Virna L. Sales

THE ROLE OF ORGAN LEVEL CONDITIONING ON THE PROMOTION OF ENGINEERED HEART VALVE TISSUE DEVELOPMENT IN-VITRO USING MESENCHYMAL STEM CELLS

We have previously shown that combined flexure and flow (CFF) augment engineered heart valve tissue formation using bone marrow-derived mesenchymal stem cells (MSC) seeded on polyglycolic acid (PGA)/poly-L-lactic acid (PLLA) blend nonwoven fibrous scaffolds (Engelmayr, et al., Biomaterials 2006; vol. 27 pp. 6083-95). In the present study, we sought to determine if these phenomena were reproducible at the organ level in a functional tri-leaflet valve. Tissue engineered valve constructs (TEVC) were fabricated using PGA/PLLA nonwoven fibrous scaffolds then seeded with MSCs. Tissue formation rates using both standard and augmented (using basic fibroblast growth factor [bFGF] and ascorbic acid-2-phosphate [AA2P]) media to enhance the overall production of collagen were evaluated, along with their relation to the local fluid flow fields. The resulting TEVCs were statically cultured for 3 weeks, followed by a 3 week dynamic culture period using our organ level bioreactor (Hildebrand et al., ABME, Vol. 32, pp. 1039-49, 2004) under approximated pulmonary artery conditions. Results indicated that supplemented media accelerated collagen formation (approximately 185% increase in collagen mass/MSC compared to standard media), as well as increasing collagen mass production from 3.90 to 4.43 pg/cell/week from 3 to 6 weeks. Using augmented media, dynamic conditioning increased collagen mass production rate from 7.23 to 13.65 pg/cell/week (88.8%) during the dynamic culture period, along with greater preservation of net DNA. Moreover, when compared to our previous CFF study, organ level conditioning increased the collagen production rate from 4.76 to 6.42 pg/cell/week (35%). Newly conducted CFD studies of the CFF specimen flow patterns suggested that oscillatory surface shear stresses were surprisingly similar to a tri-leaflet valve. Overall, we found that the use of simulated pulmonary artery conditions resulted in substantially larger collagen mass production levels and rates found in our earlier CFF study. Moreover, given the fact that the scaffolds underwent modest strains (approximately 7% max) during either CFF or physiological conditioning, the oscillatory surface shear stresses estimated in both studies may play a substantial role in eliciting MSC collagen production in the highly dynamic engineered heart valve fluid mechanical environment.

Angiotensin Type 2 Receptor Is Expressed in Murine Atherosclerotic Lesions and Modulates Lesion Evolution

Background— In the vasculature, the angiotensin type 2 (AT2) receptor (AT2R) exerts antiproliferative, antifibrotic, and proapoptotic effects. Normal adult animals have low AT2R expression; however, vascular injury and exposure to proinflammatory cytokines augment AT2R levels. We hypothesized that AT2R expression increases during initiation and progression of atherosclerosis. Methods and Results— Atherosclerotic lesions of apolipoprotein (Apo) E−/− mice contained AT2Rs, measured by real-time polymerase chain reaction and confirmed by immunohistochemistry. To test the consequences of this expression, male ApoE−/−, angiotensin II type 2 receptor-deficient (Agtr2−), and ApoE−/−, wild-type (Agtr2+) mice consumed a high-cholesterol diet from 4 weeks of age. Ten weeks later, overall area and cellular composition of aortic arch lesions did not differ significantly among genotypes. After 16 weeks, ApoE−/−/Agtr2+, but not ApoE−/−/Agtr2− mice had dramatic decreases in percent positive area of macrophages, smooth muscles, lipids, and collagen. Diminished bromodeoxyuridine incorporation and increased TUNEL staining accompanied these decreases. Conclusions— Thus, loss of AT2R during the evolution of atherosclerotic lesions augmented the extent of cellularity of atherosclerotic lesions, establishing AT2R as a modulator of atherogenesis.

