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1.  First nationwide survey of US integrated 6-year cardiothoracic surgical residency program directors 
Objective
The recently implemented integrated 6-year (I-6) format represents a significant change in cardiothoracic surgical residency training. We report the results of the first nationwide survey assessing I-6 program directors' impressions of this new format.
Methods
A 28-question web-based survey was distributed to program directors of all 24 Accreditation Council for Graduate Medical Education-accredited I-6 training programs in November 2013. The response rate was a robust 67%.
Results
Compared with graduates of traditional residencies, most I-6 program directors with enrolled residents believed that their graduates will be better trained (67%), be better prepared for new technological advances (67%), and have superior comprehension of cardiothoracic disease processes (83%). Just as with traditional program graduates, most respondents believed their I-6 graduates would be able to independently perform routine adult cardiac and general thoracic operations (75%) and were equivocal on whether additional specialty training (eg, minimally invasive, heart failure, aortic) was necessary. Most respondents did not believe that less general surgical training disadvantaged I-6 residents in terms of their career (83%); 67% of respondents would have chosen the I-6 format for themselves if given the choice. The greater challenges in training less mature and experienced trainees and vulnerability to attrition were noted as disadvantages of the I-6 format. Most respondents believed that I-6 programs represent a natural evolution toward improved residency training rather than a response to declining interest among medical school graduates.
Conclusions
High satisfaction rates with the I-6 format were prevalent among I-6 program directors. However, concerns with respect to training relatively less experienced, mature trainees were evident.
doi:10.1016/j.jtcvs.2014.04.004
PMCID: PMC4336151  PMID: 24820188
2.  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
3.  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
4.  Pathophysiology of Thoracic Aortic Aneurysm (TAA): Is it not one Uniform Aorta? Role of Embryologic Origin 
Thoracic aortic aneurysm (TAA) is a clinically silent and potentially fatal disease whose pathophysiology is poorly understood. Application of data derived from animal models and human tissue analysis of abdominal aortic aneurysms may prove misleading given current evidence of structural and biochemical aortic heterogeneity above and below the diaphragm. Genetic predisposition is more common in TAA and includes multi-faceted syndromes such as Marfan, Loeys-Dietz, and type IV Ehlers-Danlos as well as autosomal-dominant familial patterns of inheritance. Investigation into the consequences of these known mutations has provided insight into the cell signaling cascades leading to degenerative remodeling of the aortic medial extracellular matrix (ECM) with TGF-β playing a major role. Targeted research into modifying the upstream regulation or downstream effects of the TGF-β1 pathway may provide opportunities for intervention to attenuate TAA progression.
doi:10.1016/j.pcad.2013.04.002
PMCID: PMC3759819  PMID: 23993239
5.  Plasma biomarkers for distinguishing etiological subtypes of thoracic aortic aneurysm disease 
Background
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.
Methods
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).
Results
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.
Conclusions
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.
doi:10.1016/j.jtcvs.2012.12.027
PMCID: PMC4057430  PMID: 23312977
6.  Forecasting the Impact of Heart Failure in the United States 
Circulation. Heart failure  2013;6(3):606-619.
Background
Heart failure (HF) is an important contributor to both the burden and cost of national healthcare expenditures, with more older Americans hospitalized for HF than for any other medical condition. With the aging of the population, the impact of HF is expected to increase substantially.
Methods and Results
We estimated future costs of HF by adapting a methodology developed by the American Heart Association to project the epidemiology and future costs of HF from 2012 to 2030 without double counting the costs attributed to comorbid conditions. The model assumes that HF prevalence will remain constant by age, sex, and race/ethnicity and that rising costs and technological innovation will continue at the same rate. By 2030, >8 million people in the United States (1 in every 33) will have HF. Between 2012 and 2030, real (2010$) total direct medical costs of HF are projected to increase from $21 billion to $53 billion. Total costs, including indirect costs for HF, are estimated to increase from $31 billion in 2012 to $70 billion in 2030. If one assumes all costs of cardiac care for HF patients are attributable to HF (no cost attribution to comorbid conditions), the 2030 projected cost estimates of treating patients with HF will be 3-fold higher ($160 billion in direct costs).
