Hypoxia Regulates Basal and Induced DNA Synthesis and Collagen Type I Production in Human Cardiac Fibroblasts: Effects of Transforming Growth Factor-β1, Thyroid Hormone, Angiotensin II and Basic Fibroblast Growth Factor

Analysis of post-infarct ventricular remodeling consistently shows the accumulation of collagen in failing heart. The goal of this study was to gain insights into the underlying mechanisms of this event. We determined the effect of hypoxia, caused as the result of ischemia, on biological responses including cell viability, basal and growth factor-stimulated proliferative capacity and collagen type I production in cardiac fibroblasts obtained from adult human heart. The cell viability, as examined by light microscopy and analysis of DNA, did not change by hypoxia (2% oxygen). Basal level of protein synthesis, as determined by measuring the incorporation of 3H-leucine, decreased (30%, P<0.05) under hypoxia. Transforming growth factor-beta (TGF-beta1)- and thyroid hormone (T3)-induced increases in protein synthesis did not change under hypoxia. In contrast, basic fibroblast growth factor (bFGF)-stimulated protein synthesis enhanced significantly under hypoxia. Angiotensin II (Ang II)-treatment, which did not induce significant changes in protein synthesis under ambient conditions, led to moderate but significant increase under hypoxia. Basal level of DNA synthesis, as determined by measuring the incorporation of 3H-thymidine into DNA, decreased (32%, P<0.05) under hypoxia. The TGF-beta1-induced inhibition of DNA synthesis which was observed under ambient conditions was reversed [61% (P<0.005) increase under hypoxia]. Under ambient conditions, T3, Ang II and bFGF stimulated DNA synthesis and their effects were enhanced under hypoxia. Northern analysis showed a 46% (P<0.05) increase in the level of pro alpha1(l) collagen mRNA under hypoxia. The TGF-beta1-induced increase in the level of pro alpha1(l) collagen mRNA, under ambient conditions, was not observed under hypoxia. On the other hand, the T3-induced decrease in pro alpha1(l) collagen mRNA was reversed under hypoxia. Ang II- and bFGF-treatment of human cardiac fibroblasts did not cause detectable changes in the level of pro alpha1(l) collagen mRNA under ambient conditions or hypoxia. At the protein level, the amount of immunoreactive collagen type I, as determined by immunoslot blot analysis, was increased (33%, P<0.05) under hypoxia. Treatment of human cardiac fibroblasts with TGF-beta1 and T3 under ambient conditions led to diminished level of collagen type I. Under hypoxia, however, effect of both factors was reversed. The level of immunoreactive collagen type I in Ang II- and bFGF-treated cells, which was comparable to that in untreated cells under ambient conditions, remained unchanged under hypoxia. Together, these results provide evidence that hypoxia regulates growth, proliferative capacity and collagen type I production in human cardiac fibroblasts, and that although hypoxia alone may not be a stimulus for human cardiac fibroblast proliferation, it enhances growth factor-induced DNA synthesis in those cells. Furthermore, hypoxia by increasing the basal levels of collagen type I and by reversing the TGF-beta1- and T3-induced inhibition of collagen type I gene expression in human cardiac fibroblasts can enhance overall collagen type I production. Combinatorial effects of hypoxia on proliferation and collagen type I production in cardiac fibroblasts contribute to the post-infarct remodeling of the collagen matrix in failing human heart.