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1.  Pulmonary Artery Endothelial Cell Phenotypic Alterations in a Large Animal Model of Pulmonary Arteriovenous Malformations Following the Glenn Shunt 
The Annals of thoracic surgery  2013;96(4):1442-1449.
Longevity of the superior cavopulmonary connection (SCPC) is limited by the development of pulmonary arteriovenous malformations (PAVM). The goal of this study was to determine whether phenotypic changes in pulmonary artery endothelial cells (PAEC) that favor angiogenesis occur with PAVM formation.
A superior vena cava to right pulmonary artery connection was constructed in 5 pigs. Pulmonary arteries were harvested at 6-8 weeks following surgery to establish cultures of PAEC and smooth muscle cells, to determine cell proliferation, gene expression, and tubule formation. Abundance of proteins related to angiogenesis was measured in lung tissue.
Contrast echocardiography revealed right-to-left shunting, consistent with PAVM formation. While the proliferation of smooth muscle cells from the right pulmonary artery (RPA) (shunted side) and left pulmonary artery (LPA) (non- shunted side) were similar, right PAEC proliferation was significantly higher. Expression profiles of genes encoding cellular signaling proteins were higher in PAECs from the RPA vs. LPA. Protein abundance of angiopoietin-1, and Tie-2 (angiopoietin receptor) were increased in the right lung (both p<0.05). Tubule formation was increased in endothelial cells from the RPA compared to the LPA (404±16 vs. 199±71 tubules/mm2, respectively p<0.05).
These findings demonstrate that PAVMs developed in a clinically relevant animal model of SCPC. This study found that PAVM development occurred concomitantly with differential changes in PAEC proliferative ability and phenotype. Moreover, there was a significant increase in the angiopoietin/Tie-2 complex in the right lung, which may provide novel therapeutic targets to attenuate PAVM formation following a SCPC.
PMCID: PMC3934798  PMID: 23968766
2.  A Reproducible Porcine Model of Thoracic Aortic Aneurysm 
Circulation  2013;128(11 0 1):10.1161/CIRCULATIONAHA.112.000363.
Thoracic aortic aneurysms (TAAs) develop secondary to abnormal aortic extracellular matrix (ECM) remodeling, resulting in a weakened and dilated aortic wall that progressed to rupture if left unattended. Currently, no diagnostic/prognostic tests are available for detection of TAA disease. This is largely driven by the lack of a large animal model, which would permit longitudinal/mechanistic studies. Accordingly, the objective of the current study was to establish a reproducible porcine model of aortic dilatation, which recapitulates the structural and biochemical changes observed during human TAA development.
Methods and Results
Descending TAAs were induced in Yorkshire pigs (20–25 kg; n=7) through intra-adventitial injections of collagenase (5 ml, 0.35 mg/ml) and peri-adventitial application of crystalline CaCl2 (0.5 g). Three weeks post-TAA induction, aortas were harvested and tissue was collected for biochemical and histological measurements. A subset of animals underwent magnetic resonance imaging pre-operatively and at terminal surgery. Results were compared to sham-operated controls (n=6). Three weeks post-TAA induction, aortic luminal area had increased 38±13% (p=0.018 vs. control). Aortic structural changes included elastic lamellar degradation and decreased collagen content. The protein abundance of MMPs -3, -8, -9, and -12 increased in TAA tissue homogenates, while TIMPs -1, and -4 decreased.
These data demonstrate aortic dilatation, aortic medial degeneration, and alterations in MMP/TIMP abundance, consistent with TAA formation. This study establishes for the first time, a large animal model of TAA that recapitulates the hallmarks of human disease, and will provide a reproducible test-bed for examining diagnostic, prognostic, and therapeutic strategies.
PMCID: PMC3840947  PMID: 24030405
aortic disease; aneurysm; animal models of cardiovascular disease remodeling
3.  Plasma Biomarkers That Reflect Determinants of Matrix Composition Identify the Presence of Left Ventricular Hypertrophy and Diastolic Heart Failure 
Circulation. Heart failure  2011;4(3):246-256.
Chronic pressure overload (such as arterial hypertension) may cause left ventricular (LV) remodeling, alterations in cardiac function, and the development of diastolic heart failure. Changes in the composition of the myocardial extracellular matrix (ECM) may contribute to the development of pressure-overload (PO) induced LV remodeling. We hypothesized that a specific pattern of plasma biomarker expression that reflected changes in these pathophysiologic mechanisms would have diagnostic application to identify: 1-patients who have developed LV hypertrophy and 2-patients with LV hypertrophy who have developed diastolic heart failure.
Methods and Results
Plasma concentration of 17 biomarkers (MMP-1, 2, 3, 7, 8, 9, TIMP-1, 2, 3, 4, NT-proBNP, cardiotrophin, osteopontin, sRAGE, CITP, PINP, PIIINP), an echocardiogram, and 6-minute hall walk were performed on 241 referent control subjects, 144 patients with LV hypertrophy (LVH) but no evidence of heart failure, and 61 patients with LV hypertrophy and diastolic heart failure (DHF). A plasma multi-biomarker panel consisting of increased MMP-7, MMP-9, TIMP-1, PIIINP, and NT-proBNP predicted the presence of LVH with an AUC of 0.80. A plasma multi-biomarker panel consisting of increased MMP-2, TIMP-4, PIIINP and decreased MMP-8 predicted the presence of DHF with an AUC of 0.79. These multi-biomarkers panels performed better than any single biomarker including NT-proBNP, and better than using clinical co-variates alone (AUC = 0.73 for LVH, 0.68 for DHF).
Plasma biomarkers reflecting changes in ECM fibrillar collagen homeostasis, combined into a multi-biomarker panel, have discriminative value in identifying the presence of structural remodeling (LVH) and clinical disease (DHF).
