John R. Barbour

Expression of Matrix Metalloproteinases and Endogenous Inhibitors Within Ascending Aortic Aneurysms of Patients With Marfan Syndrome

Background— Marfan syndrome (MFS) is known to cause ascending thoracic aortic aneurysms (ATAAs). Transforming growth factor beta (TGF-&bgr;) has recently been implicated in this process. Imbalances between the matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) have also been shown to contribute to aneurysm formation. Whether and to what degree MMP, TIMP, and TGF-&bgr; signaling profiles are altered in ATAAs in MFS compared with non-MFS patients remains unknown. Methods and Results— ATAA samples taken during aortic replacement from age-matched MFS (n=9) and non-MFS (n=18) patients were assessed for representative subtypes of all MMP classes, all 4 known TIMPs, and type 2 TGF-&bgr; receptors (TGFBR2). Results were expressed as a percentage (mean±SEM) of reference control samples (100%; n=18) obtained from patients without ATAA. In MFS, decreased MMP-2 (76±7; P<0.05 versus control), increased MMP-12 (161±27% versus control; P<0.05), and increased MT1-MMP (248±64% versus 91±21 non-MFS and control; P<0.05) were observed. TIMP-3 (74±23%) was reduced compared with control values (P<0.05) and TIMP-2 was elevated (128±31%) compared with non-MFS (73±19%; P<0.05). In non-MFS samples, MMP-1 (70±16%), MMP-3 (77±18%), MMP-8 (75±11%), MMP-9 (69±14%), and MMP-12 (85±15%) were decreased compared with control (P<0.05). TIMPs 1 to 3 were reduced in non-MFS compared with control values (P<0.05). TGFBR2 were increased in MFS (193±32%) compared with non-MFS (95±16%) and controls (P<0.05). Conclusions— A unique MMP and TIMP portfolio was observed in ATAAs from MFS compared with non-MFS patients. In addition, MFS samples showed evidence of increased TGF-&bgr; signaling. These differences suggest disparate mechanisms of extracellular matrix remodeling between these 2 groups of patients.

Effects of Deletion of the Matrix Metalloproteinase 9 Gene on Development of Murine Thoracic Aortic Aneurysms

Background—The matrix metalloproteinases (MMPs) contribute to cardiovascular remodeling, and MMPs, such as the gelatinases (MMP-9 and MMP-2), have been identified in thoracic aortic aneurysmal (TAA) tissue, but a cause-effect relationship has not been clearly established. Accordingly, this study examined TAA progression in mice devoid of the MMP-9 gene. Methods and Results—The descending thoracic aortas of wild-type (WT) FVB (n =17) and MMP-9 gene knockout (KO, n =11) mice were exposed to 0.5 mol/L of CaCl2 for 15 minutes with terminal studies performed at 4 weeks. Aortic lumen diameter was measured using video micrometry at baseline and at 4 weeks (TAA) followed by aortic tissue analysis. In WT mice, aortic diameter increased by 138±5% at 4 weeks (P<0.05), consistent with TAA formation. In the KO mice, aortic diameter increased from baseline by 120±4% (P<0.05) but was attenuated from WT TAA values (P<0.05). Gelatin zymography performed on TAA segments confirmed the absence of MMP-9 in the KO mice but a >8-fold relative increase in the active form of MMP-2 compared with WT (P<0.05). Despite this, MMP-2 activity was relatively increased (P<0.05) and colocalized to smooth muscle cell actin in a differential pattern favoring medial distruction in the WT TAA compared with the KO TAA segments. Conclusions—These results demonstrate that MMP-9 gene deletion attenuated TAA formation despite an increase in the zymographic levels of MMP-2. These unique findings suggest that an interaction between these 2 MMPs is necessary to facilitate TAA progression.

Alterations in Aortic Cellular Constituents during Thoracic Aortic Aneurysm Development: Myofibroblast-Mediated Vascular Remodeling

The present study tested the hypothesis that changes in the resident endogenous cellular population accompany alterations in aortic collagen and elastin content during thoracic aortic aneurysm (TAA) development in a murine model. Descending thoracic aortas were analyzed at various time points (2, 4, 8, and 16 weeks) post-TAA induction (0.5 M CaCl2, 15 minutes). Aortic tissue sections were subjected to histological staining and morphometric analysis for collagen and elastin, as well as immunostaining for cell-type-specific markers to quantify fibroblasts, myofibroblasts, and smooth-muscle cells. Results were compared with reference control mice processed in the same fashion. Aortic dilatation was accompanied by changes in the elastic architecture that included: a decreased number of elastic lamellae (from 6 to 4); altered area fraction of elastin (elevated at 4 weeks and decreased at 16 weeks); and a decreased area between elastic lamellae (minimum reached at 4 weeks). Total collagen content did not change over time. Increased immunoreactivity for fibroblast and myofibroblast markers was observed at 8- and 16-week post-TAA-induction, whereas immunoreactivity for smooth-muscle cell markers peaked at 4 weeks and returned to baseline by 16 weeks. Therefore, this study demonstrated that changes in aortic elastin content were accompanied by the emergence of a subset of fibroblast-derived myofibroblasts whose altered phenotype may play a significant role in TAA development through the enhancement of extracellular matrix proteolysis.

