Hypoxia alters the synthesis and post-translational modification of collagens in human coronary artery smooth muscle cells

(250 words) Considerable data support the hypothesis that hypoxia plays a central role in the development of atherosclerotic plaques and cardiovascular disease. Hypoxia may modulate the pool of proteins within cells, including vascular smooth muscle cells of the artery wall, and drive phenotypic changes. Here we report a detailed liquid chromatography-mass spectrometry (LC-MS/MS) study of changes induced in human coronary artery smooth muscle cells (HCASMC) on transfer from an atmosphere of 20 to 1% O 2 (hypoxia) for 1 and 7 days. Exposure to 1% O 2 resulted in marked changes in the HCASMC proteome with 755 proteins (of 5,842 total) altered in abundance after 1 day, and 2874 (1234 up, 1640 down) after 7 days of hypoxia. Significant changes in specific collagens, and collagen-modifying hydroxylases and cross-linking enzymes were detected (lysyl oxidases and related species, prolyl and lysyl hydroxylases), with most of these upregulated by hypoxia. These data are consistent with significant extracellular matrix (ECM) remodeling under hypoxia and increased collagen synthesis, processing, secretion and incorporation into ECM. Analysis of protein post translational modifications (hydroxylations) indicates enhanced hydroxylation of multiple collagen peptides, particularly within the helical coil regions. Immunoblotting and ELISA data support these ECM changes. Comparison with proteomic data obtained from advanced symptomatic human carotid artery atherosclerotic plaques and control artery samples, provides evidence for a similar pattern of changes, particularly with regard to collagen-modifying and processing enzymes, consistent with the presence of hypoxic regions within human plaques. Together, the data indicate that hypoxia plays a significant role in collagen-focused ECM remodeling.