PMCID: PMC4071931  PMID: 21350055
hypertrophy; heart failure; extracellular matrix; biomarkers
4.  Plasma biomarkers for distinguishing etiological subtypes of thoracic aortic aneurysm disease 
Thoracic aortic aneurysms (TAAs) develop through an asymptomatic process resulting in gross dilatation that progresses to rupture if left undetected and untreated. If detected, TAA patients are followed over time until the risk of rupture outweighs the risk of surgical repair. Current methodologies for tracking TAA size are limited to expensive computed tomography or magnetic resonance imaging, as no acceptable population screening tools are currently available. Previous studies from this laboratory and others have identified differential protein profiles for the matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs), in ascending TAA tissue from patients with bicuspid aortic valves (BAV), versus patients with idiopathic degenerative disease and a tricuspid aortic valve (TAV). Additionally, altered microRNA (miR) expression levels have also been reported in TAAs as compared to normal aortic tissue. The objective of the present study was to identify circulating factors within the plasma that could serve as potential biomarkers for distinguishing etiological subtypes of aneurysm disease.
Ascending TAA tissue and plasma specimens were obtained from BAV (n=21) and TAV (n=21) patients at the time of surgical resection. The protein abundance of key MMPs (-1, -2, -3, -8, -9) and TIMPs (-1, -2, -3, -4), and microRNAs (-1, -21, -29a, -133a, -143, -145) was examined using a multi-analyte protein profiling system or by quantitative PCR, respectively. Results were compared to normal aortic tissue and plasma obtained from patients without aortic disease (n=10).
Significant (p < 0.05) differences in standardized miR-1 and miR-21 abundance between BAV and TAV aortic tissue samples and different tissue and plasma profiles of analyte differences from normal aorta where observed between the BAV and TAV groups. Linear regression analysis significant linear relationships in plasma and tissue measurements only for MMP-8 and TIMPs -1, -3 and -4 (p < 0.05). Receiver operator curve analysis revealed specific cassettes of analytes predictive of TAA disease. Relative to normal aorta, BAV proteolytic balance was significantly increased for MMP-1, -2 and -7, and for decreased MMP-8 and -9. In contrast, TAV proteolytic balance relative to normal aorta was significantly increased only for MMP-1 and decreased for MMP-8 and -9.
Taken together these unique data demonstrate differential plasma profiles of MMPs, TIMPs, and miRs in ascending TAA specimens from patients with BAV and TAV. These results suggest that circulating biomarkers may form the foundation for a broader platform of biomarkers capable of detecting the presence of TAA using a simple blood test and may also be useful in personalized medicine strategies to distinguish between etiological subtypes of TAAs in patients with aneurysm disease.
PMCID: PMC4057430  PMID: 23312977
5.  Differential membrane type 1 matrix metalloproteinase substrate processing with ischemia–reperfusion: Relationship to interstitial microRNA dynamics and myocardial function 
Membrane type 1 matrix metalloproteinase (MT1-MMP) is critical to a number of proteolytic and profibrotic events. However, upstream regulation of MT1-MMP with myocardial ischemia–reperfusion remains poorly understood. MicroRNAs regulate post-transcriptional events, and in silico mapping has identified a conserved sequence in MT1-MMP for microRNA-133a. This study tested the hypothesis that changes in microRNA-133a regulation occur with myocardial ischemia–reperfusion, which contributes to time- and region-dependent changes in MT1-MMP activity and processing of MT1-MMP substrates.
Yorkshire pigs (n = 12) underwent ischemia–reperfusion (90 minutes ischemia and 120 minutes reperfusion), where regional preload recruitable stroke work (sonomicrometry), interstitial MT1-MMP activity (microdialysis), Smad2 abundance (immunoblotting), and interstitial microRNA-133a (polymerase chain reaction) were determined within the ischemia–reperfusion and remote regions. Human left ventricular fibroblasts were transduced with microRNA-133a and anti–microRNA-133a (lentivirus) to determine the effects on MT1-MMP protein abundance.
With ischemia–reperfusion, regional preload recruitable stroke work decreased from steady state (139 ± 20 mm Hg to 44 ± 11 mm Hg, P <.05) within the ischemia–reperfusion region. MT1-MMP activity increased in both regions. Phosphorylated Smad2 increased within the ischemia–reperfusion region. Both in vitro and in vivo interstitial levels of microRNA-133a decreased with ischemia and returned to steady-state levels with reperfusion. In vitro transduction of microRNA-133a in left ventricular fibroblasts decreased MT1-MMP levels.
Modulation of MT1-MMP activity and microRNA-133a exportation into the myocardial interstitium occurred in the setting of acute myocardial ischemia–reperfusion. In addition, changes in microRNA-133a expression in left ventricular fibroblasts resulted in an inverse modulation of MT1-MMP abundance. Therefore, targeting of microRNA-133a represents a potentially novel means for regulating the cascade of profibrotic events after ischemia–reperfusion.
PMCID: PMC3602970  PMID: 23102905
6.  Changes in Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of MMPs Plasma Profiles in Stress-Induced Cardiomyopathy 
Journal of Cardiac Failure  2012;18(6):487-492.
Transient changes in the composition of the myocardial extracellular matrix may contribute to the ventricular systolic dysfunction in Stress-induced cardiomyopathy (SIC). We examined the changes in plasma matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) that occur early after the clinical presentation of SIC.
Methods and Results
Ten patients with SIC were enrolled. Plasma concentrations of the six major MMPs (MMP-1,-2,-3,-7,-8,-9) and all 4 tissue inhibitors of MMPs (TIMP-1,-2,-3,-4) were analyzed and compared with data from fifteen control subjects. Within 24 hours of the clinical presentation, SIC patients had lower MMP-1 levels (0.41±0.13 vs 0.70±0.13 pg/mL, p = 0.048) and MMP-8 levels (1.61±0.34 vs 4.84±1.38 pg/mL, p = 0.001) and higher TIMP-4 levels (3.06±0.40 vs 2.16±0.18 pg/mL, p =0.05) compared to control. Seven of nine SIC patients had elevated LV end-diastolic pressures, and all had normal LV end-diastolic dimensions and volumes.
Patients afflicted with SIC had MMPs and TIMPs profiles similar to those described in hypertensive heart disease and diastolic heart failure and different than the profiles following myocardial infarction. Our findings uncovered a unique biomolecular profile in SIC during the first 24 hours of presentation.
