H.E. Mewhort

Valve-Related Hemodynamics Mediate Human Bicuspid Aortopathy: Insights From Wall Shear Stress Mapping

BACKGROUND Suspected genetic causes for extracellular matrix (ECM) dysregulation in the ascending aorta in patients with bicuspid aortic valves (BAV) have influenced strategies and thresholds for surgical resection of BAV aortopathy. Using 4-dimensional (4D) flow cardiac magnetic resonance imaging (CMR), we have documented increased regional wall shear stress (WSS) in the ascending aorta of BAV patients. OBJECTIVES This study assessed the relationship between WSS and regional aortic tissue remodeling in BAV patients to determine the influence of regional WSS on the expression of ECM dysregulation. METHODS BAV patients (n = 20) undergoing ascending aortic resection underwent pre-operative 4D flow CMR to regionally map WSS. Paired aortic wall samples (i.e., within-patient samples obtained from regions of elevated and normal WSS) were collected and compared for medial elastin degeneration by histology and ECM regulation by protein expression. RESULTS Regions of increased WSS showed greater medial elastin degradation compared to adjacent areas with normal WSS: decreased total elastin (p = 0.01) with thinner fibers (p = 0.00007) that were farther apart (p = 0.001). Multiplex protein analyses of ECM regulatory molecules revealed an increase in transforming growth factor β-1 (p = 0.04), matrix metalloproteinase (MMP)-1 (p = 0.03), MMP-2 (p = 0.06), MMP-3 (p = 0.02), and tissue inhibitor of metalloproteinase-1 (p = 0.04) in elevated WSS regions, indicating ECM dysregulation in regions of high WSS. CONCLUSIONS Regions of increased WSS correspond with ECM dysregulation and elastic fiber degeneration in the ascending aorta of BAV patients, implicating valve-related hemodynamics as a contributing factor in the development of aortopathy. Further study to validate the use of 4D flow CMR as a noninvasive biomarker of disease progression and its ability to individualize resection strategies is warranted.

Monocytes Increase Human Cardiac Myofibroblast-Mediated Extracellular Matrix Remodeling Through TGF-β1

Following myocardial infarction (MI), cardiac myofibroblasts remodel the extracellular matrix (ECM), preventing mechanical complications. However, prolonged myofibroblast activity leads to dysregulation of the ECM, maladaptive remodeling, fibrosis, and heart failure (HF). Chronic inflammation is believed to drive persistent myofibroblast activity; however, the mechanisms are unclear. We assessed the influence of peripheral blood monocytes on human cardiac myofibroblast activity in a three-dimensional (3D) ECM microenvironment. Human cardiac myofibroblasts isolated from surgical biopsies of the right atrium and left ventricle were seeded into 3D collagen matrices. Peripheral blood monocytes were isolated from healthy human donors and cocultured with myofibroblasts. Monocytes increased myofibroblast activity measured by collagen gel contraction (baseline: 57.6 ± 5.9% vs. coculture: 65.2 ± 7.1% contraction; P < 0.01) and increased local ECM remodeling quantified by confocal microscopy. Under coculture conditions that allow indirect cellular interaction via paracrine factors but prevent direct cell-cell contact, monocytes had minimal effects on myofibroblast activity (17.9 ± 11.1% vs. 6.4 ± 7.0% increase, respectively; P < 0.01). When cells were cultured under direct contact conditions, multiplex analysis of the coculture media revealed an increase in the paracrine factors TGF-β1 and matrix metalloproteinase 9 compared with baseline (122.9 ± 10.1 pg/ml and 3,496.0 ± 190.4 pg/ml, respectively, vs. 21.5 ± 16.3 pg/ml and 183.3 ± 43.9 pg/ml; P < 0.001). TGF-β blockade abolished the monocyte-induced increase in cardiac myofibroblast activity. These data suggest that direct cell-cell interaction between monocytes and cardiac myofibroblasts stimulates TGF-β-mediated myofibroblast activity and increases remodeling of local matrix. Peripheral blood monocyte interaction with human cardiac myofibroblasts stimulates myofibroblast activity through release of TGF-β1. These data implicate inflammation as a potential driver of cardiac fibrosis.

