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1.  LOCALIZED TARGETING OF BIOMATERIALS FOLLOWING MYOCARDIAL INFARCTION; A FOUNDATION TO BUILD ON 
Trends in cardiovascular medicine  2013;23(8):301-311.
Acute coronary syndromes can give rise to myocardial injury-infarction (MI), which in turn promulgates a series of cellular and extracellular events that result in left ventricular (LV) dilation and dysfunction. Localized strategies focused upon interrupting this inexorable process include delivery of bioactive molecules and stem cell derivatives. These localized treatment strategies are often delivered in a biomaterial complex in order to facilitate elution of the bioactive molecules or stem cell engraftment. However, these biomaterials can impart significant and independent effects upon the MI remodeling process. In addition, significant changes in local cell and interstitial biology within the targeted MI region can occur following injection of certain biomaterials, which may hold important considerations when using these materials as matrices for adjuvant drug/cell therapies.
doi:10.1016/j.tcm.2013.04.004
PMCID: PMC3773006  PMID: 23746937
2.  Integrating the Myocardial Matrix into Heart Failure Recognition and Management 
Circulation research  2013;113(6):10.1161/CIRCRESAHA.113.300309.
In contrast to public perception, the morbidity and mortality as well as the resultant health care costs associated with chronic heart failure (HF) are increasing and arguably reaching epidemic proportions. While basic research efforts have provided unique insights into fundamental processes that govern myocardial extracellular matrix (ECM) growth and function, the translation of these findings to improved diagnostics and therapeutics for HF have not been as forthcoming. The factors that contribute to this relative paucity of new clinical tools for HF are multifactorial but likely include the need to recognize and differentiate HF phenotypes, and to couple the use of biomarkers and multimodality imaging in early translational research studies. Recognizing the classification scheme of HF with a reduced ejection fraction (HFrEF) to that of HF with a preserved ejection fraction (HFpEF)and incorporating unique and differential measurements of ECM remodeling to these specific disease processes are warranted. For example, profiling pathways of ECM degradation such as the matrix metalloproteinases (MMPs) in patients with ischemic heart disease and HFrEF can provide prognostic information in terms of risk of progression to HF. In patients with chronic hypertensive disease and HFpEF, plasma profiling indices of ECM synthesis and turnover, as well as advances in ECM imaging have been shown to provide diagnostic and prognostic utility. In terms of therapeutics, strategies which stabilize the ECM in HFrEF hold potential, whereas in contradistinction, selective antifibrotic agents may hold promise with HFpEF.
doi:10.1161/CIRCRESAHA.113.300309
PMCID: PMC3869184  PMID: 23989715
myocardial remodeling; ventricular function; diagnostics; therapeutics
3.  Membrane Associated Matrix Proteolysis and Heart Failure 
Circulation research  2013;112(1):195-208.
The extracellular matrix (ECM) is a complex entity containing a large portfolio of structural proteins, signaling molecules, and proteases. Changes in the overall integrity and activational state of these ECM constituents can contribute to tissue structure and function, which is certainly true of the myocardium. Changes in the expression patterns and activational states of a family of ECM proteolytic enzymes, the matrix metalloproteinases (MMPs), have been identified in all forms of LV remodeling and can be a contributory factor for the progression to heart failure. However, new clinical and basic research has identified some surprising and unpredicted changes in MMP profiles in LV remodeling processes, such as with pressure or volume overload, as well as with myocardial infarction. From these studies, it has become recognized that proteolytic processing of signaling molecules by certain MMP types, particularly the transmembrane MMPs, may actually facilitate ECM accumulation as well as modulate fibroblast transdifferentiation – both critical events in adverse LV remodeling. Based upon the ever increasing substrates and diversity of biological actions of MMPs, it is likely that continued research regarding the relationship of LV remodeling to this family of proteases will yield new insights into the ECM remodeling process itself as well as new therapeutic targets.
doi:10.1161/CIRCRESAHA.112.266882
PMCID: PMC4026203  PMID: 23287455
matrix metalloproteinases; heart failure; myocardial remodeling; extracellular matrix; transforming growth factor; fibroblast
4.  Analyzing Propensity Matched Zero-Inflated Count Outcomes in Observational Studies 
Journal of applied statistics  2013;41(1):10.1080/02664763.2013.834296.