PMCID: PMC3361732  PMID: 22633307
Metalloproteinases; Cardiomyopathy; Stress; Stress-induced cardiomyopathy
7.  Aortic dilatation with bicuspid aortic valves: cusp fusion correlates to matrix metalloproteinases and inhibitors 
The Annals of Thoracic Surgery  2011;93(2):457-463.
Congenital bicuspid aortic valves (BAVs) result from fusion of two valve cusps, resulting in left-noncoronary (L-N), right-left (R-L), and right-noncoronary (R-N) morphologies. BAVs predispose to ascending thoracic aortic aneurysms (ATAAs). This study hypothesized that ATAAs with each BAV morphology group possess unique signatures of matrix metalloproteinases (MMPs) and endogenous tissue inhibitors (TIMPs).
ATAA tissue from 46 BAV patients was examined for MMP/TIMP abundance and global MMP activity compared to normal aortic specimens (n=15). Proteolytic balance was calculated as the ratio of MMP abundance to a composite TIMP score (TS). Results were stratified by valve morphology group (L-N (n=6), R-L (n=31), and R-N(n=9)).
The BAV specimens (p<0.05 vs. normal aorta, 100%) displayed elevated global MMP activity (273±63%), MMP-9 (263±47%), and decreased MMP -7 (56±10%), -8 (58±11%), TIMP -1 (63±7%) and -4 (38±3%). The R-L group showed increased global MMP activity (286±89%) and MMP-9 (267±55%) with reduced MMP -7 (45±7%) -8 (68±15%), TIMP -1 (58±7%) and -4 (35±3%). The L-N group showed elevated global MMP activity (284±71%), and decreased MMP-8 (37±17%) and TIMP-4 (48±14). In the R-N group, MMP -7 (46±13%) and -8 (36±17%), and TIMP -1 (59±10) and -4 (42±5%) were decreased. The R-L group demonstrated an increased proteolytic balance for MMP-1, MMP-9, and MMP-12 relative to L-N and R-N.
Each BAV morphology group possesses a unique signature of MMPs and TIMPs. MMP/TIMP score ratios suggest that the R-L group may be more aggressive, justifying earlier surgical intervention.
PMCID: PMC3265643  PMID: 22206960
8.  Relationship Between The Temporal Profile of Plasma microRNA and Left Ventricular Remodeling In Patients Following Myocardial Infarction 
MicroRNAs (miRs) are small noncoding RNAs that recognize and bind to mRNAs and inhibit protein translation or degrade mRNA. Studies in animal models have suggested that miRs play a translational or post-translational regulatory role in myocardial growth, fibrosis, viability, and remodeling. However, whether specific temporal changes in miRs occur in patients during the LV remodeling process that follows a myocardial infarction (post-MI) remains unknown. The purpose of the current pilot study was to test the hypothesis that plasma miRs could be reliably measured in post-MI patients and that there is the relationship between temporal changes in specific miRs and post-MI LV structural remodeling.
Methods and Results
LV end-diastolic volume (EDV, echocardiography) and plasma miR were measured in age matched referent controls (CTL n=12) and post-MI patients (n=12) from day 2 through day 90 post-MI. Selected miRs (miR-1, -21, -29a, 133a, 208) were measured using quantitative rt-PCR and normalized for endogenous snRNA U6. Following MI, LVEDV increased progressively compared to CTL; this was accompanied by time dependent changes in specific miRs. For example, miR-21 initially fell 2 days post-MI (0.3±0.1 fold vs. CTL, p< 0.05), increased 5 days post-MI (2±1 fold vs. CTL, p< 0.05), and returned to CTL values at later post-MI time points. In contrast, miR-29a increased 5 days post-MI (4±1 fold vs. CTL, p< 0.05) and then fell to CTL at later time points. miR-208 increased 5 days post-MI (3±1 fold vs. CTL, p< 0.05) and remained elevated up to 90 days post-MI.
A time-dependent change in miRs occurred in post-MI patients that included an early and robust rise in miRs that have been shown to affect myocardial growth, fibrosis and viability. Thus, serially profiling miRs in the plasma of post-MI patients may hold both mechanistic and prognostic significance.
PMCID: PMC3535326  PMID: 21956146
Myocardial Infarction; Remodeling; microRNA
9.  Progressive Induction of Left Ventricular Pressure Overload in a Large Animal Model Elicits Myocardial Remodeling and a Unique Matrix Signature 
Patients with severe left ventricular (LV) pressure overload (LVPO) secondary to aortic stenosis can present with signs and symptoms of heart failure despite normal LV ejection fractions (LVEF). This process occurs, at least in part, as a result of LVPO-induced extracellular matrix (ECM) remodeling which promulgates increased LV stiffness and impaired diastolic function. However, the determinants which drive ECM remodeling in this form of LVPO remain to be fully defined.
LVPO was induced in mature pigs (n=15) by progressive ascending aortic cuff inflation (once/week/4 weeks) whereby LV mass, LVEF, and regional myocardial stiffness (rKm) were compared to referent controls (n=12). Determinants of ECM remodeling were assessed by measuring levels of mRNA expression for fibrillar collagens, matrix metalloproteinases (MMPs), and the tissue inhibitors of MMP-1 and -4 (TIMP-1, -4).
With LVPO, LV mass and rKm increased by 2- and 3-fold, respectively, compared to control, with no change in LVEF. LV myocardial collagen increased approximately 2-fold which was accompanied by reduced solubility (i.e. increased cross-linking) with LVPO, but mRNA expression for fibrillar collagen and MMPs remained relatively unchanged. In contrast, a robust increase in mRNA expression for TIMP-1 and -4 occurred with LVPO.
In a progressive model of LVPO, which recapitulates the phenotype of aortic stenosis, increased ECM accumulation and subsequently increased myocardial stiffness was not due to increased fibrillar collagen expression, but rather due to determinants of post-translational control which included increased collagen stability (thereby resistant to MMP degradation) and increased endogenous MMP inhibition. Targeting these ECM post-translational events with LVPO may hold both diagnostic and therapeutic relevance.
