Heart valve regeneration.

The valves of the heart cannot regenerate spontaneously. Therefore, heart valve disease generally necessitates surgical repair or replacement of the diseased tissue by mechanical or bioprosthetic valve substitutes in order to avoid potentially fatal cardiac or systemic consequences. Although survival and quality of life is enhanced for many patients treated surgically, currently available valve substitutes remain imperfect. This is especially the case in pediatric applications, where physiologically corrective procedures can be successfully performed, but reoperations are frequently required to replace failed valve substitutes or accommodate growth of the patient. While much work is currently underway to incrementally improve existing valve substitutes, a major impact will require radically new technologies, including tissue engineering or regeneration. The use of engineered tissue offers the potential to create a non-obstructive, non-thrombogenic tissue valve substitute containing living cells capable of providing ongoing remodeling and repair of cumulative injury to the extracellular matrix. Ideally, this would allow growth in maturing recipients. The innovative fabrication of materials and the development of sophisticated methods to repair or regenerate damaged or diseased heart valves requires integration of a diverse array of basic scientific principles and enabling technologies. Thus, heart valve tissue engineering requires an understanding of relationships of structure to function in normal and pathological valves (including mechanisms of embryological development, tissue repair and functional biomechanics), and the ability to control cell and tissue responses to injury, physical stimuli and biomaterial surfaces, through chemical, pharmacological, mechanical and potentially genetic manipulations. These approaches created by advances in cell biology raise exciting possibilities for in situ regeneration and repair of heart valves.

Dynamic and reversible changes of interstitial cell phenotype during remodeling of cardiac valves.

BACKGROUND AND AIM OF THE STUDY: The roles of cardiac valvular interstitial cells (VIC) in extracellular matrix remodeling in fetal development, adaptation and response to injury are largely unknown. METHODS: The phenotype of VIC was studied in health (normal adult human and sheep), development (fetal human and sheep), disease (human mitral valves with myxomatous degeneration), adaptation (clinical pulmonary to aortic valve autografts) and tissue-engineered heart valves matured in vitro and remodeled in vivo. Cell phenotype was assessed using expression of vimentin (V), alpha-smooth muscle actin (SMA, A), matrix metalloproteinase (MMP)-13/collagenase-3 (M), and SMemb (S). RESULTS: VIC in normal adult valves were predominantly quiescent fibroblasts immunoreactive to vimentin (89.7 +/- 2.5%), but not MMP-13 or SMemb, with only 2.5 +/- 0.4% of alpha-SMA-positive cells ('normal/quiescent' phenotype: V+/A-/M-/S-). In contrast, fetal VIC were mostly activated myofibroblasts ('developing/activated' phenotype: V+/A+/M+/S+), with 62.1 +/- 5.0% of cells staining positive for alpha-SMA. VIC in myxomatous valves, short-term autografts and engineered valves in vitro were also activated myofibroblasts with coexpression of vimentin, alpha-SMA (36.2 +/- 3.7%, 19.3 +/- 2.4%, and 60.3 +/- 9% positive cells, respectively), strong MMP-13 activity indicative of collagen remodeling, and SMemb ('remodeling/activated' phenotype: V+/A+/M+/S+). In contrast, VIC in long-term pulmonary autografts and engineered valve explants had a mostly fibroblast-like phenotype, with sparse alpha-SMA expression (6.0 +/- 1% and 5.4 +/- 1.0% positive cells) (V+/A-/M-/S-). CONCLUSION: Most VIC in normal valves were quiescent with a fibroblast-like phenotype. VIC in developing, diseased, adapting and engineered valves adjust to a dynamic environment through VIC activation and secretion of proteolytic enzymes mediating extracellular matrix remodeling ('developing/ remodeling/activated' phenotype), followed by a normalization of phenotype.

Clinical pulmonary autograft valves: pathologic evidence of adaptive remodeling in the aortic site.