Protein Precoating of Elastomeric Tissue-Engineering Scaffolds Increased Cellularity, Enhanced Extracellular Matrix Protein Production, and Differentially Regulated the Phenotypes of Circulating Endothelial Progenitor Cells

Background— Optimal cell sources and scaffold-cell interactions remain unanswered questions for tissue engineering of heart valves. We assessed the effect of different protein precoatings on a single scaffold type (elastomeric poly (glycerol sebacate)) with a single cell source (endothelial progenitor cells). Methods and Results— Elastomeric poly (glycerol sebacate) scaffolds were precoated with laminin, fibronectin, fibrin, collagen types I/III, or elastin. Characterized ovine peripheral blood endothelial progenitor cells were seeded onto scaffolds for 3 days followed by 14 days incubation. Endothelial progenitor cells were CD31+, vWF+, and α-SMA- before seeding confirmed by immunohistochemistry and immunoblotting. Both precoated and uncoated scaffolds demonstrated surface expression of CD31+ and vWF+, α-SMA+ cells and were found in the “interstitium” of the scaffold. Protein precoating of elastomeric poly (glycerol sebacate) scaffolds revealed significantly increased cellularity and altered the phenotypes of endothelial progenitor cells, which resulted in changes in cellular behavior and extracellular matrix production. Moreover, mechanical flexure testing demonstrated decreased effective stiffness of the seeded scaffolds compared with unseeded controls. Conclusions— Scaffold precoating with extracellular matrix proteins can allow more precise “engineering” of cellular behavior in the development of tissue engineering of heart valves constructs by altering extracellular matrix production and cell phenotype.

Development of microfluidics as endothelial progenitor cell capture technology for cardiovascular tissue engineering and diagnostic medicine

We have developed a unique microfluidic platform capable of capturing circulating endothelial progenitor cells (EPCs) by understanding surface chemistries and adhesion profiles. The surface of a variable‐shear‐stress microfluidic device was conjugated with 6 different antibodies [anti‐CD34, ‐CD31, ‐vascular endothelial growth factor receptor‐2 (VEGFR‐2), ‐CD146, ‐CD45, and ‐von Willebrand factor (vWF)] designed to match the surface antigens on ovine peripheral blood‐derived EPCs. Microfluidic analysis showed a shear‐stress‐dependent decrease in EPC adhesion on attached surface antigens. EPCs exhibited increased adhesion to antibodies against CD34, VEGFR‐2, CD31, and CD146 compared to CD45, consistent with their endothelial cell‐specific surface profile, when exposed to a minimum shear stress of 1.47 dyn/cm2. Bone‐marrow‐derived mesenchymal stem cells and artery‐derived endothelial and smooth muscle cells were used to demonstrate the specificity of the EPC microfluidic device. Coated hematopoietic specific‐surface (CD45) and granular vWF antibodies, as well as uncoated bare glass and substrate (1% BSA), were utilized as controls. Microfluidic devices have been developed as an EPC capture platform using immobilized antibodies targeted as EPC surface antigens. This EPC chip may provide a new and effective tool for addressing challenges in cardiovascular disease and tissue engineering.—Plouffe, B. D., Kniazeva, T., Mayer, J. E., Jr., Murthy, S. K., Sales, V. L. Development of microfluidics as endothelial progenitor cell capture technology for cardiovascular tissue engineering and diagnostic medicine. FASEB J. 23, 3309–3314 (2009). www.fasebj.org

Transforming Growth Factor-β1 Modulates Extracellular Matrix Production, Proliferation, and Apoptosis of Endothelial Progenitor Cells in Tissue-Engineering Scaffolds