PMCID: PMC3241904  PMID: 22056365
10.  Selective microRNA Suppression in Human Thoracic Aneurysms: Relationship of miR-29a to Aortic Size and Proteolytic Induction 
Increasing evidence points to a direct role for altered microRNA (miRNA or miR) expression levels in cardiovascular remodeling and disease progression. While alterations in miR expression levels have been directly linked to cardiac hypertrophy, fibrosis, and remodeling, their role in regulating gene expression during thoracic aortic aneurysm (TAA) development has yet to be explored.
Methods and Results
The present study examined miR expression levels in aortic tissue specimens collected from patients with ascending TAAs by quantitative real-time PCR, and observed decreased miR expression (miRs -1, -21, -29a, -133a, and -486) as compared to normal aortic specimens. A significant relationship between miR expression levels (miRs -1, -21, -29a, and -133a) and aortic diameter was identified; as aortic diameter increased, miR expression decreased. Using a bioinformatics approach, members of the matrix metalloproteinase (MMP) family, proteins involved in TAA development, were examined for putative miR binding sites. MMP-2 and MMP-9 were identified as potential targets for miR-29a and miR-133a respectively, and MMP-2 was subsequently verified as a miR-29a target in vitro. A significant inverse relationship between miR-29a and total MMP-2 was then identified in the clinical TAA specimens.
These findings demonstrate altered miR expression patterns in clinical TAA specimens, suggesting that the loss of specific miR expression may allow for the elaboration of specific MMPs capable of driving aortic remodeling during TAA development. Importantly, these data suggest that these miRs have biological and clinical relevance to the behavior of TAAs, and may provide significant targets for therapeutic and diagnostic applications.
PMCID: PMC3246193  PMID: 22010139
aneurysm; thoracic aorta; microRNA; MMP; remodeling
11.  Targeted Regional Injection of Biocomposite Microspheres Alters Post Myocardial Infarction Remodeling and Matrix Proteolytic Pathways 
Circulation  2011;124(11 Suppl):S35-S45.
While localized delivery of biocomposite materials, such as calcium hydroxyapatite (CHAM), have been demonstrated to potentially attenuate adverse LV remodeling post-myocardial infarction (MI), the underlying biological mechanisms for this effect remain unclear. This study tested the hypothesis that targeted CHAM injections would alter proteolytic pathways (matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs)), and be associated with parameters of post-MI LV remodeling.
Methods and Results
MI was induced in adult sheep followed by 20 targeted injections of a total volume of 1.3 mL (n=6) or 2.6 mL of CHAM (n=5), or saline (n=13), and LV end-diastolic volume (EDV) and MMP/TIMP profiles in the MI region were measured at 8 weeks post-MI. LV EDV decreased with 2.6 mL CHAM vs MI Only (105.4±7.5 vs 80.6±4.2 respectively, p<0.05) but not with 1.3 mL CHAM (94.5±5.0, p=0.32). However, MI thickness increased by 2-fold in both CHAM groups compared to MI Only (p<0.05). MMP-13 increased 40-fold in the MI Only group (p<0.05) but fell by over 6-fold in both CHAM groups (p<0.05). MMP-7 increased approximately 1.5-fold in the MI Only group (p<0.05) but decreased to referent control values in both CHAM groups in the MI region (p<0.05). Collagen content was reduced by approximately 30% in the CHAM groups compared to MI Only (p<0.05).
Differential effects on LV remodeling and MMP/TIMP profiles occurred with CHAM. Thus, targeted injections of a biocomposite material can favorably affect the post-MI remodeling process and therefore holds promise as a treatment strategy in and of itself, or as a matrix with potentially synergistic effects with localized pharmacologic or cellular therapies.
PMCID: PMC3218562  PMID: 21911817
Infarct Expansion; Matrix Metalloproteinase; Tissue Inhibitor of Matrix Metalloproteinases
12.  Targeted Imaging of the Spatial and Temporal Variation of Matrix Metalloproteinase Activity in Porcine Model of Post-Infarct Remodeling: Relationship to Myocardial Dysfunction 
Matrix metalloproteinases (MMPs) are known to modulate left ventricular (LV) remodeling after a myocardial infarction (MI). However, the temporal and spatial variation of MMP activation and their relationship to mechanical dysfunction post MI remains undefined.
Methods and Results
MI was surgically induced in pigs (n=23) and cine MR and dual isotope hybrid SPECT/CT imaging obtained using thallium-201 (201Tl) and a technetium-99m labeled MMP targeted tracer (99mTc-RP805) at 1, 2 and 4 weeks post MI along with controls (n=5). Regional myocardial strain was computed from MR images and related to MMP zymography and ex vivo myocardial 99mTc-RP805 retention. MMP activation as assessed by in vivo and ex vivo 99mTc-RP805 imaging/retention studies was increased nearly 5-fold within the infarct region at 1 week post-MI and remained elevated up to 1 month post-MI. The post-MI change in LV end-diastolic volumes was correlated with MMP activity (y=31.34e0.48x, p=0.04). MMP activity was increased within the border and remote regions early post-MI, but declined over 1 month. There was a high concordance between regional 99mTc-RP805 uptake and ex vivo MMP-2 activity.
A novel, multimodality non-invasive hybrid SPECT/CT imaging approach was validated and applied for in vivo evaluation of MMP activation in combination with cine MR analysis of LV deformation. Increased 99mTc-RP805 retention was seen throughout the heart early post-MI and was not purely a reciprocal of 201Tl perfusion. 99mTc-RP805 SPECT/CT imaging may provide unique information regarding regional myocardial MMP activation and predict late post-MI LV remodeling.
PMCID: PMC3140564  PMID: 21505092
MMP; LV remodeling; spatiotemporal imaging
13.  Continuous Localized Monitoring of Plasmin Activity Identifies Differential and Regional Effects of the Serine Protease Inhibitor Aprotinin: Relevance to Antifibrinolytic Therapy 
Antifibrinolytic therapy, such as the use of the serine protease inhibitor aprotinin, was a mainstay for hemostasis following cardiac surgery. However, aprotinin was empirically dosed, and while the pharmacological target was the inhibition of plasmin activity (PLact) this was never monitored, off-target effects occurred, and led to withdrawn from clinical use. The present study developed a validated fluorogenic-microdialysis method to continuously measure PLact and tested the hypothesis that standardized clinical empirical aprotinin dosing would impart differential and regional effects on PLact.