Conclusions
The estimated prevalence and cost of care for HF will increase markedly because of aging of the population. Strategies to prevent HF and improve the efficiency of care are needed.
doi:10.1161/HHF.0b013e318291329a
PMCID: PMC3908895  PMID: 23616602
AHA Scientific Statements; heart failure
7.  Myosin Cross-Bridge Dynamics in Patients With Hypertension and Concentric Left Ventricular Remodeling 
Circulation. Heart failure  2012;5(6):803-811.
Background
Hypertension (HTN) causes concentric left ventricular (LV) remodeling, defined as an increased relative wall thickness or overt LV hypertrophy, and associated diastolic dysfunction. HTN and concentric remodeling are also common precursors to heart failure with a preserved ejection fraction (EF). It is not known if the myofilament contributes to diastolic dysfunction in patients with concentric remodeling.
Methods and Results
Intra-operative myocardial biopsies were obtained in 15 male patients undergoing coronary bypass grafting (CBG), all with normal LV EF and wall motion. Eight patients had a history of HTN and concentric remodeling. Seven without HTN or remodeling served as controls. Myocardial strips were dissected and demembranated with detergent. Isometric tension was measured and sinusoidal length perturbation analysis performed at sarcomere length 2.2μm and pCa 8 –4.5. Sinusoidal analysis provides estimates of cross-bridge dynamics, including rate constants of attachment and detachment and cross-bridge attachment time (ton). The normalized isometric tension-pCa relation was similar in HTN and controls. However, ton was significantly prolonged at submaximal [Ca2+] (pCa ≥ 6.5) in HTN patients. Analysis of protein phosphorylation revealed ~25% reduction in phosphorylation of troponin I in HTN patients (P < 0.05).
Conclusions
Compared with controls, patients with HTN and concentric remodeling display prolonged ton at submaximal [Ca2+] without a change in the tension-pCa relation. Prolonged ton implicates altered cross-bridge dynamics as a cause of slowed relaxation in these patients. This finding was associated with reduced phosphorylation of troponin I, suggesting decreased phosphorylation of protein kinase A/G sites as a mechanism.
doi:10.1161/CIRCHEARTFAILURE.112.968925
PMCID: PMC3505876  PMID: 23014131
diastole; heart failure; hypertrophy; myocardium; remodeling
8.  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
9.  Aortic dilatation with bicuspid aortic valves: cusp fusion correlates to matrix metalloproteinases and inhibitors 
The Annals of Thoracic Surgery  2011;93(2):457-463.
Background
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).
Methods
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)).
Results
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.
Conclusion
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.
doi:10.1016/j.athoracsur.2011.09.057
PMCID: PMC3265643  PMID: 22206960
10.  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
11.  Selective microRNA Suppression in Human Thoracic Aneurysms: Relationship of miR-29a to Aortic Size and Proteolytic Induction 
Background
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.
Conclusions
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.
doi:10.1161/CIRCGENETICS.111.960419
PMCID: PMC3246193  PMID: 22010139
aneurysm; thoracic aorta; microRNA; MMP; remodeling
12.  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
13.  Intraoperative Diagnosis of Stanford Type A Dissection by Transesophageal Echocardiogram in a Patient Presenting for Renal Transplantation 
A 48-year-old patient with hypertensive end-stage renal disease presented for cadaveric renal transplantation. On physical exam, a previously undocumented diastolic murmur was heard loudest at the left lower sternal border. The patient had a history of pericardial effusions and reported “a feeling of chest fullness” when lying flat. As such, a transesophageal echocardiogram (TEE) was performed after induction of anesthesia to evaluate the pericardial space and possibly determine the etiology and severity of the new murmur. The TEE revealed a Stanford Type A aortic dissection. The renal transplant was cancelled (organ reassigned within region), and the patient underwent an urgent ascending and proximal hemiarch aortic replacement. This case demonstrates the importance of a thorough physical exam and highlights the utility of TEE for noncardiac surgical cases.
