Controlling matrix formation and cross-linking by hypoxia in cardiovascular tissue engineering

Abstract

In vivo functionality of cardiovascular tissue engineered constructs requires in vitro control of tissue development to obtain a well developed extracellular matrix (ECM). We hypothesize that ECM formation and maturation is stimulated by culturing at low oxygen concentrations. Gene expression levels of monolayers of human vascular-derived myofibroblasts, exposed to 7, 4, 2, 1, and 0.5% O(2) (n = 9 per group) for 24 h, were measured for vascular endothelial growth factor (VEGF), procollagen α1(I) and α1(III), elastin, and cross-link enzymes lysyl oxidase (LOX) and lysyl hydroxylase 2 (LH2). After 4 days of exposure to 7, 2, and 0.5% O(2) (n = 3 per group), protein synthesis was evaluated. All analyses were compared with control cultures at 21% O(2). Human myofibroblasts turned to hypoxia-driven gene expression, indicated by VEGF expression, at oxygen concentrations of 4% and lower. Gene expression levels of procollagen α1(I) and α1(III) increased to 138 ± 26 and 143 ± 19%, respectively, for all oxygen concentrations below 4%. At 2% O(2), LH2 and LOX gene expression levels were higher than control cultures (340 ± 53 and 136 ± 29%, respectively), and these levels increased even further with decreasing oxygen concentrations (611 ± 176 and 228 ± 45%, respectively, at 0.5% O(2)). Elastin gene expression levels remained unaffected. Collagen synthesis and LH2 protein levels increased at oxygen concentrations of 2% and lower. Oxygen concentrations below 4% induce enhanced ECM production by human myofibroblasts. Implementation of these results in cardiovascular tissue engineering approaches enables in vitro control of tissue development.

Low Oxygen Concentrations Impair Tissue Development in Tissue-Engineered Cardiovascular Constructs

“…The primer sequences have been previously described. 13 Evaluation of the tissue culture environment…”

“…12 To investigate whether culturing at even lower oxygen concentrations could further improve tissue formation, we have recently investigated the effect of culturing at oxygen concentrations of 7% and lower on human vascular-derived cells. 13 At gene expression level, oxygen levels below 4% O 2 were beneficial for collagen type I and III, and the cross-link enzymes lysyl oxidase (LOX) and lysyl hydroxylase 2 (LH2). At protein level, overall collagen synthesis and LH2 protein amount increased at 2% and 0.5% O 2 compared with 21% O 2 .…”

“…Based on the two-dimensional (2D) cell study, we expect cells exposed to oxygen concentrations below 4% to increase collagen and collagen cross-link production. 13 To translate cell to tissue level, a finite element model has been developed in which the oxygen gradient within the tissues was determined. For the experimental work, tissue-engineered (TE) cardiovascular constructs derived from human vascular-derived cells seeded onto a rapid degrading scaffold were cultured at 7%, 4%, 2%, and 0.5% O 2 and compared with control cultures at 21% O 2 at gene expression and tissue level.…”

Central stiffening in adulthood linked to aberrant aortic remodeling under suboptimal intrauterine conditions

“…In fact, central arterial stiffening due to altered composition of the ECM is a strong and independent predictor of CVD (1,7). Given that elastin content is fixed upon conclusion of developmental remodeling (28,40) and that fetal hypoxemia alters known regulators of ECM deposition (15,44), permanent central arterial stiffening may arise from aberrant arterial formation in utero in fetuses growing under placental insufficiency.…”

Cardioprotection via adaptation to hypoxia: expanding the timeline and targets?

“…Hypoxia has been shown to stimulate collagen synthesis by cultured human vascular-derived myofibroblasts [24]. In contrast, the effects on matrix metalloproteinases have been mixed: several studies reported increases in MMP-2 synthesis in cultured rodent fibroblasts [1,13], whereas exposure of cultured human myofibroblasts to chronic hypoxia attenuated MMP-2 activation [20].…”