OBJECTIVE: We studied the pathologic features, cellular phenotypes, and matrix remodeling of clinical pulmonary-to-aortic valve transplants functioning up to 6 years. METHODS: Nine autografts and associated vascular walls early (2-10 weeks) and late (3-6 years) postoperatively were examined by using routine morphologic methods and immunohistochemistry. In 4 cases autograft and homograft cusps were obtained from the same patients. RESULTS: Autografts had near-normal trilaminar cuspal structure and collagen architecture and viable valvular interstitial and endothelial cells throughout the time course. In contrast, cusps of homografts used to replace the pulmonary valves in the same patients were devitalized. In early autograft explants, 19.3% +/- 2.4% of cuspal interstitial cells were myofibroblasts expressing alpha-actin. In contrast, myofibroblasts comprised only 6.0% +/- 1.1% of cells in late explants and 2.5% +/- 0.4% and 4.6% +/- 0.8% of cells in normal pulmonary and aortic valves, respectively (P <.05). In early autografts only 12.0% +/- 4.6% of endothelial cells expressed the systemic arterial endothelial cell marker EphrinB2, whereas later explants had 85.6% +/- 5.4% of endothelial cells expressing EphrinB2 (P <.05). In early autografts 43.8% +/- 8.8% of interstitial cells expressed metalloproteinase 13, whereas late autografts had 11.4% +/- 2.7% of interstitial cells expressing matrix metalloproteinase 13 (P <.05). Collagen content in autografts was comparable with that of normal valves and was higher than that seen in homograft valves (P <.005). However, autograft walls were damaged, with granulation tissue (early) and scarring, with focal loss of normal smooth muscle cells, elastin, and collagen (late). CONCLUSIONS: The structure of pulmonary valves transplanted to the systemic circulation evolved toward that of normal aortic valves. Key processes in this remodeling included onset of a systemic endothelial cell phenotype and reversible plasticity of fibroblast-like valvular interstitial cells to myofibroblasts.

Genetically determined resistance to collagenase action augments interstitial collagen accumulation in atherosclerotic plaques.

BACKGROUND: We hypothesized that collagenolytic activity produced by activated macrophages contributes to collagen loss and the subsequent instability of atheromatous lesions, a common trigger of acute coronary syndromes. However, no direct in vivo evidence links collagenases with the regulation of collagen content in atherosclerotic plaques. METHODS AND RESULTS: To test the hypothesis that collagenases influence the structure of atheromata, we examined collagen accumulation in atherosclerotic lesions of apolipoprotein E-deficient mice (apoE-/-) that express collagenase-resistant collagen-I (ColR/R/apoE-/-, n=12) or wild-type collagen-expressing mice (Col+/+/apoE-/-, n=12). Aortic atheromata of both groups had similar sizes and numbers of macrophages, a major source of collagenases. However, aortic intimas from ColR/R/apoE-/- mice contained fewer smooth muscle cells, a source of collagen, probably because of decreased migration or proliferation or increased cell death. Despite reduced numbers of smooth muscle cells, atheromata of ColR/R/apoE-/- mice contained significantly more intimal collagen than did those of Col+/+/apoE-/- mice. CONCLUSIONS: These results establish that collagenase action regulates plaque collagen turnover and smooth muscle cell accumulation.

Tissue-engineered microvessels on three-dimensional biodegradable scaffolds using human endothelial progenitor cells.

Tissue engineering may offer patients new options when replacement or repair of an organ is needed. However, most tissues will require a microvascular network to supply oxygen and nutrients. One strategy for creating a microvascular network would be promotion of vasculogenesis in situ by seeding vascular progenitor cells within the biopolymeric construct. To pursue this strategy, we isolated CD34(+)/CD133(+) endothelial progenitor cells (EPC) from human umbilical cord blood and expanded the cells ex vivo as EPC-derived endothelial cells (EC). The EPC lost expression of the stem cell marker CD133 but continued to express the endothelial markers KDR/VEGF-R2, VE-cadherin, CD31, von Willebrand factor, and E-selectin. The cells were also shown to mediate calcium-dependent adhesion of HL-60 cells, a human promyelocytic leukemia cell line, providing evidence for a proinflammatory endothelial phenotype. The EPC-derived EC maintained this endothelial phenotype when expanded in roller bottles and subsequently seeded on polyglycolic acid-poly-l-lactic acid (PGA-PLLA) scaffolds, but microvessel formation was not observed. In contrast, EPC-derived EC seeded with human smooth muscle cells formed capillary-like structures throughout the scaffold (76.5 +/- 35 microvessels/mm(2)). These results indicate that 1) EPC-derived EC can be expanded in vitro and seeded on biodegradable scaffolds with preservation of endothelial phenotype and 2) EPC-derived EC seeded with human smooth muscle cells form microvessels on porous PGA-PLLA scaffolds. These properties indicate that EPC may be well suited for creating microvascular networks within tissue-engineered constructs.