Determining the effectiveness of different treatments from observational data, which are characterized by imbalance between groups due to lack of randomization, is challenging. Propensity matching is often used to rectify imbalances among prognostic variables. However, there are no guidelines on how appropriately to analyze group matched data when the outcome is a zero inflated count. In addition, there is debate over whether to account for correlation of responses induced by matching, and/or whether to adjust for variables used in generating the propensity score in the final analysis. The aim of this research is to compare covariate unadjusted and adjusted zero-inflated Poisson models that do and do not account for the correlation. A simulation study is conducted, demonstrating that it is necessary to adjust for potential residual confounding, but that accounting for correlation is less important. The methods are applied to a biomedical research data set.
doi:10.1080/02664763.2013.834296
PMCID: PMC3843491  PMID: 24298197
Poisson; count data; propensity matching; random effects; zero inflation
5.  Injectable and bioresponsive hydrogels for on-demand matrix metalloproteinase inhibition 
Nature materials  2014;13(6):653-661.
Inhibitors of matrix metalloproteinases (MMPs) have been extensively explored to treat pathologies where excessive MMP activity contributes to adverse tissue remodeling. While MMP inhibition remains a relevant therapeutic target, MMP inhibitors have not translated to clinical application due to the dose-limiting side effects following systemic administration of the drugs. Here, we describe the synthesis of a polysaccharide-based hydrogel that can be locally injected into tissues and releases a recombinant tissue inhibitor of MMPs (rTIMP-3) in response to MMP activity. Specifically, rTIMP-3 is sequestered in the hydrogels through electrostatic interactions and is released as crosslinks are degraded by active MMPs. Targeted delivery of the hydrogel/rTIMP-3 construct to regions of MMP over-expression following a myocardial infarction (MI) significantly reduced MMP activity and attenuated adverse left ventricular remodeling in a porcine model of MI. Our findings demonstrate that local, on-demand MMP inhibition is achievable through the use of an injectable and bioresponsive hydrogel.
doi:10.1038/nmat3922
PMCID: PMC4031269  PMID: 24681647
6.  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.
Background:
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.
Methods:
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.
Results:
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).
Conclusions:
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.
doi:10.1016/j.athoracsur.2013.05.075
PMCID: PMC3934798  PMID: 23968766
7.  A Reproducible Porcine Model of Thoracic Aortic Aneurysm 
Circulation  2013;128(11 0 1):10.1161/CIRCULATIONAHA.112.000363.
Background
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.
Conclusions
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.
doi:10.1161/CIRCULATIONAHA.112.000363
PMCID: PMC3840947  PMID: 24030405
aortic disease; aneurysm; animal models of cardiovascular disease remodeling
8.  GENE TARGETING IN ISCHEMIC HEART DISEASE AND FAILURE: TRANSLATIONAL AND CLINICAL STUDIES 
Biochemical pharmacology  2012;85(1):1-11.
Alternative and innovative targeted strategies hold relevance in improving the current treatments for ischemic heart disease (IHD). One potential treatment modality, gene targeting, may provide a unique alternative to current IHD therapies. The principal function of gene targeting in IHD is to augment the expression of an endogenous gene through amplification of an exogenous gene, delivered by a plasmid or a viral vector to enhance myocardial perfusion, and limit the long-term sequelae. The initial clinical studies of gene targeting in IHD were focused upon induction of angiogenic factors and the outcomes were equivocal. Nevertheless, significant advancements have been made in viral vectors, mode of delivery, and potentially relevant targets for IHD. Several of these advancements, particularly with a focus on translational large animal studies, are the focus of this review. The development of novel vectors with prolonged transduction efficiency and minimal inflammation, coupled with hybrid perfusion-mapping delivery devices, and improving the safety of vector use and efficacy of gene systems are but a few of the exciting progresses that are likely to proceed to clinical studies in the near future.
