Type-2 diabetes-induced changes in vascular extracellular matrix gene expression: Relation to vessel size
© Song and Ergul; licensee BioMed Central Ltd. 2006
Received: 06 December 2005
Accepted: 17 February 2006
Published: 17 February 2006
Hyperglycemia-induced changes in vascular wall structure contribute to the pathogenesis of diabetic microvascular and macrovascular complications. Matrix metalloproteinases (MMP), a family of proteolytic enzymes that degrade extracellular matrix (ECM) proteins, are essential for vascular remodeling. We have shown that endothelin-1 (ET-1) mediates increased MMP activity and associated vascular remodeling in Type 2 diabetes. However, the effect of Type 2 diabetes and/or ET-1 on the regulation of ECM and MMP gene expression in different vascular beds remains unknown.
Aorta and mesenteric artery samples were isolated from control, Type 2 diabetic Goto-Kakizaki (GK) rats and GK rats treated with ETA antagonist ABT-627. Gene expression profile of MMP-2, MMP-9, MT1-MMP, fibronectin, procollagen type 1, c-fos and c-jun, were determined by quantitative real-time (qRT) PCR. In addition, aortic gene expression profile was evaluated by an ECM & Adhesion Molecules pathway specific microarray approach.
Analysis of the qRT-PCR data demonstrated a significant increase in mRNA levels of MMPs and ECM proteins as compared to control animals after 6 weeks of mild diabetes. Futhermore, these changes were comparable in aorta and mesentery samples. In contrast, treatment with ETA antagonist prevented diabetes-induced changes in expression of MMPs and procollagen type 1 in mesenteric arteries but not in aorta. Microaarray analysis provided evidence that 27 extracellular matrix genes were differentially regulated in diabetes. Further qRT-PCR with selected 7 genes confirmed the microarray data.
These results suggest that the expression of both matrix scaffold protein and matrix degrading MMP genes are altered in macro and microvascular beds in Type 2 diabetes. ETA antagonism restores the changes in gene expression in the mesenteric bed but not in aorta suggesting that ET-1 differentially regulates microvascular gene expression in Type 2 diabetes.
Changes in vascular wall structure occur in diabetes and contribute to both micro- and macrovascular complications. Previous studies in streptozosin (STZ)-induced model of Type 1 diabetes documented increased intimal proliferation and medial thickness as well as extracellular matrix (ECM) deposition in microvessels such as mesenteric arteries as early as 3 weeks of experimental diabetes [1–4]. Vascular remodeling and hypertrophy associated with augmented expression of dedifferentiation markers of vascular smooth muscle cells also occur in larger vessels like aorta . While these studies provided evidence for diabetes-induced alterations in ECM synthesis and vascular structure of an experimental model of Type 1 diabetes that is characterized by highly elevated blood glucose levels, to what extent mild-to-modest hyperglycemia as seen in Type 2 diabetes influences the gene expression of ECM proteins associated with vascular remodeling and whether there are differences in micro vs macrovascular bed are not fully understood.
Vascular ECM proteins such as collagen type 1 and 3, fibronectin and thrombospondins not only function as scaffolding proteins but also involved in matrix signaling by interacting with integrin family of proteins and triggering growth-promoting signals. ECM displays a very dynamic equilibrium where there is constant synthesis, degradation and reorganization. Turnover of matrix proteins are regulated by matrix metalloproteinases (MMPs) . While decreased MMP activity is generally believed to contribute to ECM accumulation in diabetic kidney and in vascular tissue from patients with diabetes, we and others have recently reported that there is an early activation of MMPs in hypertension and diabetes [7–9]. However, transcriptional regulation of ECM proteins and MMPs in different vascular beds and specifically in Type 2 diabetes remains to be determined.
Vasoactive factors including endothelin-1 (ET-1) and angiotensin II are involved in diabetic vascular remodeling as evidenced by studies that demonstrated attenuation of these responses by blockade of these systems in both experimental and clinical diabetes. For example, Gilbert and colleagues reported that ETA receptor antagonism prevents mesenteric vascular hypertrophy in Type 1 diabetes . Another study provided evidence that blockade of ET-1 action inhibits ECM deposition in the aorta as well . We recently reported that ET-1 levels are elevated and an ETA antagonist prevents ECM deposition and MMP activation in middle cerebral arteries but not in the kidney of Goto-Kakizaki (GK) rats, a non-obese Type 2 diabetes model [9, 10]. Thus, this study was designed to test the hypothesis that there is a differential regulation of MMP activation in micro vs macrovessels in Type 2 diabetes and ET-1 contributes to this process.