Tetrandrine reverses human cardiac myofibroblast activation and myocardial fibrosis

Tetrandrine (TTD) is a calcium channel blocker with documented antifibrotic actions. In this study, for the first time, we identified that TTD can directly prevent in vitro human cardiac myofibroblast activation and limit in vivo myocardial fibrosis. In vitro, cardiac myofibroblasts from human atrial biopsies (N = 10) were seeded in three-dimensional collagen matrices. Cell-collagen constructs were exposed to transforming growth factor-β1 (10 ng/ml), with or without TTD (1 and 5 μM) for 48 h. Collagen gel contraction, myofibroblast activation (α-smooth muscle actin expression), expression of profibrotic mRNAs, and rate of collagen protein synthesis were compared. TTD decreased collagen gel contraction (79.7 ± 1.3 vs 60.1 ± 8.9%, P < 0.01), α-smooth muscle actin expression (flow cytometry), collagen synthesis ([(3)H]proline incorporation), and collagen mRNA expression. Cell viability was similar between groups (annexin positive cells: 1.7 vs. 1.4%). TTD inhibited collagen gel contraction in the presence of T-type and L-type calcium channel blockers, and the intracellular calcium chelator BAPTA-AM (15 μM), suggesting that the observed effects are not mediated by calcium homeostasis. In vivo, Dahl salt-sensitive hypertensive rats were treated with variable doses of TTD (by intraperitoneal injection over 4 wk) and compared with untreated controls (N = 12). Systemic blood pressure was monitored by tail cuff. Myocardial fibrosis and left ventricular compliance were assessed by histology and passive pressure-volume analysis. Myocardial fibrosis was attenuated compared with untreated controls (%collagen area: 9.4 ± 7.3 vs 2.1 ± 1.0%, P < 0.01). Left ventricular compliance was preserved. In conclusion, TTD reverses human cardiac myofibroblast activation and myocardial fibrosis, independent of calcium channel blockade.

SURGICAL APPLICATION OF A NOVEL BIOMATERIAL ENHANCES POST-MI FUNCTIONAL RECOVERY BY STIMULATING VASCULOGENESIS THROUGH AN ACTIVE BIO-INDUCTIVE MECHANISM

BACKGROUND: Epicardial infarct repair (EIR) using a bioinductive extracellular matrix (ECM) biomaterial is a novel surgical approach that targets the infarcted myocardium to enhance myocardial repair and prevent ischemic HF. We previously demonstrated that EIR prevents maladaptive LV remodeling and improves post-MI functional recovery. It is unclear if these benefits are a consequence of passive biomechanical infarct restraint or secondary to active paracrine signals from within the biomaterial (bio-inductive effect). In this study we outline the mechanisms by which EIR enhances myocardial repair following ischemic injury. METHODS AND RESULTS: Epicardial infarct repair performed using a biologically active ECM-biomaterial (CorMatrix-ECM, CorMatrix Cardiovascular Inc., GA, USA) was compared to EIR performed using the same ECM-biomaterial biologically inactivated by gluteraldehyde fixation. The active (N1⁄44) or inactive (N1⁄46) ECM-biomaterial was surgically applied to the epicardial surface of the infarcted myocardium following permanent coronary artery ligation in a rat model. Using a conductance catheter, indices of cardiac performance were quantified by pressure volume loop analysis 14-weeks posttreatment. EIR with either biomaterial resulted in functional recovery above sham-treated (N1⁄44) animals (ejection fraction: 42.43 13.09% vs. 25.15 6.99%, respectively; P1⁄40.035), however we observed a trend toward greater functional improvement in active EIR-treated animals as compared to inactive EIR-treated animals (ejection fraction: 47.13 15.45% vs. 30.50 13.40%, respectively; P1⁄40.16; stroke work: 10618 1548mmHg*uL vs. 8148 2514mmHg*uL, respectively; P1⁄40.17). Increased vascularity was observed within the infarcted myocardium of active EIR-treated animals as compared to inactive EIR-treated animals (11.8 5.6 vs. 3.7 3.9 blood vessels per high power field, respectively; P1⁄40.0008) potentially resulting in increased microvascular perfusion accounting for the functional recovery observed. Increased epicardial thickness, indicative of epicardial activation, was also observed in active EIR-treated animals as compared to inactive EIR-treated animals (4.6 1.6 vs. 2.9 0.7 fold increase relative to sham-treated animals, respectively; P1⁄40.0012). An increase in the number of epicardium derived progenerator cells (EDPCs), identified by Wilms’ tumor-1 expression and nuclearization of b-catenin was also observed in active EIR-treated animals, specifically in the infarcted myocardium indicative of increased epithelial to mesenchymal transition. Vascular structures positive for both nuclear b-catenin and a-SMA identified within the infarcted myocardium of active biomaterial-treated animals indicate that EDPCs undergo EMT and differentiate into vascular smooth muscle cells (VSMCs) through vasculogenesis (Figure). CONCLUSION: These data suggest that EIR with a biologically active biomaterial enhances myocardial repair through a bio-inductive mechanism beyond passive infarct restraint. The epicardial application of ECM-biomaterial stimulates epicardial progenitor cell mobilization and increases vasculogenesis to enhance functional recovery post-MI.