doi:10.1155/2011/263561
PMCID: PMC3505951  PMID: 23213602
14.  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
15.  Cellular phenotype transformation occurs during thoracic aortic aneurysm development 
Objective
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1016/j.jtcvs.2009.12.033
PMCID: PMC2901416  PMID: 20219212
aneurysm; extracellular matrix; remodeling; phenotype; fibroblast
16.  Differential Effect of Wall Tension on Matrix Metalloproteinase Promoter Activation in the Thoracic Aorta 
The Journal of surgical research  2009;160(2):333-339.
Background
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.
Results
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.
Conclusions
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.
doi:10.1016/j.jss.2008.12.033
PMCID: PMC2859117  PMID: 19375723
vascular remodeling; wall tension; thoracic aorta; matrix metalloproteinase
17.  Differential Matrix Metalloproteinase Levels in Adenocarcinoma and Squamous Cell Carcinoma of the Lung 
Objective
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.
Methods
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.
Results
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).
Conclusion
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.
doi:10.1016/j.jtcvs.2009.12.016
PMCID: PMC2844342  PMID: 20304142
matrix metalloproteinases; lung cancer; multiplex; recurrence
18.  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
19.  Differential effects of mechanical and biological stimuli on matrix metalloproteinase promoter activation in the thoracic aorta 
Circulation  2009;120(11 Suppl):S262-S268.
Background
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.
Conclusions
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.
doi:10.1161/CIRCULATIONAHA.108.843581
PMCID: PMC2774261  PMID: 19752377
Metalloproteinases; Aorta; Wall stress; Angiotensin
20.  Transforming growth factor-beta signaling in thoracic aortic aneurysm development: a paradox in pathogenesis 
Journal of vascular research  2008;46(2):119-137.
Thoracic aortic aneurysms (TAAs) are potentially devastating, and due to their asymptomatic behavior, pose a serious health risk characterized by the lack of medical treatment options and high rates of surgical morbidity and mortality. Independent of the inciting stimuli (biochemical/mechanical), TAA development proceeds by a multifactorial process influenced by both cellular and extracellular mechanisms, resulting in alterations of the structure and composition of the vascular extracellular matrix (ECM). While the role of enhanced ECM proteolysis in TAA formation remains undisputed, little attention has been focused on the upstream signaling events that drive the remodeling process. Recent evidence highlighting the dysregulation of transforming growth factor-beta (TGF-β) signaling in ascending TAAs from Marfan syndrome patients has stimulated an interest in this intracellular signaling pathway. However, paradoxical discoveries have implicated both enhanced TGF-β signaling and loss of function TGF-β receptor mutations, in aneurysm formation; obfuscating a clear functional role for TGF-β in aneurysm development. In an effort to elucidate this subject, TGF-β signaling and its role in vascular remodeling and pathology will be reviewed, with the aim of identifying potential mechanisms of how TGF-β signaling may contribute to the formation and progression of TAA.
doi:10.1159/000151766
PMCID: PMC2645475  PMID: 18765947
TGF-β; aneurysm; signal transduction; extracellular matrix; remodeling
21.  Regional Heterogeneity within the Aorta: Relevance to Aneurysm Disease 
Vascular remodeling within the aorta results in a loss of structural integrity with consequent aneurysm formation. This degradation is more common in the abdominal aorta, but also occurs above the diaphragm in the thoracic aorta. Conventionally, the aorta has been considered a large vascular conduit with uniform cellular and extracellular structure and function. Evidence is accumulating, however, to suggest that variations exist between the thoracic and abdominal aorta, thereby demonstrating regional heterogeneity. Further pathophysiologic studies of aortic dilation in each of these regions have identified disparities in atherosclerotic plaque deposition, vessel mechanics, protease profiles, and cell signaling pathways. Improved understanding of this spatial heterogeneity may promote evolution in the management of aneurysm disease through computational models of aortic wall stress, imaging of proteolytic activity, targeted pharmacologic treatment, and the application of region-specific gene therapy.