Animal and tissue preparation
All experiments were performed on male Wistar (Harlan, Indianapolis, IN) and Goto-Kakizaki (in-house bred, derived from the Tampa colony) rats . The animals were housed at the Medical College of Georgia animal care facility that is approved by the American Association for Accreditation of Laboratory Animal Care and study was approved by the Institutional Animal Care and Use Committee. Animals were fed standard rat chow and tap water ad-libitum. During housing, drinking water measurements, weight, and blood glucose measurements were performed twice weekly. At 12 weeks of age, when all GK animals became overtly diabetic, telemetry transmitters for blood pressure measurements were implanted as previously reported . After a 2 week-recovery period, control and diabetic animals were administered the ETA selective antagonist, ABT-627 (5 mg/kg/day) in drinking water, or vehicle only . Treatment was maintained until the time of sacrifice at 18 weeks of age. Animals were anesthetized with sodium pentobarbital and exsanguinated via the abdominal aorta. Upon sacrifice, the mesenteric bed was harvested, third order mesenteric arteries and thoracic aorta were isolated and immediately put into RNAlater™ (Ambion, Austin, TX, USA) for storage at -80°C.
RNA isolation and cDNA synthesis
Total RNA extraction was carried out using the RNeasy® Mini kit (Qiagen Inc., Valencia, CA) according to manufacturer's instructions. RNA quality from each sample was assured by the A260/280 absorbance ratio and by electrophoresis of 1.2% agarose formaldehyde gel. 1.0–2.0 μg of total RNA was reverse transcribed into single strand cDNA using MuLV reverse transcriptase (Applied Biosystems, Foster City, CA, USA). RT reaction was carried out for 60 min at 42°C and 5 min at 95°C in a thermocycler.
Primer design and qRT-PCR
List of primers used in qRT-PCR
Expect Size (bp)
GTC TTC CCC TTC GTC TTC CT
ACC CCA CTT CTT GTC AGC GT
TGT CCC AGA TAA GCC CAG AA
TAT TCC TCA CCC GCC AGA AC
AGC CCA GAA CAC CAT TCC TAC
ATG CCT GCT TCA CCA CAT TC
GCA CAG GGG AAG AAA AGG AG
TTG AGT GGA TGG GAG GAG AG
GTG CCA AGG TGG AAA TCA GAG
AAG GTT GAA GGA AAC GAG CGA
Procollagen 1 (F)
AAG GGT GAG ACA GGC GAA CAA
Procollagen 1 (R)
TTG CCA GGA GAA CCA GCA GAG
GTG GTG GAA GGC GTA TCG AGT TT
GTG TGA TGC CAG AAG CAG ATC CA
CCT AGC TGA ACT GCA TAG CCA GAA
AAG TTG CTG AGG TTG GCG TAG A
cDNA gene expression array analysis
The expression profile of extracellular matrix & adhesion molecules genes was analyzed using the non-radioactive GEArray Q series Mouse gene array (MM-010N, SuperArray Bioscience Corp., Frederick, MD). This array membrane is composed of 96 extracellular matrix & adhesion molecules genes, a plasmid pUC18 negative control, and four housekeeping genes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A, ribosomal protein L13a, and β-actin. Biotinylated cDNA probes were denatured and hybridized to extracellular matrix & adhesion molecules gene-specific cDNA fragments spotted on the membranes. After pre-hybridization with GEAhyb Hybridization Solution (SuperArray) of denatured salmon sperm DNA (Invitrogen). The array membrane was hybridized with denatured cDNA probes overnight at 55°C. Following washing the membrane twice with 2 × SSC, 1% SDS and twice with 0.1 × SSC, 0.5% SDS for 15 min at 55°C each, the membrane was blocked with GEAblocking Solution Q (SuperArray) for 40 min and incubated with alkaline phosphatase-conjugated streptavidin for 10 min at room temperature. Chemiluminescent detection was performed using CDP-Star substrate. The results were analyzed with ScanAlyze and GEArray Analyzer. The relative expression levels of different genes were estimated by comparing its signal intensity with that of internal control β-actin.
RT-PCR results were reported as relative gene expression and the fold change in target genes was determined by 2-ΔΔCt method, where -ΔΔCt = (CtTarget - CtActin)GK - (CtTarget - CtActin)control and Ct value = the cycle number that crosses signal threshold. To evaluate the effect of ABT-627 treatment on gene expression profile in the diabetic group, fold change was determined as (CtTarget - CtActin)GK - (CtTarget - CtActin)GK + ABT-627. Group comparisons (C vs GK or GK vs GK +ABT-627) were performed using Student's t-test. For Array studies, relative gene expression was analyzed using the SAM (Statistical Analysis of Microarrays) software .
Results and discussion
Metabolic parameters of Control Wistar and GK rats.