doi:10.1016/j.bcp.2012.08.018
PMCID: PMC3575101  PMID: 22935384
gene therapy; myocardial infarction; ischemia; plasmid; adenovirus; adeno-associated virus
9.  Myocardial Remodeling With Aortic Stenosis and Following Aortic Valve Replacement- Mechanisms and Future Prognostic Implications 
Aortic valve stenosis is a common cause of left ventricular pressure overload, a pathologic process that elicits myocyte hypertrophy and alterations in extracellular matrix composition, both of which contribute to increases in left ventricular stiffness. However, clinical and animal studies suggest that increased myocardial extracellular matrix fibrillar collagen content occurs later in the time course of left ventricular pressure overload at a time coincident with severe abnormalities in diastolic function followed by the development of symptomatic heart failure. Aortic valve replacement remains the most effective treatment for elimination of chronic pressure overload secondary to aortic stenosis but has traditionally been recommended only after the onset of clinical symptoms. However, long-term follow-up of symptomatic aortic stenosis patients after aortic valve replacement suggests that valve replacement may not result in complete reversal of the maladaptive changes that occur within the myocardial extracellular matrix secondary to the pressure overload state. Quite the contrary, residual left ventricular extracellular matrix abnormalities such as these are likely responsible for persistent abnormalities in diastolic function and increased morbidity and mortality after aortic valve replacement. Thus, defining the mechanisms and pathways responsible for regulating the myocardial extracellular matrix during the natural history of aortic stenosis may provide a means by which to detect crucial structural milestones and thereby permit more precise identification of the development of maladaptive left ventricular remodeling.
doi:10.1016/j.jtcvs.2011.04.044
PMCID: PMC3210937  PMID: 21762938
10.  Changes in the Myocardial Interstitium and Contribution to the Progression of Heart Failure 
Heart failure clinics  2012;8(1):7-20.
SYNOPSIS
The myocardial interstitium is highly organized and orchestrated whereby small disruptions in composition, spatial relationships, or content lead to altered myocardial systolic and/or diastolic performance. These changes in extracellular matrix structure and function are important in the progression to heart failure in pressure overload hypertrophy (POH), dilated cardiomyopathy (DCM), and ischemic heart disease. The myocardial interstitium is not a passive entity, but rather a complex and dynamic microenvironment which represents an important structural and signaling system within the myocardium.
doi:10.1016/j.hfc.2011.08.012
PMCID: PMC3227393  PMID: 22108723
interstitium; extracellular matrix; heart failure; pressure overload hypertrophy; dilated cardiomyopathy; myocardial infarction
11.  Progressive Induction of Left Ventricular Pressure Overload in a Large Animal Model Elicits Myocardial Remodeling and a Unique Matrix Signature 
Background
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.
Methods
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).
Results
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.
Conclusions
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.
doi:10.1016/j.jtcvs.2011.09.032
PMCID: PMC3241904  PMID: 22056365
12.  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.
Background
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).
Conclusions
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).
doi:10.1161/CIRCHEARTFAILURE.110.958199
PMCID: PMC4071931  PMID: 21350055
hypertrophy; heart failure; extracellular matrix; biomarkers
13.  Diversity of Myocardial Interstitial Proteolytic Pathways; Gene Deletion Reveals Unexpected Consequences 
Circulation  2011;124(19):2052-2055.
doi:10.1161/CIRCULATIONAHA.111.064071
PMCID: PMC3229095  PMID: 22064956
14.  Hemodynamics and Myocardial Blood Flow Patterns Following Placement of a Cardiac Passive Restraint Device in a Model of Dilated Cardiomyopathy 
Background
The present study examined a cardiac passive restraint device which applies epicardial pressure (HeartNetTM Implant) in a clinically relevant model of dilated cardiomyopathy (DCM) to determine effects on hemodynamic and myocardial blood flow patterns.