doi:10.1016/j.jtcvs.2008.06.027
PMCID: PMC2679174  PMID: 19026791
thoracic aorta; abdominal aorta; aortic aneurysm; matrix metalloproteinase
22.  Endothelin-A Receptor Inhibition Following Cardiopulmonary Bypass: Cytokines and Receptor Activation 
The Annals of thoracic surgery  2008;86(5):1576-1583.
Background
Basic studies have suggested that cross-talk exists between the endothelin-A receptor (ET-AR) and tumor necrosis factor signaling pathway. This study tested the hypothesis that administration of an ET-AR antagonist at the separation from cardiopulmonary bypass would alter the tumor necrosis factor activation in the early post operative period.
Methods
Patients (n = 44) were randomized to receive bolus infusion of vehicle, 0.1, 0.5, 1, or 2 mg/kg of the ET-AR antagonist (sitaxsentan) at the separation from cardiopulmonary bypass (n=9, 9, 9, 9, and 8 respectively). Plasma levels of tumor necrosis factor-α and soluble tumor necrosis factor receptor 1 and 2 were measured.
Results
Compared to the vehicle group at 24 hours, plasma levels of tumor necrosis factor-α and tumor necrosis factor receptor 2 (indicative of receptor activation) were reduced in the 1 mg/kg ET-AR antagonist group (by ~13 pg/mL and ~0.5 ng/mL respectively; p<0.05). Plasma tumor necrosis factor receptor I levels also decreased (by ~1 ng/mL) following infusion of the higher doses of the ET-AR antagonist and remained lower (by ~3 ng/mL) at 24 hours post infusion (p<0.05). In addition, a dose effect was observed between the ET-AR antagonist and these indices of tumor necrosis factor activation (p<0.01).
Conclusions
This study demonstrated a mechanistic relationship between the ET-AR and tumor necrosis factor receptor activation in the post cardiac surgery period. Thus, in addition to the potential cardiovascular effects, a selective ET-AR antagonist can modify other biological processes relevant to the post cardiac surgery setting. (word count:245/250)
doi:10.1016/j.athoracsur.2008.06.076
PMCID: PMC2671963  PMID: 19049753
Cardiopulmonary Bypass (CPB); Inflammation; Cytokines
23.  DIFFERENTIAL EFFECTS OF EPSILON-AMINOCAPROIC ACID AND APROTININ ON MATRIX METALLOPROTEINASE RELEASE IN PATIENTS FOLLOWING CARDIOPULMONARY BYPASS 
This study examined whether differential effects of two agents commonly used for hemostatic purposes during cardiac surgery, aprotinin or epsilon-aminocaproic acid (EACA), exist with respect to elevations in proinflammatory interleukins (ILs) and matrix metalloproteinases (MMPs) in patients undergoing coronary artery bypass surgery. Sixty patients were prospectively randomized to receive either aprotinin (1×106 KIU;n=30) or EACA (5gIV;n=30) and blood samples were obtained for IL and MMP levels just before induction of anesthesia (Baseline), 10 minutes after separation from cardiopulmonary bypass (Post) and 6 Hours after surgery (6 Hours). IL-6 was increased at Post in the EACA group and increased further at 6 hours. In the aprotinin group, IL-6 was significantly increased only at 6 Hours. MMP subtypes associated with inflammation, MMP-8 and -9 were increased in the EACA group at Post and remained elevated at 6 Hours. Thus, differential effects on IL and MMP release occurred between aprotinin and EACA, indicative of different mechanisms of action independent of hemostatic effects.
doi:10.1097/FJC.0b013e318168400a
PMCID: PMC2666004  PMID: 18427286
matrix metalloproteinases; interleukins; cardiac surgery; aprotinin; epsilon-aminocaproic acid
24.  DYNAMIC CHANGES IN MATRIX METALLOPROTIENASE ACTIVITY WITHIN THE HUMAN MYOCARDIAL INTERSTITIUM DURING MYOCARDIAL ARREST AND REPERFUSION 
Circulation  2008;118(14 Suppl):S16-S23.