Pigs (30 kg) were instrumented with microdialysis probes to continuously measure PLact in myocardial, kidney and skeletal muscle compartments (deltoid) and then randomized to High Dose aprotinin administration (2 mKIU load/ 0.5 mKIU/hr infusion; n=7), Low Dose aprotinin administration (1 mKIU load/ 0.250 mKIU/hr infusion; n=6). PLact was compared to time matched vehicle (n=4), and PLact was also measured in plasma by an in-vitro fluorogenic method. Aprotinin suppressed PLact in the myocardium and kidney at both High and Low Doses- indicative that both doses exceeded a minimal concentration necessary for PLact inhibition. However, differential effects of aprotinin on PLact were observed in the skeletal muscle indicative of different compartmentalization of aprotinin.
Using a large animal model and a continuous method to monitor regional plasmin activity, these unique results demonstrated that an empirical aprotinin dosing protocol causes maximal and rapid suppression in the myocardium and kidney and in turn would likely increase the probability of off-target effects and adverse events. Further, this proof of principle study demonstrated that continuous monitoring of determinants of fibrinolysis may provide a novel approach for managing fibrinolytic therapy.
PMCID: PMC3079882  PMID: 21502925
aprotinin; plasmin inhibition; fibrinolysis; plasma; interstitial; microdialysis probe
14.  Determinants of extracellular matrix remodelling are differentially expressed in paediatric and adult dilated cardiomyopathy 
European Journal of Heart Failure  2010;13(3):271-277.
The left ventricular phenotype of idiopathic dilated cardiomyopathy (DCM) can appear similar in paediatric and adult patients. However, the aetiology of paediatric DCM is usually idiopathic, and often leads an aggressive clinical course. A structural underpinning of DCM is extracellular matrix changes, which are determined by a balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). This study tested the hypothesis that different MMP/TIMP profiles occur in paediatric and adult DCM patients.
Methods and results
Left ventricular samples from paediatric (age 9 ± 5 years; n = 10) and adult (age 62 ± 3 years; n = 20) DCM (at time of transplant) were subjected to an MMP/TIMP multiplex array and immunoassay in order to measure the MMP subclasses; collagenases (MMP-8, -13), gelatinases (MMP-2, -9), stromelysin/matrilysin (MMP-3, -7), membrane type (MT1-MMP), as well as for the four known TIMPs. MMP-8 and -9 levels increased by over 150% (P < 0.05), whereas MMP-3 and -7 levels decreased by over 30% (P < 0.05) in paediatric DCM when compared with adult DCM. TIMP-1 and -2 levels increased two-fold (P < 0.05), but TIMP-3 fell by 41% (P < 0.05) in paediatric DCM. Myocardial levels of specific interleukins (IL-1beta, IL-2, IL-8) were increased by approximately 50% in paediatric DCM.
These unique findings demonstrated that a specific MMP/TIMP profile occurs in paediatric DCM when compared with adult DCM, and that local cytokine induction may contribute to this process. These distinct differences in the determinants of myocardial matrix structure and function may contribute to the natural history of DCM in children.
PMCID: PMC3041467  PMID: 21147820
Cardiomyopathy; Extracellular matrix; Matrix metalloproteinase; Paediatrics
15.  Circulating Matrix Metalloproteinase Levels Following Ventricular Septal Defect Repair in Infants 
Congenital heart surgery initiates a complex inflammatory response that can influence the post-operative course. However, broad integration of the cytokine and proteolytic cascades (matrix metalloproteinases: MMPs), which may contribute to post-operative outcomes has not been performed.
Using a low volume (50 – 60 μL), high sensitivity, multiplex approach a panel of cytokines (IL-2, -4, -6, -8, -10, TNFα, IL-1β, GM-CSF) and MMPs (MMP-2, -3, -7, -8, -9, -12, -13) were serially measured in patients (n=9) pre-operatively and post-VSD repair. Results were correlated with outcomes such as inotropic requirement, oxygenation, and fluid balance. Serial changes in peri-operative plasma levels of the cytokines and MMPs exhibited distinct temporal profiles. Plasma levels of IL-2, -8, -10, and MMP-9 peaked within 4 hours, while MMP-3 and MMP-8 levels remained elevated at 24 and 48 hours following cross-clamp removal. Area-under-the-curve analysis of early cytokine levels were associated with major clinical variables, including inverse correlations between early IL-10 levels and cumulative inotrope requirement at 48 hours (r: −0.85, p<0.005) and late MMP-7 levels and cumulative fluid balance (r: −0.90, p<0.001).
The unique findings of this study were that serial profiling a large array of cytokines and proteolytic enzymes following congenital heart surgery can provide insight into relationships between changes in bioactive molecules to early postoperative outcomes. Specific patterns of cytokine and MMP release may hold significance as biomarkers for predicting and managing the post-operative course following congenital heart surgery.
PMCID: PMC2972374  PMID: 20561637
Ventricular septal defect repair; Inflammation; Cytokines; Matrix metalloproteinases
16.  Cellular phenotype transformation occurs during thoracic aortic aneurysm development 
Thoracic aortic aneurysms (TAAs) result from dysregulated remodeling of the vascular extracellular matrix (ECM) which may occur as a result of altered resident cellular function. The present study tested the hypothesis that aortic fibroblasts undergo a stable change in cellular phenotype during TAA formation.
Primary murine aortic fibroblasts were isolated from normal and TAA-induced aortas (4-wks post-induction with 0.5M CaCl2 15 min) by outgrowth method. Normal and TAA cultures were examined using a focused PCR array to determine fibroblast-specific changes in gene expression in the absence and presence of biological stimulation (endothelin-1, phorbol-12-myristate-13-acetate, angiotensin II). The relative expression of 38 genes, normalized to 4 housekeeping genes, was determined and genes displaying a minimum 2-fold increase/decrease or genes with significantly different normalized Ct values were considered to have altered expression.