GK + ABT-627
Body weight (g)
502 ± 11
359 ± 7*
379 ± 29
Blood glucose (mg/dl)
116 ± 5
239 ± 23*
211 ± 18
Mean Arterial Pressure (MAP, mm Hg)
101 ± 2
110 ± 7
112 ± 1
qRT-PCR-based fold changes in gene expression in control vs diabetic GK rats treated with or without ABT-627.
C vs GK
GK vs GK+ABT627
C vs GK
GK vs GK+ABT627
Microarray analysis of fold-changes in aortic gene expression in diabetes
Cell Adhesion Molecules
Extracellular Matrix Proteins
Catenin a-like 1
In conclusion, mild diabetes can stimulate a gene expression pattern that promotes remodeling of both micro and macrovessels. While ET-1 contributes to the alterations in gene expression profile in the microvasculature via activation of the ETA receptor subtype, changes in macrovascular gene expression are independent of ET-1.
This work was supported by grants from NIH (HL076236-01, DK074385), American Diabetes Association Research grant and Pfizer Atorvastatin Research Award to Adviye Ergul. The authors wish to thank Abbott Laboratories for the ABT627 compound.
- Rumble JR, Cooper ME, Cox AJ, Soulis T, Wu L, Youssef S, Jasik M, Jerums G, Gilbert RE: Vascular hypertrophy in experimental diabetes: Role of advanced glycation end products. J Clin Invest. 1997, 99 (5): 1016-1027.PubMed CentralView ArticlePubMedGoogle Scholar
- Cooper ME, Rumble J, Komers R, Du HC, Jandeleit K, Chou ST: Diabetes-associated mesenteric vascular hypertrophy is attenuated by angiotensin-converting enzyme inhibition. Diabetes. 1994, 43 (10): 1221-1228.View ArticlePubMedGoogle Scholar
- Vranes D, Cooper ME, Dilley RJ: Cellular mechanisms of diabetic vascular hypertrophy. Microvascular Research. 1999, 57: 8-18. 10.1006/mvre.1998.2107.View ArticlePubMedGoogle Scholar
- Gilbert RE, Rumble JR, Cao Z, Cox AJ, van Eeden P, Allen TJ, Kelly DJ, Cooper ME: Endothelin receptor antagonism ameliorates mast cell infiltration, vascular hypertrophy, and epidermal growth factor expression in experimental diabetes. Circ Res. 2000, 86: 158-165.View ArticlePubMedGoogle Scholar
- Fukuda G, Khan ZA, Barbin YP, Farhangkhoee H, Tilton RG, Chakrabarti S: Endothelin-mediated remodeling in aortas of diabetic rats. Diabetes Metab Res Rev. 2005, 21 (4): 367-375. 10.1002/dmrr.527.View ArticlePubMedGoogle Scholar
- Visse R, Nagase H: Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res. 2003, 92 (8): 827-839. 10.1161/01.RES.0000070112.80711.3D.View ArticlePubMedGoogle Scholar
- Ammarguellat FZ, Gannon PO, Amiri F, Schiffrin EL: Fibrosis, matrix metalloproteinases, and inflammation in the heart of DOCA-salt hypertensive rats: role of ET(A) receptors. Hypertension. 2002, 39 (2 Pt 2): 679-684. 10.1161/hy0202.103481.View ArticlePubMedGoogle Scholar
- Ergul A, Portik-Dobos V, Giulumian AD, Molero MM, Fuchs LC: Stress upregulates arterial matrix metalloproteinase expression and activity via endothelin A receptor activation. Am J Physiol Heart. 2003, 285 (5): H2225-2232.View ArticleGoogle Scholar
- Harris AK, Hutchinson JR, Sachidanandam K, Johnson MH, Dorrance AM, Stepp DW, Fagan SC, Ergul A: Type 2 diabetes causes remodeling of cerebrovasculature via differential regulation of matrix metalloproteinases and collagen synthesis: role of endothelin-1. Diabetes. 2005, 54 (9): 2638-2644.View ArticlePubMedGoogle Scholar
- Portik-Dobos V, Harris AK, Song W, Hutchinson J, Johnson MH, Imig JD, Pollock DM, Ergul A: Endothelin antagonism prevents early EGFR transactivation but not increased matrix metalloproteinase activity in diabetes. Am J Physiol. 2006, 290 (2): R435-441.Google Scholar
- Standaert ML, Sajan MP, Miura A, Kanoh Y, Chen HC, Farese RV, Farese RV: Insulin-induced activation of atypical protein kinase C, but not protein kinase B, is maintained in diabetic (ob/ob and Goto-Kakazaki) liver. Contrasting insulin signaling patterns in liver versus muscle define phenotypes of type 2 diabetic and high fat-induced insulin-resistant states. J Biol Chem. 2004, 279 (24): 24929-24934. 10.1074/jbc.M402440200.View ArticlePubMedGoogle Scholar
