Mimecan and cardiac extracellular matrix integrity Lucas Van Aelst, MD Department of Internal Medicine, University Hospitals Leuven, Leuven, Belgium

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1 Mimecan and cardiac extracellular matrix integrity Lucas Van Aelst, MD Department of Internal Medicine, University Hospitals Leuven, Leuven, Belgium VIB-Vesalius Research Center, KULeuven, Leuven, Belgium

2 What is mimecan? Synonym: Osteoglycin (OGN) Part of the Small Leucin-Rich ProteoGlycans (SLRP) Other members: biglycan, decorin, fibromodulin, lumican Phylogenetic analysis and chromosomal organization of various human SLRP classes. Schaefer, Iozzo J Biol Chem 2008;283:21305

3 Small Leucin-Rich Proteoglycans Important for regulation of matrix structure, cell cycle and growth actions Multiple signaling pathways evoked by SLRPs Schaefer, Iozzo J Biol Chem 2008;283:21305 Iozzo J Biol Chem 1999;274:18843

4 Expression level Matricellular proteins Modulate cell-matrix interactions and cell function, without participating in structural scaffold of the extracellular matrix. Members: CCN family members (CCN1-6), Osteonectin (SPARC), Osteopontin (OPN), Tenascins (TN-C, TN-X), Thrombospondins (TSP-1, TSP-2) B I R T H Tissue repair, Tumour growth Time Prenatal Growth Adult life Pathology

5 Known non-cardiac functions of mimecan Studied in: 1990: Bone formation (Bentz et al., J Biol Chem 265:5024), 1997: Atherosclerosis (Shanahan et al., Arterioscler Thromb Vasc Biol 17:2437), 1998: Eye & corneal transparency (Liu et al., J Biol Chem 273:22584), 2005: Pituitary tumours (Hu et al., J Clin Endocrin Metab 90;6657), 2008: Cochlea & auditory phenotype (Williamson et al., Hear Res 237:57), 2008: Metastasis (Cui et al., Acta Biochim Biophys Sin 40:349), 2009: Arteriogenesis (Kampmann et al., Mol Cell Biochem 322:15), 2009: Differentiation SCLC(-)/NSCLC(+) (Zhang et al., Oncol Rep 22:1057), 2010: Metabolic syndrome (Pravenec. Methods Mol Biol 597:415), 2010: (Pre)term labour (Romero et al., J Matern Fetal Neonatal Med 23:261).

6 Osteoglycin protein expression in human heart disease CAD: Isolated coronary artery disease; AS: Concentric hypertrophy secondary to aortic stenosis; HF: Ischemic heart failure and eccentric hypertrophy; HTN: Hypertensive heart disease Petretto et al., Nat Genet 2008;40(5):546

7 Does mimecan regulate the integrity of the cardiac extracellular matrix after myocardial infarction?

8 Relative expression level (A.U) Increased expression of mimecan in infarcted area mrna: Myocardial infarction in mice, sacrificed after 3d, 7d, 14d p = 0,05 p < 0,05 p < 0,05 p < 0,05 p = NS Sham 3d post-mi 7d post-mi 14d post-mi Van Aelst, unpublished

9 Relative expression level (A.U) PreOGN OGN GAPDH Protein: Increased expression of mimecan in infarcted area B Infarcted Area B Remote Area S I R 3,5 3 2,5 2 1,5 1 0,5 0 p < 0,05 p < 0,05 p = NS Sham 3d post-mi 14d post-mi Infarcted Area Remote Area Van Aelst, unpublished

10 Infarct Size (%) Survival Increased mortality in the absence of mimecan Myocardial infarction, mimecan-ko vs. WT mice, 14 days Male WT (n = 7) Female WT (n = 14) Female KO (n = 8) * Similar infarct size in WT vs. KO Male KO (n = 13) 10 0 Days after myocardial infarction * p < 0,05 WT KO Van Aelst, unpublished

11 Cardiac rupture and dilatation in the absence of mimecan in male mice Mimecan WT, MI, 3d Mimecan KO, MI, 3d Van Aelst, unpublished

12 LVIDd - length (mm) Fractional shortening (%) No difference in diastolic dimension nor cardiac function 14d after MI in WT vs KO mice WT KO WT KO WT: n = 14 KO: n = 8 WT KO WT KO Baseline 14d after MI Baseline 14d after MI Van Aelst, unpublished

13 CD 45 count/mm² No significant differences in inflammatory and basic structural histological analyses between WT and KO animals 750 WT, 14d KO, 14d 500 Infarct Thickness (µm) 316 ± ± Res. Necrotic Area (%) 4,9 ± 0,5 4,0 ± 0,9 0 WT KO Mac3 count/mm² 131 ± ± 35 CD31 count/mm² 151 ± ± 20 CD45, WT CD45, KO Van Aelst, unpublished

14 Collagen Deposition (%) No obvious difference in total collagen content in the infarcted area between WT and KO mice WT KO 10 0 WT KO Van Aelst, unpublished

15 Impaired collagen cross-linking in the infarct in the absence of mimecan MIMECAN WILD TYPE MIMECAN KNOCKOUT % Thick/thin collagen fibers: 33 ± 3.4 % Thick/thin collagen fibers: 18 ± 4.3* *P<0.01 Van Aelst, unpublished

16 LVIDd (mm) Ejection Fraction (%) Mimecan treatment prevents cardiac dilatation and dysfunction I.V. injection of AdV Mimecan, 7 d before MI in WT mice high mimecan plasma/cardiac levels Vector: Group: RR5 - no OGN OGN - no Sham MMI MMI Sham p < 0,05 p < 0,05 RR5 RR5 - MMIOGN - MI MMI MI n = 5 n = 5 n = 10 n = 10 Vector: Group: RR5 RR5 - no OGN OGN - no Sham MMI Sham MMI RR5- MMI MI OGN- MMI MI Van Aelst, unpublished

17 Conclusion: Matricellular proteins modulate cell-matrix interactions and cell function, without participating in the structural scaffold of the extracellular matrix (ECM). SLRPs, a recently described class of collagen-associated matrix proteins, have a role in collagen fibrillogenesis and a direct and indirect effect on cell growth. As such, they can be classified as matricellular proteins. Mimecan KO male mice die between 3d and 10d after MI (rupture). In mimecan KO female mice, there is no significant difference in mortality and cardiac function as compared to WT female mice; there is a difference in collagen quality between mimrcan WT and KO female animals. Adenoviral overexpression of mimecan improves cardiac function after MI. Further in vitro studies are needed to establish the role of mimecan in cardiac ECM integrity after myocardial infarction.

18 Acknowledgements: Katholieke Universiteit Leuven P. Carai P. Carmeliet J. d Hooge M. Swinnen* D. Vanhoutte* P. Veulemans Funding and Grants: Imperial College London L. Colman S. Cook Maastricht University S. Heymans S. Jochems* A. Papageorgiou M. Schellings* B. Schroen R. Van Leeuwen W. Verhesen