At steady state TAA fibroblasts revealed elevated expression of several MMPs (Mmp2, Mmp11, Mmp14), collagen genes/elastin (Col1a1, Col1a2, Col3a1, Eln), and other matrix proteins, as well as decreased expression of Mmp3, Timp3, and Ltbp1. Moreover, gene expression profiles in TAA fibroblasts were different than normal fibroblasts after equivalent biological stimuli.
This study demonstrated for the first time that isolated primary aortic fibroblasts from TAA-induced mice possess a unique and stable gene expression profile, and when challenged with biological stimuli, induce a transcriptional response that is different from normal aortic fibroblasts. Together, these data suggest that aortic fibroblasts undergo a stable phenotypic change during TAA development which may drive the enhancement of ECM proteolysis in TAA progression.
PMCID: PMC2901416  PMID: 20219212
aneurysm; extracellular matrix; remodeling; phenotype; fibroblast
17.  Chronic Localized High Frequency Electrical Stimulation Within the Myocardial Infarct: Effects on Matrix Metalloproteinases and Regional Remodeling 
Circulation  2010;122(1):20-32.
Disruption of the balance between matrix metalloproteinases (MMP) and MMP inhibitors (TIMPs) within a myocardial infarct (MI) contribute to left ventricular (LV) wall thinning and changes in regional stiffness at the MI region. This study tested the hypothesis that a targeted regional approach through localized high frequency stimulation (LHFS) using low amplitude electrical pulses instituted within a formed MI scar would alter MMP/TIMP levels and prevent MI thinning.
At 3 wks following MI, pigs were randomized for LHFS (n=7, 240bpm, 0.8V, 0.05ms pulses) or unstimulated (UNSTIM, n=10). At 4 wks post-MI, LV wall thickness (echo, 0.89±0.07 vs 0.67±0.08 cm, p<0.05) and regional stiffness (piezoelectric crystals, 14.70±2.08 vs 9.11±1.24, p<0.05) were higher with LHFS than UNSTIM. In vivo interstitial MMP activity (fluorescent substrate cleavage, 943±59 vs 1210±72 units, p<0.05) in the MI region was lower with LHFS than in UNSTIM. In the MI region, MMP-2 levels were lower, while TIMP-1 and collagen levels were higher with LHFS than in UNSTIM (all p<0.05). Transforming growth factor-β (TGF-β) receptor 1 and phosphorylated SMAD-2/3 levels within the MI region were higher with LHFS than in UNSTIM. Electrical stimulation (4Hz) of isolated fibroblasts resulted in a reduction of MMP-2 and MT1-MMP levels, but increased TIMP-1 levels compared to unstimulated fibroblasts.
These unique findings demonstrate that LHFS of the MI region altered LV wall thickness and material properties, likely due to reduced regional MMP activity. Thus, LHFS may provide a novel means to favorably modify LV remodeling post-MI.
PMCID: PMC2946370  PMID: 20566951
Myocardial infarction; extracellular matrix; remodeling; matrix metalloproteinases
18.  Effects of Aprotinin or Tranexamic Acid on Proteolytic / Cytokine Profiles in Infants Following Cardiac Surgery 
The Annals of thoracic surgery  2010;89(6):1843-1852.
Following cardiopulmonary bypass (CPB), elaboration of cytokines, and subsequent interstitial proteases induction, such as matrix metalloproteinases (MMPs), can result in a complex postoperative course. The serine protease inhibitor, aprotinin, which has been used in congenital heart surgery putatively for modulating fibrinolysis, is now unavailable, necessitating the use of lysine analogues, such as tranexamic acid (TXA). The present study tested the hypothesis that distinctly different plasma profiles of signaling molecules and proteases, would be differentially affected following the administration of aprotinin or TXA in the context of congenital cardiac surgery and CPB.
37 Patients (age 4.8±0.3 mos) undergoing corrective surgery for ventricular septal defect (VSD) and Tetralogy of Fallot (TOF) received either aprotinin (n=22), or TXA (n=15). Using a high throughput multiplex suspension immunoassay, plasma was serially quantified for cytokines and MMPs: before aprotinin or TXA (baseline), after separation from CPB, and 4, 12, 24, and 48 hours post-CPB.
Tumor necrosis factor-alpha (TNF) increased initially following CPB in both the aprotinin and TXA groups, but at 24 and 48 hours post-CPB was approximately 50% lower in the aprotinin group (p<0.05). IIL-10 levels were 3-fold higher in the TXA group compared to the aprotinin group immediately post-CBP (p<0.05). Plasma levels of MMP types associated with inflammation, MMP-8 and -9, were 2-fold higher in the late post-CPB period in the TXA group when compared to the aprotinin group.
Following VSD or TOF repair in children, cytokine induction occurs, which is temporally related to the emergence of a specific MMP profile. Moreover, these unique findings demonstrated differential effects between the serine protease inhibitor aprotinin, and the lysine analogue TXA with respect to cytokine and MMP induction in the early post-operative period. The different cytokine-proteolytic profile between these anti-fibrinolytics may in turn influence biological processes in the post-operative period.
PMCID: PMC3100188  PMID: 20494037
19.  Differential Effect of Wall Tension on Matrix Metalloproteinase Promoter Activation in the Thoracic Aorta 
The Journal of surgical research  2009;160(2):333-339.
Vascular remodeling relies upon extracellular matrix restructuring by the matrix metalloproteinases (MMPs). Induction of MMP-2 and MMP-9 by biological signaling molecules has been defined, but whether a mechanical stimulus such as elevated wall tension may generate MMP promoter activation remains unknown. Accordingly, this study examined whether MMP promoter activation would occur as a function of wall tension.
Materials and Methods
The MMP-2 or MMP-9 promoter sequences were fused to the reporter gene lacZ and inserted into the mouse genome. Thoracic aortic rings were harvested (6 preparations/construct) and maintained under physiological conditions at predetermined tension values corresponding to 0, 70, 85 and 100 mmHg for 3 hours. Relative gene expression of lacZ, directly reflecting MMP promoter activity, was then quantified by QPCR.
