Noninvasive targeted imaging of matrix metalloproteinase activation in a murine model of postinfarction remodeling.

BACKGROUND: Time-dependent activation of matrix metalloproteinases (MMPs) after myocardial infarction (MI) contributes to adverse left ventricular (LV) remodeling; however, noninvasive methods to monitor this process serially are needed. METHODS AND RESULTS: MMP-targeted radiotracers were developed that displayed selective binding kinetics to the active MMP catalytic domain. Initial nonimaging studies were performed with a (111)In-labeled MMP-targeted radiotracer ((111)In-RP782) and negative control compound ((111)In-RP788) in control mice (Ctrl) and in mice 1 week after surgically induced MI. Localization of (111)In-RP782 was demonstrated within the MI by microautoradiography. A 334+/-44% increase (P<0.001 versus Ctrl) in relative retention of (111)In-RP782 was confirmed by gamma well counting of myocardium. Subsequent high-resolution dual-isotope planar and hybrid micro-single-photon emission computed tomography/CT imaging studies with an analogous 99mTc-labeled MMP-targeted radiotracer (99mTc-RP805) and 201Tl demonstrated favorable biodistribution and clearance kinetics of 99mTc-RP805 for in vivo cardiac imaging, with robust retention 1 to 3 weeks after MI in regions of decreased 201Tl perfusion. Gamma well counting yielded a similar approximately 300% increase in relative myocardial retention of 99mTc-RP805 in MI regions (Ctrl, 102+/-9%; 1 week, 351+/-77%; 2 weeks, 291+/-45%; 3 weeks, 292+/-41%; P<0.05 versus Ctrl). Myocardial uptake in the MI region was also significantly increased approximately 5-fold when expressed as percentage injected dose per gram tissue. There was also a significant 2-fold increase in myocardial activity in remote regions relative to control mice, suggesting activation of MMPs in regions remote from the MI. CONCLUSIONS: This novel noninvasive targeted MMP radiotracer imaging approach holds significant diagnostic potential for in vivo localization of MMP activation and tracking of MMP-mediated post-MI remodeling.

Time-dependent changes in myocardial structure following discrete injury in mice deficient of matrix metalloproteinase-3.

Myocardial scars from radiofrequency (RF) ablation can increase in size in the post-injury period, resulting in remodeling of the extracellular matrix (ECM). The matrix metalloproteinases (MMPs) contribute to adverse myocardial remodeling following injury. However, the role of specific MMP types in RF scar enlargement remains unclear. One MMP type, MMP-3, degrades a wide range of ECM substrates and can activate other MMPs. This project examined LV remodeling in wild type (WT) and MMP-3 deficient (mmp-3-/-) mice following RF injury. RF lesions (0.5 mm probe, 80 degrees C, 30 s) were created on the LV epicardium of WT (C57/BL6) and mmp-3-/- mice and were terminally studied at 1 h, 3, 7, and 28 days post-RF (n=10 each). Heart mass indexed to tibial length (mg/mm) was similar in the WT and mmp-3-/- mice at 1 h (8.1+/-0.3 vs. 7.6+/-0.3), but lower in the mmp-3-/- mice at 28 days post-RF (11.9+/-0.4 vs. 10.5+/-0.4, P<0.05). Scar volumes were greater in the mmp-3-/- mice at 3 days, but similar in the two groups at 28 days. Immunohistochemical localization showed fewer macrophages and lymphocytes at the scar border at 3 days in the mmp-3-/- hearts, but similar staining for these cells in WT and mmp-3-/- hearts at 7 and 28 days post-RF. Post-RF, the early increase in scar volume was accelerated in mmp-3-/- mice and associated with abnormal inflammatory cell infiltration/migration to the area of injury. These findings define a mechanistic role for MMP-3 in RF scar expansion and provide a temporal window during which interruption of MMP-3 activation may impair post-RF myocardial wound healing.

Age-dependent changes in myocardial matrix metalloproteinase/tissue inhibitor of metalloproteinase profiles and fibroblast function.

OBJECTIVE: To evaluate the effects of aging on left ventricular (LV) geometry, collagen levels, matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) abundance, and myocardial fibroblast function. METHODS: Young (3-month-old; n=28), middle-aged (MA; 15-month-old; n=17), and old (23-month-old; n=16) CB6F1 mice of both sexes were used in this study. Echocardiographic parameters were measured; collagen, MMP, and TIMP levels were determined for both the soluble and insoluble protein fractions; and fibroblast function was evaluated. RESULTS: LV end-diastolic dimensions and wall thickness increased in both MA and old mice, accompanied by increased soluble protein and decreased insoluble collagen. Immunoblotting revealed differential MMP/TIMP profiles. Compared to MA levels, MMP-3, MMP-8, MMP-9, MMP-12, and MMP-14 increased, and TIMP-3 and TIMP-4 decreased in the insoluble fraction of old mice, suggesting increased extracellular matrix (ECM) degradative capacity. Fibroblast proliferation was blunted with age. CONCLUSION: This study, for the first time, identified specific differences in cellular and extracellular processes that likely contribute to age-dependent ECM remodeling.

Effects of deletion of the tissue inhibitor of matrix metalloproteinases-1 gene on the progression of murine thoracic aortic aneurysms.