- Williams JM, Pollock JS, Pollock DM: Arterial pressure response to the antioxidant tempol and ETB receptor blockade in rats on a high-salt diet. Hypertension. 2004, 44 (5): 770-775. 10.1161/01.HYP.0000144073.42537.06.View ArticlePubMedGoogle Scholar
- Tusher VG, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A. 2001, 98 (9): 5116-5121. 10.1073/pnas.091062498.PubMed CentralView ArticlePubMedGoogle Scholar
- Galis ZS, Khatri JJ: Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly. Circ Res. 2002, 90 (3): 251-262.PubMedGoogle Scholar
- Portik-Dobos V, Anstadt MP, Hutchinson J, Bannan M, Ergul A: Evidence for a matrix metalloproteinase induction/activation system in arterial vasculature and decreased synthesis and activity in diabetes. Diabetes. 2002, 51: 3063-3068.View ArticlePubMedGoogle Scholar
- Ergul A, Portik-Dobos V, Hutchinson J, Franco J, Anstadt MP: Downregulation of vascular matrix metalloproteinase inducer and activator proteins in hypertensive patients. Am J Hypertens. 2004, 17 (9): 775-782. 10.1016/j.amjhyper.2004.06.025.View ArticlePubMedGoogle Scholar
- Rumble JR, Cooper ME, Soulis T, Cox A, Wu L, Youssef S, Jasik M, Jerums G, Gilbert RE: Vascular hypertrophy in experimental diabetes. Role of advanced glycation end products. J Clin Invest. 1997, 99 (5): 1016-1027.PubMed CentralView ArticlePubMedGoogle Scholar
- Gilbert RE, Cox A, McNally PG, Wu LL, Dziadek M, Cooper ME, Jerums G: Increased epidermal growth factor in experimental diabetes related kidney growth in rats. Diabetologia. 1997, 40 (7): 778-785. 10.1007/s001250050749.View ArticlePubMedGoogle Scholar
- Wu S, Hopfner RL, McNeill JR, Wilson TW, Gopalakrishnan V: Altered paracrine effect of endothelin in blood vessels of the hyperinsulinemic, insulin resistant obese Zucker rat. Cardiovasc Res. 2000, 45: 994-1000. 10.1016/S0008-6363(99)00417-4.View ArticlePubMedGoogle Scholar
- Katakam PV, Pollock JS, Pollock DM, Ujhelyi MR, Miller AW: Enhanced endothelin-1 response and receptor expression in small mesenteric arteries of insulin-resistant rats. Am J Physiol. 2001, 280 (2): H522-527.Google Scholar
- Hattori Y, Kasai K, Nakamura T, Emodo T, Shimoda S-I: Effects of glucose and insulin on immunoreactive endothelin-1 release from cultured bovine endothelial cells. Metabolism. 1991, 40: 165-169. 10.1016/0026-0495(91)90168-V.View ArticlePubMedGoogle Scholar
- Schroen B, Heymans S, Sharma U, Blankesteijn WM, Pokharel S, Cleutjens JP, Porter JG, Evelo CT, Duisters R, van Leeuwen RE: Thrombospondin-2 is essential for myocardial matrix integrity: increased expression identifies failure-prone cardiac hypertrophy. Circ Res. 2004, 95 (5): 515-522. 10.1161/01.RES.0000141019.20332.3e.View ArticlePubMedGoogle Scholar
- Bornstein P, Agah A, Kyriakides TR: The role of thrombospondins 1 and 2 in the regulation of cell-matrix interactions, collagen fibril formation, and the response to injury. Int J Biochem Cell Biol. 2004, 36 (6): 1115-1125. 10.1016/j.biocel.2004.01.012.View ArticlePubMedGoogle Scholar
- Spinale FG: Cell-matrix signaling and thrombospondin: another link to myocardial matrix remodeling. Circ Res. 2004, 95 (5): 446-448. 10.1161/01.RES.0000142315.88477.42.View ArticlePubMedGoogle Scholar
- Yang Z, Kyriakides TR, Bornstein P: Matricellular proteins as modulators of cell-matrix interactions: adhesive defect in thrombospondin 2-null fibroblasts is a consequence of increased levels of matrix metalloproteinase-2. Mol Biol Cell. 2000, 11 (10): 3353-3364.PubMed CentralView ArticlePubMedGoogle Scholar
- Kyriakides TR, Zhu YH, Smith LT, Bain SD, Yang Z, Lin MT, Danielson KG, Iozzo RV, LaMarca M, McKinney CE: Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis. J Cell Biol. 1998, 140 (2): 419-430. 10.1083/jcb.140.2.419.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.