MMP-2 promoter activity decreased to 0.42±0.11 at 0 mmHg and increased to 1.57±0.24 fold at 100 mmHg (p<0.05), whereas MMP-9 was unaffected.
Using unique transgenic constructs with homology to human MMP promoters, this study demonstrated that a physiologically relevant mechanical stimulus was sufficient to differentially induce MMP promoter activation.
PMCID: PMC2859117  PMID: 19375723
vascular remodeling; wall tension; thoracic aorta; matrix metalloproteinase
20.  Plasma Profiling Determinants of Matrix Homeostasis in Pediatric Dilated Cardiomyopathy 
Cardiology in the young  2010;21(1):52-61.
Dilated cardiomyopathy is an important cause of heart failure in both children and adults, but is more progressive in children. In adult dilated cardiomyopathy, left ventricular remodeling is associated with changes in plasma levels of matrix metalloproteinases, and tissue inhibitor of metalloproteinases. Plasma matrix metalloproteinases, and tissue inhibitors of metalloproteinase changes in pediatric dilated cardiomyopathy has not been examined. The present study developed a low blood volume, high sensitivity assay to test the hypothesis that unique and differential plasma matrix metalloproteinases and tissue inhibitors of metalloproteinase profile exists in pediatric dilated cardiomyopathy patients.
A systemic blood sample (1 milliliter) was obtained from 7 children with dilated cardiomyopathy (age 8 plus or minus 7 years), and 26 age-matched normal volunteers. Using a high throughput multiplex suspension immunoassay, plasma levels were quantified for: collagenases (matrix metalloproteinase-8), gelatinases (matrix metalloproteinase-2, -9), lysins (matrix metalloproteinase-3, -7), and tissue inhibitor of metalloproteinases-1, -2, and -4. Matrix metalloproteinase to tissue inhibitor of metalloproteinases ratios were also calculated. Plasma matrix metalloproteinase-2, -7, -8 and -9 levels were increased by greater than 2-fold in dilated cardiomyopathy patients than normals ( with p less than 0.05). Dilated cardiomyopathy patients also had significantly higher tissue inhibitor of metalloproteinases-1 and -4 (298 percent and 230 percent; with p less than 0.05).
These unique findings demonstrated that a specific plasma matrix metalloproteinase/tissue inhibitors of metalloproteinase profile occurs in pediatric dilated cardiomyopathy when compared to normal children. These distinct differences in the determinants of myocardial matrix structure and function may contribute to the natural history of dilated cardiomyopathy in children, and may provide a novel biomarker platform in pediatric dilated cardiomyopathy.
PMCID: PMC3093970  PMID: 20977821
21.  Differential Matrix Metalloproteinase Levels in Adenocarcinoma and Squamous Cell Carcinoma of the Lung 
The matrix metalloproteinases (MMPs) have been implicated in the aggressive course of non-small cell lung cancer (NSCLC). However, there are a large number of MMP subtypes with diverse proteolytic substrates and different induction pathways. This study tested the hypothesis that a differential MMP profile would exist between NSCLC and normal lung and that MMP patterns would differ between NSCLC histologic type.
NSCLC samples and remote normal samples were obtained from patients with stage I or II NSCLC with either squamous cell (n=22) or adenocarcinoma (n=19) histology. Absolute concentrations for each of the MMP subclasses; collagenases (MMP-1, 8, -13), gelatinases (MMP-2,-9), lysins (MMP-2, -7) and elastase (MMP-12) were determined by a calibrated and validated multiplex suspension array.
Overall, MMP levels were significantly increased in NSCLC compared to normal. For example, MMP-1 and MMP-7 increased by approximately 10 fold in NSCLC (p<0.05). Moreover, a different MMP portfolio was observed between NSCLC histologic types. For example MMP-1,-8,-9 and -12 increased by over 4-fold in squamous cell versus adenocarcinoma (p<0.05). In those patients who recurred within 3 years of resection, 3-fold higher levels of MMP-8 and -9 were observed (p<0.05).
Increased levels of a number of MMP types occur with NSCLC, but the MMP profile was distinctly different between histologic types and in those patients with recurrence. These different MMP profiles may be important in the mechanistic basis for the natural history of different NSCLC types, as well as identifying potential prognostic and therapeutic targets.
PMCID: PMC2844342  PMID: 20304142
matrix metalloproteinases; lung cancer; multiplex; recurrence
22.  Caspase Inhibition Modulates Left Ventricular Remodeling Following Myocardial Infarction through Cellular and Extracellular Mechanisms 
Myocyte death occurs by necrosis and caspase-mediated apoptosis in myocardial infarction (MI). In-vitro studies suggest caspase activation causes myocardial contractile protein degradation without inducing apoptosis. Thus, caspase activation may evoke left ventricular (LV) remodeling through independent processes post-MI. The effects of caspase activation on LV geometry post-MI remain unclear. This project applied pharmacologic caspase inhibition (CASPI) to a porcine model of MI.
Methods and Results
Pigs (34kg) were instrumented in order to induce 60min of coronary artery occlusion followed by reperfusion and a 7-day follow-up period. Upon reperfusion, the pigs were randomized to saline (n=12) or CASPI (n=10, IDN6734, 6mg/kg IV, then 6mg/kg/hr for 24hrs). Plasma troponin-I values were reduced with CASPI compared to saline at 24hrs post-MI (133±15 vs 189±20ng/mL, respectively, p<0.05). LV end-diastolic area (echocardiography) and inter-regional length (sonomicrometry) increased from baseline in both groups but were attenuated with CASPI by 40% and 90%, respectively (p<0.05). Myocyte length was reduced with CASPI compared to saline (128±3 vs 141±4μm, respectively, p<0.05). Plasma free-pro-MMP-2 values increased from baseline with CASPI (27±6%, p<0.05) indicative of reduced conversion to active MMP-2. Separate in-vitro studies demonstrated that activated caspase species cleaved pro-MMP-2 yielding active MMP-2 forms and that MMP activity was increased in the presence of activated caspase-3.
