad inhibitor in order to identify the involved signaling pathways.12
Materials and methods Materials and reagents AGE were purchased from Calbiochem (Temecula, CA, USA), which was prepared by reacting bovine serum albumin with glycoaldehyde under sterile conditions as described previously.13 A neutralizing anti-RAGE antibody was purchased from R&D systems (Minneapolis, MN, USA). Antibodies against phospho-smad2/3 and smad2/3 were from Cell Signaling Technology (Beverly, MA, USA). SB431542 was from Gene Operation (Ann Arbor, MI, USA). Rabbit anti-TGF-β1 and matrix metalloproteinases-2 (MMP-2) antibodies were from Epitomics (Burlingame, CA, USA). P16 was from Santa Cruz Biotechnology (Dallas, TX, USA). Senescence-βGalactosidase Staining kit was from Beyotime Institute of Biotechnology (Shanghai, China). Fetal bovine serum as well as Dulbecco’s modified Eagle’s medium were from Grand Island Biological Company (Gibco, Grand Island, NY, USA). The protein quantification assay kit was purchased from Bio-Rad (Hercules, CA, USA).
Cell culture Animal handling and use complied with the Guide for the Care and Use of Laboratory Animals published by the USA National Institutes of Health (NIH Publication No.85-23, revised 1996), and were approved by the Animal Care and Use Committee of Nanjing Medical University. Cardiac fibroblasts were isolated from neonatal Sprague–Dawley rats (1- to 3-day-old) as described previously.14 Hearts were carefully extracted, and then isolated ventricles were incubated in collagenase II solutions for further digestion. The homogenized tissue was allowed to adhere to the 100-mm dish in culture media (Dulbecco’s modified Eagle’s medium) containing 10% fetal bovine serum for 2 h at 37°C. CF were separated from cardiomyocytes by differential adhesion to the dish during this process. Cells were washed twice with phosphate-buffered saline, trypsinized and passaged twice. Cells at passage 3 were used for the present study, and were divided into four groups: control; AGE (200 μg/mL); RAGE-Ab (2 μg/ mL) + AGE (200 μg/mL; A + RAGE-Ab); SB431542 © 2015 Japan Geriatrics Society
(10 μmol/L) + AGE (200 μg/mL; A + SB) and cultured for 48 h. Nε-carboxy-methyl-lysine (CML) was the major form of AGE in vivo. In order to address the “gap” difference in the concentrations between the present culture condition (200 μg/mL of bovine-derived AGE) and the reported concentrations of AGE in humans, the CML concentration was measured by an enzyme-linked immunosorbent assay kit (Uscn Life Science, Wuhan, China). Bovine-derived AGE was characterized by higher CML concentration compared with the unmodified bovine serum albumin (2.97 nmol/mg protein in bovinederived AGE vs 0.15 nmol/mg protein in unmodified bovine serum albumin). We therefore examined the effects of 200 μg/mL bovine-derived AGE (the CML concentration in 200 μg/mL AGE was 0.59 nmol/mL (121.31 mg/L) on cardiac fibroblasts, whose CML concentrations are representative of those found in the plasma of aged diabetic patients.15
Western blot analysis For western blot, the protein samples were extracted from whole cells according to the procedure, and total protein (30 μg) was fractionated on sodium dodecylsulfatepolyacrylamide gel, and transferred to polyvinylidenefluoride (PVDF) membranes as described previously.9 After transfer, the membranes were blocked with 5% non-fat milk, and incubated overnight with primary antibodies against TGF-β, p-smad2/3, smad2/3, MMP-2, p16 and glyceraldehyde 3-phosphate dehydrogenase. Then the membranes were washed with tris buffered saline tween-20 (TBST), and incubated with anti-rabbit HRP-conjugated secondary antibodies for two hours at 4°C, washed with TBST, and visualized by ECL reagent (Life Technology Company, Rockford, lL, USA), then detected by Odyssey Infrared Imaging System (LI-COR Inc, Lincoln, Nebraska, USA). Protein of interest signal was normalized versus glyceraldehyde 3-phosphate dehydrogenase.
Beta-galactosidase activity Beta-galactosidase (SA-β-Gal) activity is detected by Senescence-β-Galactosidase assay chemically at pH 6.0 wash according to the instructions (Senescence-βGalactosidase Staining kit; Beyotime Institute of Biotechnology, Wuhan, China). Briefly, the cells were grown in six-well plates, interfered for 72 h, fixed with 5% fixative solution for 15 min at room temperature; washed three times with phosphate-buffered saline; and incubated in staining solution containing X-gal at 37°C overnight. Cells were photographed by ordinary optical microscope at ×10 magnification, and the proportions of SA-β-Gal positive cells were shown as a percentage of the total number of cells quantified in each dish. The data were expressed as the mean of triplicates ± SD. | 523
M Fang et al.
Statistical analysis All data are shown as mean ± SD. Statistical analyses were carried out with GraphPad Prism 5.0 software (Graphpad Software, Inc., San Diego, CA, USA). Oneway ANOVA was used to compare the difference in those groups. Statistical significance was determined at a P value of