Regular ArticlesAlterations in Aortic Cellular Constituents during Thoracic Aortic Aneurysm Development: Myofibroblast-Mediated Vascular Remodeling

The present study tested the hypothesis that changes in the resident endogenous cellular population accompany alterations in aortic collagen and elastin content during thoracic aortic aneurysm (TAA) development in a murine model. Descending thoracic aortas were analyzed at various time points (2, 4, 8, and 16 weeks) post-TAA induction (0.5 M CaCl2, 15 minutes). Aortic tissue sections were subjected to histological staining and morphometric analysis for collagen and elastin, as well as immunostaining for cell-type-specific markers to quantify fibroblasts, myofibroblasts, and smooth-muscle cells. Results were compared with reference control mice processed in the same fashion. Aortic dilatation was accompanied by changes in the elastic architecture that included: a decreased number of elastic lamellae (from 6 to 4); altered area fraction of elastin (elevated at 4 weeks and decreased at 16 weeks); and a decreased area between elastic lamellae (minimum reached at 4 weeks). Total collagen content did not change over time. Increased immunoreactivity for fibroblast and myofibroblast markers was observed at 8- and 16-week post-TAA-induction, whereas immunoreactivity for smooth-muscle cell markers peaked at 4 weeks and returned to baseline by 16 weeks. Therefore, this study demonstrated that changes in aortic elastin content were accompanied by the emergence of a subset of fibroblast-derived myofibroblasts whose altered phenotype may play a significant role in TAA development through the enhancement of extracellular matrix proteolysis.

Altered Transforming Growth Factor-Beta Signaling in a Murine Model of Thoracic Aortic Aneurysm

Objective: Thoracic aortic aneurysms (TAAs) develop by a multifactorial process involving maladaptive signaling pathways that alter the aortic vascular environment. Transforming growth factor-beta (TGF-β) has been implicated in regulating the structure and composition of the extracellular matrix by differential activation of various intracellular signaling pathways. However, whether and to what degree TGF-β signaling contributes to TAA development remains unclear. Accordingly, the hypothesis that alterations in TGF-β signaling occur during aneurysm formation was tested in a murine model of TAA. Methods: TAAs were surgically induced in mice (C57BL/6J) and aortas were analyzed at predetermined time points (1, 2, and 4 weeks post-TAA induction). Quantitative real-time PCR (QPCR) was performed to evaluate the expression of 84 relevant TGF-β superfamily genes, and the protein levels of key signaling intermediates were measured by immunoblotting. Results were compared to unoperated reference control mice. Results: QPCR revealed increased expression of TGF-β superfamily ligands (Gdf-2, -6, -7, Inhba), ligand inhibitors (Bmper, Chrd, Gsc), and transcriptional regulators (Dlx2, Evi1), among other genes (Cdkn2b, Igf1, IL-6). Protein levels of TGF-β receptorII, Smad2, Smad1/5/8, phospho-Smad1/5/8, and Smurf1 were increased from control values post-TAA induction. Both TGF-β receptorI and Smad4 were decreased from control values, while ALK-1 levels remained unchanged. Conclusions: These alterations in the TGF-β pathway suggest a mechanism by which primary signaling is switched from a TGF-βRI/Smad2-dependent response, to an ALK-1/Smad1/5/8 response, representing a significant change in signaling outcome, which may enhance matrix degradation.

Contents Vol. 45, 2008

U.H. von Andrian, Boston, Mass. J.E. Brayden, Burlington, Vt. G. Breier, Dresden N.J. Brown, Sheffi eld G. Clough, Southampton M.J. Davis, Columbia, Mo. M.G.A. oude Egbrink, Maastricht J.C. Frisbee, Morgantown, W.Va. C.J. Garland, Bath M. Gassmann, Zürich T. Gloe, Munich M. Gollasch, Berlin T.M. Griffi th, Cardiff A.M. Heagerty, Manchester P. Hellstrand, Lund D. Henrion, Angers C. Hill, Canberra M.A. Hill, Columbia, Miss. V.W. van Hinsbergh, Leiden Y. Huang, Shatin, Hong Kong V.H. Huxley, Columbia, Mo. J.D. Imig, Augusta, Ga. W.F. Jackson, Kalamazoo, Mich. A. Koller, Valhalla, N.Y. I. Laher, Vancouver B.L. Langille, Toronto T.M. Lincoln, Birmingham, Ala. L. Lindbom, Stockholm J. Lopez-Barneo, Sevilla R.M. Lynch, Tucson, Ariz. J.M. Marshall, Birmingham S. Massberg, Boston, Mass. J.C.I. McGrath, Glasgow A.C. Newby, Bristol H. Nilsson, Aarhus A.R. Pries, Berlin I.H. Sarelius, Rochester, N.Y. E.L. Schiff rin, Montréal G.W. Schmid-Schönbein, La Jolla, Calif. S.M. Schwartz, Seattle, Wash. S.S. Segal, New Haven, Conn. A.C. Shore, Exeter U. Simonsen, Aarhus L. Sorokin, Muenster D.W. Stepp, Augusta, Ga. A. Tedgui, Paris J.E. Tooke, Exeter E. Vicaut, Paris B.R. Wamhoff , Charlottesville, Va. C. Webb, Augusta, Ga. C. de Wit, Luebeck Founded 1964 as ‘Angiologica’ by M. Comèl and L. Laszt (1964–1973) continued as ‘Blood Vessels’ by J.A. Bevan (1974–1991) continued as ‘Journal of Vascular Research’ by M.J. Mulvany (1991–2002)