From stem cells to viable autologous semilunar heart valve.

BACKGROUND: An estimated 275,000 patients undergo heart valve replacement each year. However, existing solutions for valve replacement are complicated by the morbidity associated with lifelong anticoagulation of mechanical valves and the limited durability of bioprostheses. Recent advances in tissue engineering and our understanding of stem cell biology may provide a lifelong solution to these problems. METHODS AND RESULTS: Mesenchymal stem cells were isolated from ovine bone marrow and characterized by their morphology and antigen expression through immunocytochemistry, flow cytometry, and capacity to differentiate into multiple cell lineages. A biodegradable scaffold was developed and characterized by its tensile strength and stiffness as a function of time in cell-conditioned medium. Autologous semilunar heart valves were then created in vitro using mesenchymal stem cells and the biodegradable scaffold and were implanted into the pulmonary position of sheep on cardiopulmonary bypass. The valves were evaluated by echocardiography at implantation and after 4 months in vivo. Valves were explanted at 4 and 8 months and examined by histology and immunohistochemistry. Valves displayed a maximum instantaneous gradient of 17.2+/-1.33 mm Hg, a mean gradient of 9.7+/-1.3 mm Hg, an effective orifice area of 1.35+/-0.17 cm2, and trivial or mild regurgitation at implantation. Gradients changed little over 4 months of follow-up. Histology showed disposition of extracellular matrix and distribution of cell phenotypes in the engineered valves reminiscent of that in native pulmonary valves. CONCLUSIONS: Stem-cell tissue-engineered heart valves can be created from mesenchymal stem cells in combination with a biodegradable scaffold and function satisfactorily in vivo for periods of >4 months. Furthermore, such valves undergo extensive remodeling in vivo to resemble native heart valves.

The independent role of cyclic flexure in the early in vitro development of an engineered heart valve tissue.

Tissue engineered heart valves (TEHV) are being investigated as an alternative to current non-viable prosthetic valves and valved conduits. Studies suggest that pulse duplicator bioreactors can stimulate TEHV development. In the current study, a model system was used to determine if cyclic flexure, a major mode of heart valve deformation, has independent effects on TEHV cell and extracellular matrix (ECM) development. Ovine vascular smooth muscle cells (SMC) were seeded for 30 h onto strips of non-woven 50:50 polyglycolic acid (PGA) and poly-L-lactic acid (PLLA) scaffold. After 4 days of incubation, SMC-seeded and unseeded scaffolds were either maintained under static conditions (static group), or subjected to unidirectional cyclic three-point flexure at a physiological frequency and amplitude in a bioreactor (flex group) for 3 weeks. After seeding or incubation, the effective stiffness (E) was measured, with SMC-seeded scaffolds further characterized by DNA, collagen, sulfated glycosaminoglycan (S-GAG), and elastin content, as well as by histology. The seeding period was over 90% efficient, with a significant accumulation of S-GAG, no significant change in E, and no collagen detected. Following 3 weeks of incubation, unseeded scaffolds exhibited no significant change in E in the flex or static groups. In contrast, E of SMC-seeded scaffolds increased 429% in the flex group (p<0.01) and 351% in the static group (p<0.01), with a trend of increased E, a 63% increase in collagen (p<0.05), increased vimentin expression, and a more homogenous transmural cell distribution in the flex versus static group. Moreover, a positive linear relationship (r2=0.996) was found between the mean E and mean collagen concentration. These results show that cyclic flexure can have independent effects on TEHV cell and ECM development, and may be useful in predicting the mechanical properties of TEHV constructed using novel scaffold materials.

Molecular basis of cell membrane estrogen receptor interaction with phosphatidylinositol 3-kinase in endothelial cells.

OBJECTIVE: Nontranscriptional signaling mechanisms mediate some of the biological effects of estrogen, such as the rapid actions on the blood vessels. By interacting with phosphatidylinositol 3-kinase (PI3K), estrogen receptor (ER) alpha leads to activation of protein kinase Akt and to subsequent increase in endothelial nitric oxide synthase activity. Because PI3K is mainly a cytoplasmic complex, we studied the cellular site of interaction between this enzyme and ERalpha, and we dissected the molecular mechanisms that mediate this interaction. METHODS AND RESULTS: By using cultured human saphenous vain endothelial cells, we found that cell membrane-bound ERalpha colocalizes with PI3K and may be responsible for PI3K activation. Furthermore, we characterized the subsequent steps in the activation of the PI3K/Akt signaling cascade, comparing the molecular events that follow insulin or estradiol activation of PI3K. CONCLUSIONS: We provide novel evidence for an important role of nonnuclear estrogen receptor in rapid, nontranscriptional responses of human endothelial cells to estrogen.

NFATc1 mediates vascular endothelial growth factor-induced proliferation of human pulmonary valve endothelial cells.

Mice deficient for the transcription factor NFATc1 fail to form pulmonary and aortic valves, a defect reminiscent of some types of congenital human heart disease. We examined the mechanisms by which NFATc1 is activated and translocated to the nucleus in human pulmonary valve endothelial cells to gain a better understanding of its potential role(s) in post-natal valvular repair as well as valve development. Herein we demonstrate that activation of NFATc1 in human pulmonary valve endothelial cells is specific to vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2. VEGF-induced NFATc1 nuclear translocation was inhibited by either cyclosporin A or a calcineurin-specific peptide inhibitor; these findings suggest that VEGF stimulates NFATc1 nuclear import in human pulmonary valve endothelial cells by a calcineurin-dependent mechanism. Importantly, both cyclosporin A and the calcineurin-specific peptide inhibitor reduced VEGF-induced human pulmonary valve endothelial cell proliferation, indicating a functional role for NFATc1 in endothelial growth. In contrast, VEGF-induced proliferation of human dermal microvascular and human umbilical vein endothelial cells was not sensitive to cyclosporin A. Finally, NFATc1 was detected in the endothelium of human pulmonary valve leaflets by immunohistochemistry. These results suggest VEGF-induced NFATc1 activation may be an important mechanism in cardiac valve maintenance and function by enhancing endothelial proliferation.