Methods
DCM was established in 10 pigs (3 weeks atrial pacing, 240 beats per minute). Hemodynamic parameters and regional left ventricle (LV) blood flow were measured under baseline conditions and following acute HeartNet (Paracor Medical Inc, Sunnyvale, CA) placement. Measurements were repeated following adenosine infusion, allowing maximal coronary vasodilation and coronary flow reserve determination.
Results
LV dilation and systolic dysfunction occurred relative to baseline as measured by echocardiography. LV end diastolic dimension increased and LV fractional shortening decreased (3.8±0.1 vs 6.1±0.2cm and 31.6±0.5 vs 16.2±2.1%, both p<0.05 respectively) consistent with the DCM phenotype. The HeartNet was successfully deployed without arrhythmias and a computed median mid-LV epicardial pressure of 1.4 mmHg was applied by the HeartNet throughout the cardiac cycle. Acute HeartNet placement did not adversely affect steady state hemodynamics. With the HeartNet in place, coronary reserve was significantly blunted.
Conclusions
In a large animal model of DCM, the cardiac passive restraint device did not appear to adversely affect basal resting myocardial blood flow. However, following acute HeartNet placement, LV maximal coronary reserve was blunted. These unique results suggest that cardiac passive restraint devices which apply epicardial transmural pressure can alter myocardial blood flow patterns in a DCM model. Whether this blunting of coronary reserve holds clinical relevance with chronic passive restraint device placement remains unestablished.
doi:10.1016/j.jtcvs.2010.09.065
PMCID: PMC3134533  PMID: 21397269
15.  Targeted Regional Injection of Biocomposite Microspheres Alters Post Myocardial Infarction Remodeling and Matrix Proteolytic Pathways 
Circulation  2011;124(11 Suppl):S35-S45.
Background
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).
Conclusions
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.
doi:10.1161/CIRCULATIONAHA.111.035774
PMCID: PMC3218562  PMID: 21911817
Infarct Expansion; Matrix Metalloproteinase; Tissue Inhibitor of Matrix Metalloproteinases
16.  Gender Differences in Non-Ischemic Myocardial Remodeling: Are They Due to Estrogen Modulation of Cardiac Mast Cells and/or Membrane Type 1 Matrix Metalloproteinase 
SUMMARY
This review is focused on gender differences in cardiac remodeling secondary to sustained increases in cardiac volume (VO) and generated pressure (PO). Estrogen has been shown to favorably alter the course of VO-induced remodeling. That is, the VO-induced increased extracellular matrix proteolytic activity and mast cell degranulation responsible for the adverse cardiac remodeling in males and ovariectomized rodents do not occur in intact premenopausal females. While less is known regarding the mechanisms responsible for female cardioprotection in PO-induced stress, gender differences in remodeling have been reported indicating the ability of premenopausal females to adequately compensate. In view of the fact that, in male mice with PO, mast cells have been shown to play a role in the adverse remodeling suggests favorable estrogen modification of mast cell phenotype may also be responsible for cardioprotection in females with PO. Thus, while evidence is accumulating regarding premenopausal females being cardioprotected; there remains the need for in-depth studies to identify critical downstream molecular targets that are under the regulation of estrogen and relevant to cardiac remodeling. Such studies would result in the development of therapy which provides cardioprotection while avoiding the adverse effects of systemic estrogen delivery.
doi:10.1007/s00424-013-1229-9
PMCID: PMC3654075  PMID: 23417570
17.  Continuous Localized Monitoring of Plasmin Activity Identifies Differential and Regional Effects of the Serine Protease Inhibitor Aprotinin: Relevance to Antifibrinolytic Therapy 
Background
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.
Methods/Results
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.
Conclusions
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.
doi:10.1097/FJC.0b013e31820b7df1
PMCID: PMC3079882  PMID: 21502925
aprotinin; plasmin inhibition; fibrinolysis; plasma; interstitial; microdialysis probe
18.  Circulating Matrix Metalloproteinase Levels Following Ventricular Septal Defect Repair in Infants 
Background
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.