CASPI attenuated regional and global LV remodeling post-MI and altered viable myocyte geometry. Caspases increased MMP activity in-vitro while CASPI modified conversion of MMP-2 to the active form in-vivo. Taken together, the results of the present study suggest that the elaboration of caspases post-MI likely contribute to LV remodeling through both cellular and extracellular mechanisms.
PMCID: PMC2880531  PMID: 20147844
myocardial infarction; remodeling; caspase inhibition; left ventricular
23.  Mesenchymal Cell Transplantation and Myocardial Remodeling Following Myocardial Infarction 
Circulation  2009;120(11 Suppl):S220-S229.
Targeted delivery of mesenchymal precursor cells (MPCs) can modify left ventricular (LV) cellular and extracellular remodeling following myocardial infarction (MI). However, whether and to what degree LV remodeling may be affected by MPC injection post-MI, and whether these effects are concentration dependent remain unknown.
Methods and Results
Allogeneic MPCs were expanded from sheep bone marrow, and direct intra-myocardial injection was performed within the borderzone region one hour following MI induction (coronary ligation) in sheep at the following concentrations: 25×106 (25 M, n=7), 75 ×106 (75 M, n=7), 225 ×106 (225 M, n=10), 450 ×106 (450 M, n=8), and MPC free media only (MI Only, n=14). LV end diastolic volume increased in all groups but was attenuated in the 25 and 75 M groups. Collagen content within the borderzone region was increased in the MI Only, 225, and 450 M groups. Whereas plasma ICTP, an index of collagen degradation, was highest in the 25 M group. Within the borderzone region matrix metalloproteinases (MMPs) and MMP tissue inhibitors (TIMPs) also changed in a MPC concentration dependent manner. For example, borderzone levels of MMP-9 were highest in the 25 M group when compared to the MI Only and other MPC treatment group values.
MPC injection altered collagen dynamics, MMP, and TIMP levels in a concentration dependent manner, and thereby influenced indices of post-MI LV remodeling. However, the greatest effects with respect to post-MI remodeling were identified at lower MPC concentrations, thus suggesting a therapeutic threshold exists for this particular cell therapy.
PMCID: PMC2752314  PMID: 19752372
Infarct Expansion; Stem Cells; Matrix Metalloproteinase; Tissue Inhibitor of Matrix Metalloproteinase; Fibrosis
24.  Differential effects of mechanical and biological stimuli on matrix metalloproteinase promoter activation in the thoracic aorta 
Circulation  2009;120(11 Suppl):S262-S268.
The effect of multiple integrated stimuli on vascular wall expression of matrix metalloproteinases (MMPs) remains unknown. Accordingly, this study has examined the influence of the vasoactive peptide angiotensin II (AngII) on wall tension-induced promoter activation of MMP-2, MMP-9, and membrane type-1 MMP (MT1-MMP).
Methods and Results
Thoracic aortic rings harvested from transgenic reporter mice containing the MMP-2, MMP-9, or MT1-MMP promoter sequence fused to a reporter gene were subjected to three hours of wall tension at 70, 85, or 100 mmHg with or without 100nM AngII. Total RNA was harvested from the aortic rings, and reporter gene transcripts were quantified by QPCR to measure MMP promoter activity. MT1-MMP promoter activity was increased at both 85 and 100 mmHg compared to baseline tension of 70 mmHg, while treatment with AngII stimulated MT1-MMP promoter activity to the same degree at all tension levels (p<0.05). Elevated tension and AngII displayed a potential synergistic enhancement of MMP-2 promoter activation at 85 and 100mmHg, while the same stimuli caused a decrease in MMP-9 promoter activity (p<0.05) at 100 mmHg.
This study has demonstrated that exposure to a relevant biological stimulus (AngII) in the presence of elevated tension modulated MMP promoter activation. Furthermore, these data suggest that a mechanical-molecular set point exists for the induction of MMP promoter activation, and that this set point can be adjusted up or down by a secondary biological stimulus. Together, these results may have significant clinical implications toward the regulation of hypertensive vascular remodeling.
PMCID: PMC2774261  PMID: 19752377
Metalloproteinases; Aorta; Wall stress; Angiotensin
25.  Cardiac Restricted Over-Expression of Membrane Type-1 Matrix Metalloproteinase in Mice: Effects on Myocardial Remodeling with Aging 
Circulation. Heart failure  2009;2(4):351-360.
The direct consequences of a persistently increased myocardial expression of the unique matrix metalloproteinase (MMP), membrane type-1 (MT1-MMP) on myocardial remodeling remained unexplored.
Cardiac restricted MT1-MMPexp was constructed in mice using the full length human MT1-MMP gene ligated to the myosin heavy chain promoter, which yielded approximately a 200% increase in MT1-MMP when compared to age/strain matched wild type mice (WT). LV function and geometry was assessed by echocardiography in 3 month (“young”) WT (n=32) and MT1-MMPexp (n=20) mice, and compared to 14 month (“middle age”) WT (n=58) and MT1-MMPexp (n=35) mice. LV end-diastolic volume was similar between the WT and MT1-MMPexp young groups as was LV ejection fraction. In the middle age WT mice, LV end-diastolic volume and ejection fraction was similar to young WT mice. However, in the MT1-MMPexp middle age mice, LV end-diastolic volume was approximately 43% higher and LV ejection fraction 40% lower (both p<0.05). Moreover, in the middle age MT1-MMPexp mice, myocardial fibrillar collagen increased by nearly 2-fold and was associated with an approximate 3-fold increase in the processing of the pro-fibrotic molecule, latency-associated transforming growth factor binding protein. In a second study, 14 day survival following myocardial infarction was significantly lower in middle aged MT1-MMPexp mice.
Persistently increased myocardial MT1-MMP expression, in and of itself, caused LV remodeling, myocardial fibrosis, dysfunction and reduced survival following myocardial injury. These findings suggest that MT1-MMP plays a mechanistic role in adverse remodeling within the myocardium.
PMCID: PMC2743030  PMID: 19808359
matrix; myocardial remodeling; ventricular function; aging

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