Background— Valvular endothelial cells and circulating endothelial progenitor cells (EPCs) can undergo apparent phenotypic change from endothelial to mesenchymal cell type. Here we investigated whether EPCs can promote extracellular matrix formation in tissue engineering scaffolds in response to transforming growth factor (TGF)-β1. Method and Results— Characterized ovine peripheral blood EPCs were seeded onto poly (glycolic acid)/poly (4-hydroxybutyrate) scaffolds for 5 days. After seeding at 2×106 cells/cm2, scaffolds were incubated for 5 days in a roller bottle, with or without the addition of TGF-β1. After seeding at 15×106 cells/cm2, scaffolds were incubated for 10 days in a roller bottle with or without the addition of TGF-β1 for the first 5 days. Using immunofluorescence and Western blotting, we demonstrated that EPCs initially exhibit an endothelial phenotype (ie, CD31+, von Willebrand factor+, and α–smooth muscle actin (SMA)−) and can undergo a phenotypic change toward mesenchymal transformation (ie, CD31+ and α-SMA+) in response to TGF-β1. Scanning electron microscopy and histology revealed enhanced tissue formation in EPC-TGF-β1 scaffolds. In both the 10- and 15-day experiments, EPC-TGF-β1 scaffolds exhibited a trend of increased DNA content compared with unstimulated EPC scaffolds. TGF-β1–mediated endothelial to mesenchymal transformation correlated with enhanced expression of laminin and fibronectin within scaffolds evidenced by Western blotting. Strong expression of tropoelastin was observed in response to TGF-β1 equal to that in the unstimulated EPC. In the 15-day experiments, TGF-β1–stimulated scaffolds revealed dramatically enhanced collagen production (types I and III) and incorporated more 5-bromodeoxyuridine and TUNEL staining compared with unstimulated controls. Conclusions— Stimulation of EPC-seeded tissue engineering scaffolds with TGF-β1 in vitro resulted in a more organized cellular architecture with glycoprotein, collagen, and elastin synthesis, and thus noninvasively isolated EPCs coupled with the pleiotropic actions of TGF-β1 could offer new strategies to guide tissue formation in engineered cardiac valves.

Evolving Indications for Tricuspid Valve Surgery

Opinion statementMore attention has been paid to the mitral valve (MV) than the tricuspid valve (TV), and this relative paucity of data has led to confusion regarding the timing of TV surgery. We review the American College of Cardiology/American Heart Association and European Society of Cardiology guidelines to identify areas of concordance (severe tricuspid regurgitation [TR] in a patient undergoing mitral valve surgery); discordance (less than severe TR but with markers for late TR recurrence such as pulmonary hypertension, a dilated TV annulus, atrial fibrillation, permanent transtricuspid pacing wires and others); and disagreement (surgery for primary TR). We provide our perspective from Northwestern University on these issues and where the guidelines are silent (TR in patients undergoing non-mitral valve operations). Finally, we review recent publications on the results of TV repair and replacement. Although there have been scant publications in the past, there have been more useful publications in recent years to guide our decision making.

Stem Cell–Derived, Tissue-Engineered Pulmonary Artery Augmentation Patches In Vivo

BACKGROUND Reconstruction of the right ventricular outflow tract is a frequently encountered component of many congenital cardiac repairs. We sought to tissue engineer pulmonary artery augmentation patches from retrovirally labeled endothelial progenitor and mesenchymal stem cells and determine the persistence of the seeded cells in vivo. METHODS Autologous ovine endothelial progenitor and mesenchymal stem cells were labeled with a retroviral vector encoding green and red fluorescent proteins, coseeded onto biopolymers, and cultured for 5 days. The tissue-engineered patches were implanted into the main pulmonary artery with 1, 2, 4, and 6 week in vivo maturation (n = 8). In vivo evaluation included ultrasonography and angiography, with preimplant and explanted specimens evaluated using histologic examination and immunofluorescence. RESULTS Echocardiography at each time demonstrated laminar pulmonary artery flow without a pressure gradient across the replaced segment. Pulmonary angiography did not exhibit stenosis or aneurysmal change. Gross appearance of all explanted patches showed progressive tissue formation with increased length of time in vivo. Retrovirally labeled cellular persistence was 96%, 82%, 85%, and 66% at 1, 2, 4, and 6 weeks after implantation, respectively. Early in the in vivo remodeling period, the number of green fluorescent protein-positive endothelial progenitor cells was 1.6 fold greater than the red fluorescent protein-positive mesenchymal stem cells. As in vivo remodeling continued, red fluorescent protein-expressing mesenchymal stem cells were expressed 1.2 to 1.7 times that of the green fluorescent protein-positive endothelial progenitor cells. CONCLUSIONS The data demonstrate the successful creation of an anatomically functional, autologous tissue-engineered pulmonary artery using coseeded progenitor cell sources. Labeled implanted stem cells persisted in the engineered construct, suggesting that in vitro seeding is necessary to engineer tissue. This study demonstrates an effective method to track multiple cell types after implantation.