OBJECTIVE: The cause of thoracic aortic aneurysms (TAAs) is poorly understood. Previous work has suggested an association between development of aortic aneurysms and matrix metalloproteinase (MMP) activity. We hypothesized that removal of the primary endogenous aortic MMP inhibitor (TIMP) through TIMP-1 gene deletion will increase TAA progression. METHODS AND RESULTS: The descending thoracic aortas of wild-type 129 SvE and TIMP-1 gene knockout (TIMP-1-/-) mice were exposed to 0.5 mol/L CaCl2 for 15 minutes, with terminal studies performed at 4 or 8 weeks. TAA lumen diameter was measured using confocal microscopy and normalized to the ascending aorta. In addition, sections were studied with in situ zymography and immunohistochemistry staining for MMP-9. Both wild-type [TAA/ascending ratio (mean+/-SEM): control, 0.85+/-0.02 (n=14); 4 weeks, 1.00+/-0.03 (n=13); 8 weeks, 1.05+/-0.10 (n=9)] and TIMP-1-/- [control, 0.98+/-0.04 (n=11); 4 weeks, 1.10+/-0.03 (n =21); 8 weeks, 1.22+/-0.09 (n=10)] groups developed aneurysms at 4 and 8 weeks compared with their respective controls (P<0.05). TIMP-1-/- animals developed larger aneurysms than the corresponding wild-type group (P<0.05). Aneurysms in the TIMP-1-/- group were larger at 8 weeks than at 4 weeks (P<0.05), which was not seen in the wild-type aneurysm groups. Both groups showed presence of MMP-9 in 4 and 8 weeks, most prominently in the adventitia and outer media. In situ zymographic activity was increased in the 8-week TIMP-1-/- group compared with wild-type. CONCLUSIONS: Deletion of the TIMP-1 gene results in increased and continued progression of aneurysm formation compared with wild-type mice in a unique TAA model caused at least in part by an alteration in the balance between gelatinase activity and its endogenous inhibition. Therapeutic strategies aimed at modifying MMP activity may reduce or prevent the progression of TAAs.

Myocardial remodeling after discrete radiofrequency injury: effects of tissue inhibitor of matrix metalloproteinase-1 gene deletion.

Discrete myocardial lesions created through the delivery of radiofrequency (RF) energy can expand; however, the mechanisms have not been established. Matrix metalloproteinases (MMPs) play an important role in myocardial remodeling, and MMP activity can be regulated by the tissue inhibitors of the metalloproteinases (TIMPs). This study examined the role of TIMP-1 in postinjury myocardial remodeling. Lesions were created on the left ventricular (LV) epicardium of wild-type (WT, 8-12 wk, 129SVE) and age-matched TIMP-1 gene-deficient (timp-1(-/-)) mice through the delivery of RF current (80 degrees C, 30 s). Heart mass, LV scar volumes, and collagen content were measured at 1 h and 3, 7, and 28 days postinjury (n = 10 each). Age-matched, nonablated mice were used as reference controls (n = 5). Heart mass indexed to tibial length increased in WT and timp-1(-/-) mice but was greater in the timp-1(-/-) mice by 7 days. Scar volumes increased in a time-dependent manner in both groups but were higher in the timp-1(-/-) mice than the WT mice at 7 days (1.48 +/- 0.09 vs. 1.20 +/- 0.11 mm(3).mg(-1).mm, P < 0.05) and remained higher at 28 days. In the remote myocardium, wall thickness was greater and relative collagen content was lower in the timp-1(-/-) mice at 28 days postinjury. Discrete myocardial RF lesions expand in a time-dependent manner associated with myocyte hypertrophy remote to the scar. Moreover, postinjury myocardial remodeling was more extensive with TIMP-1 gene deletion. Thus TIMP-1 either directly or through modulation of MMP activity may regulate myocardial remodeling following infliction of a discrete injury.

A murine model of thoracic aortic aneurysms.

BACKGROUND: The mechanisms of thoracic aortic aneurysm (TAA) formation are poorly understood, mainly due to the lack of a useful and reproducible model. Accordingly, the goal of this study was to test the hypothesis that abluminal calcium chloride (CaCl(2)) application could create TAAs in the mouse. MATERIALS AND METHODS: Adult 129/SvE mice (n = 8) were anesthetized and their thoracic aortas exposed via left thoracotomy. CaCl(2) (0.5M) was applied to the distal descending thoracic aorta for 15 min followed by chest closure. At 4 weeks, the perfusion-fixed aorta was harvested from the root to the renal arteries. Diameter measurements were made using confocal microscopy, and wall thickness was measured from hematoxylin and eosin-stained sections. RESULTS: The control (n = 15) distal descending thoracic aortic diameter was 0.60 +/- 0.04 mm and increased by 25% (0.76 +/- 0.06 mm, P < 0.05) following CaCl(2) treatment. Control aortic wall thickness was 48 +/- 9 mum and decreased by 47% in corresponding CaCl(2)-exposed segments (25 +/- 8 mum, P < 0.05). The diameter and wall thickness of the ascending aorta (used as an internal control) were not significantly different between groups. Picrosirius red staining of the TAA showed adventitial collagen breakdown and disruption of lamellar organization. CONCLUSIONS: We conclude that abluminal application of CaCl(2) to the thoracic aorta reliably produces dilation, wall-thinning, and disruption of mural architecture, the hallmark signs of aneurysm formation. To our knowledge, these findings describe for the first time the generation of a reproducible model of isolated TAA formation in a murine system.