Background
Past studies have clearly established that matrix metalloproteinases (MMPs) contribute to adverse myocardial remodeling with ischemia and reperfusion. However, these studies measured MMP levels in extracted samples, and therefore whether and to what degree actual changes in interstitial MMP activity occur within the human myocardium in the context of ischemia/reperfusion remained unknown.
Methods
The present study directly quantified MMP interstitial activity (MMPact) within the myocardium of patients (n=14) undergoing elective cardiac surgery during steady-state conditions, as well as during and following an obligatory period of myocardial arrest and reperfusion achieved by cardiopulmonary bypass (CPB). Interstitial MMP activity was continuously monitored using a validated MMP fluorogenic substrate, a microdialysis system placed within the myocardium, and in-line fluorescent detection system.
Results
MMP activity, as measured by fluorescent emission, reached a stable steady state level by 10 minutes following deployment of the microdialysis system. During initiation of CPB, MMP activity increased by 20% from baseline values (p<0.05), and then rapidly fell with cardiac arrest and longer periods of CPB. However, with restoration of myocardial blood flow and separation from CPB, MMP interstitial activity increased by over 30% from baseline (p<0.05).
Conclusions
The present study directly demonstrated that MMP proteolytic activity exists within the human myocardial interstitium and is a dynamic process under conditions such as myocardial arrest and reperfusion.
doi:10.1161/CIRCULATIONAHA.108.786640
PMCID: PMC2663795  PMID: 18824748
microdialysis; cardiac surgery; fluorogenic substrates; extracellular matrix
25.  Altered Transforming Growth Factor-beta Signaling in a Murine Model of Thoracic Aortic Aneurysm 
Journal of vascular research  2008;45(6):457-468.
Objective
Thoracic aortic aneurysms (TAAs) develop by a multifactorial process involving maladaptive signaling pathways which alter the aortic vascular environment. Transforming growth factor-beta (TGF-β) has been implicated in regulating the structure and composition of the extracellular matrix by differential activation of various intracellular signaling pathways. However, whether and to what degree TGF-β signaling contributes to TAA development remains unclear. Accordingly, the hypothesis that alterations in TGF-β signaling occur during aneurysm formation was tested in a murine model of TAA.
Methods
TAAs were surgically induced in mice (C57BL/6J) and aortas were analyzed at predetermined time points (1-, 2-, and 4- wks post-TAA induction). Quantitative real-time PCR (QPCR) was performed to evaluate the expression of 84 relevant TGF-β superfamily genes, and the protein levels of key signaling intermediates were measured by immunoblotting. Results were compared to unoperated reference control mice.
Results
QPCR revealed increased expression of TGF-β superfamily ligands (Gdf -2, -6, -7, Inhba), ligand inhibitors (Bmper, Chrd, Gsc), and transcriptional regulators (Dlx2, Evi1), among other genes (Cdkn2b, Igf1, IL-6). Protein levels of TGF-β receptorII, Smad2, Smad1/5/8, phospho-Smad1/5/8, and Smurf1 were increased from control values post-TAA induction. Both TGF-β receptorI and Smad4 were decreased from control values, while ALK-1 levels remained unchanged.
Conclusions
These alterations in the TGF-β pathway suggest a mechanism by which primary signaling is switched from a TGF-βRI/Smad2-dependent response, to an ALK-1/Smad1/5/8 response, representing a significant change in signaling outcome which may enhance matrix degradation.
doi:10.1159/000127437
PMCID: PMC2574785  PMID: 18434745
TGF-β; aneurysm; signal transduction; extracellular matrix; remodeling

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