Methods/Results
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).
Conclusions
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.
doi:10.1016/j.jtcvs.2010.05.014
PMCID: PMC2972374  PMID: 20561637
Ventricular septal defect repair; Inflammation; Cytokines; Matrix metalloproteinases
19.  Myxomavirus-Derived Serpin Prolongs Survival and Reduces Inflammation and Hemorrhage in an Unrelated Lethal Mouse Viral Infection 
Lethal viral infections produce widespread inflammation with vascular leak, clotting, and bleeding (disseminated intravascular coagulation [DIC]), organ failure, and high mortality. Serine proteases in clot-forming (thrombotic) and clot-dissolving (thrombolytic) cascades are activated by an inflammatory cytokine storm and also can induce systemic inflammation with loss of normal serine protease inhibitor (serpin) regulation. Myxomavirus secretes a potent anti-inflammatory serpin, Serp-1, that inhibits clotting factor X (fX) and thrombolytic tissue- and urokinase-type plasminogen activators (tPA and uPA) with anti-inflammatory activity in multiple animal models. Purified serpin significantly improved survival in a murine gammaherpesvirus 68 (MHV68) infection in gamma interferon receptor (IFN-γR) knockout mice, a model for lethal inflammatory vasculitis. Treatment of MHV68-infected mice with neuroserpin, a mammalian serpin that inhibits only tPA and uPA, was ineffective. Serp-1 reduced virus load, lung hemorrhage, and aortic, lung, and colon inflammation in MHV68-infected mice and also reduced virus load. Neuroserpin suppressed a wide range of immune spleen cell responses after MHV68 infection, while Serp-1 selectively increased CD11c+ splenocytes (macrophage and dendritic cells) and reduced CD11b+ tissue macrophages. Serp-1 altered gene expression for coagulation and inflammatory responses, whereas neuroserpin did not. Serp-1 treatment was assessed in a second viral infection, mouse-adapted Zaire ebolavirus in wild-type BALB/c mice, with improved survival and reduced tissue necrosis. In summary, treatment with this unique myxomavirus-derived serpin suppresses systemic serine protease and innate immune responses caused by unrelated lethal viral infections (both RNA and DNA viruses), providing a potential new therapeutic approach for treatment of lethal viral sepsis.
doi:10.1128/AAC.02594-12
PMCID: PMC3754305  PMID: 23774438
20.  Early Post-Operative Outcomes and Blood Product Utilization in Adult Cardiac Surgery- The Post Aprotinin Era 
Circulation  2011;124(11 Suppl):S62-S69.
Background
Aprotinin was a commonly utilized pharmacological agent for homeostasis in cardiac surgery but was discontinued resulting in the extensive use of lysine analogues. This study tested the hypothesis that early post-operative adverse events and blood product utilization would affected in this post-aprotinin era.
Methods/Results
Adult patients (n=781) undergoing coronary artery bypass (CABG), valve replacement, or both from November 1, 2005-October 31, 2008 at a single institution were included. Multiple logistic regression modeling and propensity scoring were performed on 29 pre-operative and intra-operative variables in patients receiving aprotinin (n=325) or lysine analogues (n=456). The propensity adjusted relative risk (RR;95% confidence interval;CI) for the intra-operative use of packed red blood cells (RR:0.75;CI:0.57–0.99), fresh frozen plasma (RR:0.37;0.21–0.64), and cryoprecipitate (RR:0.06;CI:0.02–0.22) were lower in the aprotinin versus lysine analogue group (all p<0.05). The risk for mortality (RR:0.53;CI:0.16–1.79) and neurological events (RR:0.87;CI:0.35–2.18) remained similar between groups, whereas a trend for reduced risk for renal dysfunction was observed in the aprotinin group.