Lipid lowering reduces oxidative stress and endothelial cell activation in rabbit atheroma.

BACKGROUND: Lipid lowering may reduce acute coronary events in patients in part by reducing vascular inflammation. Oxidative stress induces endothelial cell (EC) expression of vascular cell adhesion molecule 1 (VCAM-1) and monocyte chemoattractant protein 1 (MCP-1) and reduces levels of atheroprotective NO, leading to monocyte recruitment and macrophage accumulation. This study tested the hypothesis that lipid lowering decreases oxidative stress and improves EC functions related to inflammatory cell accumulation. METHODS AND RESULTS: Rabbits consumed an atherogenic diet for 4 months to produce atheroma, followed by a purified chow diet for 16 months. Atherosclerotic aortas from hypercholesterolemic rabbits produced high levels of reactive oxygen species. Oxidized LDL (oxLDL) accumulated in atheroma underlying ECs that overexpress VCAM-1. In contrast, few if any ECs in atheroma stained for endothelial NO synthase (eNOS). Lipid lowering reduced reactive oxygen species production, oxLDL accumulation, and plasma levels of anti-oxLDL IgG. After lipid lowering, VCAM-1 and MCP-1 expression decreased, eNOS expression increased, and ECs exhibited a more normal ultrastructure. CONCLUSIONS: These results establish that lipid lowering can reduce oxidative stress and EC activation in vivo. These mechanisms may contribute to improvement in endothelial function and plaque stabilization observed clinically.

Evolution of cell phenotype and extracellular matrix in tissue-engineered heart valves during in-vitro maturation and in-vivo remodeling.

BACKGROUND AND AIM OF THE STUDY: Contemporary tissue valves are non-viable, and unable to grow, repair or remodel. It was postulated that tissue-engineered heart valves (TEHV) fabricated from autologous cells and a biodegradable scaffold could yield a dynamic progression of cell phenotype and extracellular matrix (ECM), in vitro and in vivo, and ultimately recapitulate native valve microscopic architecture. METHODS: Trileaflet valve constructs were fabricated from poly-4-hydroxybutyrate-coated polyglycolic acid seeded with ovine endothelial and carotid artery medial cells, cultured in vitro for 4-14 days in a pulse duplicator, implanted as pulmonary valves in five lambs, and explanted at 4-20 weeks. ECM composition and collagen architecture were examined by histology (including Movat pentachrome stain and picrosirius red under polarized light), and cell phenotype by immunohistochemistry. RESULTS: Cells from in-vitro constructs (14 days) were activated myofibroblasts, with strong expression of alpha-actin (microfilaments), vimentin (intermediate filaments) and SMemb (non-muscle myosin produced by activated mesenchymal cells). Cells from in-vivo explants at 16-20 weeks were fibroblast-like, with predominant vimentin expression and undetectable levels of alpha-actin (similar to native valve). Collagen elaboration and cellular expression of MMP-13 (collagenase 3) were evident in vitro at 14 days. In-vivo explants had increased collagen accumulation and strong MMP-13 expression at 4-8 weeks, but less activation (decreased expression of SMemb) and patchy endothelial cells at 16-20 weeks. Moreover, the ECM architecture of 16- to 20-week explanted TEHV resembled that of native valves. CONCLUSION: Cell phenotype and ECM in TEHV prepared in vitro and implanted in vivo are dynamic, and reflect the ability of a vital tissue to remodel and, potentially, to grow.

Selective matrix metalloproteinase inhibition reduces left ventricular remodeling but does not inhibit angiogenesis after myocardial infarction.

BACKGROUND: Broad inhibition of matrix metalloproteinases (MMPs) attenuates left ventricular remodeling after myocardial infarction (MI). However, it is not clear if selective MMP inhibition strategies will be effective or if MMP inhibition will impair angiogenesis after MI. METHODS AND RESULTS: We used a selective MMP inhibitor (MMPi) that does not inhibit MMP-1 in rabbits, which, like humans but unlike rodents, express MMP-1 as a major collagenase. On day 1 after MI, rabbits were randomized to receive either inhibitor (n=10) or vehicle (n=8). At 4 weeks after MI, there were no differences in infarct size or collagen fractional area. However, MMPi reduced ventricular dilation. The increase in end-diastolic dimension from day 1 to week 4 was 3.1+/-0.5 mm for vehicle versus 1.3+/-0.3 mm for MMPi (P<0.01). The increase in end-systolic dimension was 2.8+/-0.5 mm for vehicle and 1.3+/-0.4 mm for MMPi (P<0.05). Furthermore, MMPi reduced infarct wall thinning; the minimal infarct thickness was 0.8+/-0.1 mm for vehicle and 1.6+/-0.3 mm for MMPi (P<0.05). Interestingly, the MMPi group had increased numbers of vessels in the subendocardial layer of the infarct; the number of capillaries was increased in the subendocardial layer (46+/-4 vessels/field versus 17+/-3 vessels/field for vehicle; P<0.001), and the number of arterioles was also increased (4.0+/-0.8 vessels/field versus 2.0+/-0.4 vessels/field for vehicle; P<0.05). CONCLUSIONS: MMP inhibition attenuates left ventricular remodeling even when the dominant collagenase MMP-1 is not inhibited; furthermore, this selective MMP inhibition appears to increase rather than decrease neovascularization in the subendocardium.