Understanding the C-pulse device and its potential to treat heart failure.

The Sunshine Heart C-Pulse (C-Pulse; Sunshine Heart Inc., Tustin, CA) device is an extra-aortic implantable counterpulsation pump designed as a non-blood contacting ambulatory heart assist device, which may provide relief from symptoms for class II–III congestive heart failure patients. It has a comparable hemodynamic augmentation to intra-aortic balloon counterpulsation devices. The C-Pulse cuff is implanted through a median sternotomy, secured around the ascending aorta, and pneumatically driven by an external system controller. Pre-clinical studies in the acute pig model, and initial temporary clinical studies in patients undergoing off-pump coronary bypass surgery have shown substantial increase in diastolic perfusion of the coronary vessels, which translated to a favorable improvement in ventricular function. A U.S. prospective multi-center trial to evaluate the safety and efficacy of the C-Pulse in class III patients with moderate heart failure is now in progress.

Durability of Functional Tricuspid Valve Repair

Current tricuspid repair techniques have variable and disappointing durability. The authors of several studies have shown the superiority of ring (rather than suture or pericardial) annuloplasties; however, others suggest equal or superior results with suture or pericardial repair techniques. Indeed, recurrent significant tricuspid regurgitation has been reported consistently after repair; it is therefore unclear which technique provides the best long-term outcomes and in which patients. In this study, we evaluated the outcomes of different tricuspid repairs regarding durability and analyzed the risk factors for repair failure. We also presented our current approach to surgical management of functional tricuspid regurgitation on the basis of recent studies and our experience treating patients with heart failure.

Integrating principles of developmental biology in tissue engineering of heart valves

Approximately eight out of every thousand infants are born with congenital heart disease. The majority of these defects involve malformations or absence of the pulmonary valve and main pulmonary arteries. Although repair early in life is possible, the procedure often requires the use of a replacement valve. Current replacement options include prosthetic and bioprosthetic valves; however, these replacements have limited durability and are subject to calcification, thrombosis and a lack of growth potential [1]. Children, therefore, face an ongoing morbidity due to the limitations of these replacement valves and must endure multiple follow-up operations in order to place progressively larger valves to accommodate their growth. Tissue engineering offers the potential to create a living valve with the capacity for growth and self-repair, and which is resistant to infection. Tissue engineering is defined as an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain or improve function [2]. This approach is based on seeding autologous cells onto biodegradable scaffolds or decelullarized biological matrices in order to provide temporary structural support and organization until the cells synthesize their own extracellular matrix. Previous studies by our group and others have demonstrated in vivo results culminating in the creation of single-valve leaflets [3], vascular grafts [4,5] and trileaflet-valved conduits [6–11]. These tissue-engineered heart valve (TEHV) structures have been created using vascular cells [3,4,6–9], umbilical bloodderived endothelial progenitor cells [11] and noncirculating bone marrow-derived mesenchymal stem cells [12,13] as cell sources. Our group and others have also demonstrated results in preliminary in vivo experiments. However, many questions remain unanswered: • What is the optimal cell source? • How will the scaffold material influence tissue growth and allow favorable scaffold cell and extracellular matrix interactions? • What in vitro conditions provide the most cell growth? • What is the timeframe for in vivo maturation? The ideal heart valve replacement would not only be biocompatible, readily available and incredibly durable, but also have the potential for growth [14]. Although the ultimate goal of TEHV is to recapitulate the matrix and cells found in the native tissue, variabilities exist within the potential strategies and sources of cells. A widely accepted paradigm of tissue engineering comprises of a scaffold that is preseeded with cells, followed by an in vitro stage of tissue formation typically conducted in a bioreactor (that recapitulates a physiological metabolic and mechanical environment) and, following subsequent implantation of the construct, an in vivo stage of tissue growth and remodeling [15].