Conclusions
In the post-aprotinin era with the exclusive use of lysine analogues, the relative risk of early post-operative outcomes such as mortality and renal dysfunction have not improved, but the risk for the intra-operative use of blood products has increased. Thus, improvements in early post-operative outcomes have not been realized with the discontinued use of aprotinin, but rather increased blood product utilization has occurred with the attendant costs and risks inherent with this strategy.
doi:10.1161/CIRCULATIONAHA.110.002543
PMCID: PMC3181091  PMID: 21911820
22.  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.
Objective
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1016/j.athoracsur.2010.02.069
PMCID: PMC3100188  PMID: 20494037
23.  Plasma Profiling Determinants of Matrix Homeostasis in Pediatric Dilated Cardiomyopathy 
Cardiology in the young  2010;21(1):52-61.
Objective
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.
Methods/Results
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).
Conclusions
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.
doi:10.1017/S1047951110001381
PMCID: PMC3093970  PMID: 20977821
24.  Caspase Inhibition Modulates Left Ventricular Remodeling Following Myocardial Infarction through Cellular and Extracellular Mechanisms 
Background
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.
Conclusion
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.
doi:10.1097/FJC.0b013e3181d4ca66
PMCID: PMC2880531  PMID: 20147844
myocardial infarction; remodeling; caspase inhibition; left ventricular
25.  Selective Endothelin-1 Receptor type-A Inhibition in Cardiac Surgery Subjects with Pre-Existing LV Dysfunction: Influence on Early Post-Operative Hemodynamics 
Background and Objective
A robust release of endothelin-1-1 (ET) with subsequent ETA subtype receptor (ET-AR) activation occurs in patients following cardiac surgery requiring cardiopulmonary bypass (CPB). Increased ET-AR activation has been identified in patients with poor LV function (reduced ejection fraction; EF). Accordingly, this study tested the hypothesis that a selective ET-AR antagonist (ET-ARA) administered peri-operatively would favorably affect post-CPB hemodynamic profiles in patients with a pre-existing poor LVEF.
Methods and Results
Patients (n=29; 66±2 yrs) with a reduced LVEF (37±2%) were prospectively randomized, in a blinded fashion, at the time of elective coronary revascularization and/or valve replacement requiring CPB, to infusion of the highly-selective and potent ET-ARA, sitaxsentan at 1 or 2 mg/kg (IV bolus; n=9, 10 respectively) or vehicle (saline; n=10). Infusion of the ET-ARA/vehicle was performed immediately prior to separation from CPB and again at 12 hrs post-CPB. ET and hemodynamic measurements were performed at baseline, at separation from CPB (Time 0) and at 0.5, 6, 12, 24 hrs post-CPB. Baseline plasma ET (4.0±0.3 fmol/mL) was identical across all 3 groups, but when compared to pre-operative, baseline values obtained from age matched subjects with a normal LVEF (n=37;LVEF>50%), were significantly increased (2.9±0.2 fmol/mL, p<0.05) Baseline systemic (SVR; 1358±83 d·s·cm-5) and pulmonary (PVR; 180±23 d·s·cm-5) vascular resistance were equivalent in all 3 groups. As a function of Time 0, SVR changed in an equivalent fashion in the post-CPB period, but a significant ET-ARA effect was observed for PVR (ANOVA; p<0.05). For example at 24 hrs post-CPB, PVR increased by 40 d.scm-5 in the vehicle group, but directionally decreased by over 40 d·s·cm-5 in the 2 mg/kg ETARA group (p<0.05). Total adverse events were equivalently distributed across the ET-ARA/placebo groups.
Conclusions
These unique findings demonstrated that infusion of an ET-ARA in high risk cardiac surgery patients was not associated with significant hemodynamic compromise. Moreover, ET-ARA favorably affected PVR in the early post-operative period. Thus, the ET-AR serves as a potential pharmacological target for improving outcomes following cardiac surgery in patients with compromised LV function.
doi:10.1016/j.jtcvs.2009.11.046
PMCID: PMC2827656  PMID: 20074751
Endothelin-1; receptor antagonist; cardiac surgery; systolic dysfunction

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