P721Transgenic mice over-expressing lysyl oxidase exhibit reduced neointimal thickening after carotid artery ligation

Abstract

Purpose: Lysyl oxidase (LOX) is an extracellular matrix-modifying enzyme that could play a critical role in vascular remodelling. We have developed a transgenic mouse model that over-expresses LOX in vascular smooth muscle cells (VSMC) to clarify whether LOX could regulate VSMC phenotype and vascular remodelling. Methods: We designed a construct in which the full-length cDNA of human LOX was under the control of the SM22α proximal promoter and generated a transgenic mouse model (TgLOX) by conventional methods. LOX expression was assessed by real-time PCR, western-blot and immunohistochemistry and LOX activity by a fluorimetric assay. Collagen content and assembly were also analyzed. [3H]-thymidine incorporation into DNA, was used as an index of mitogenic activity and the carotid artery ligation model allowed to evaluate neointimal formation in TgLOX mice. Results: The SM22α proximal promoter drove the expression of a transgene containing the human LOX cDNA. Two stable transgenic lines, phenotypically indistinguishable, were generated. Transgene expression followed the expected SMC-specific pattern. In TgLOX mice, real-time PCR and immunohistochemistry evidenced a strong expression of LOX in the media from aorta and carotid arteries, coincident with a higher proportion of mature collagen. VSMC isolated from TgLOX mice expressed high levels of LOX pro-enzyme, which was properly secreted and processed into mature and bioactive LOX. [3H]-thymidine incorporation into DNA was significantly reduced in cells from TgLOX mice. Transgenic VSMC also exhibited low levels of both SMemb (marker of SMC phenotypic switching) and PCNA (marker of cell proliferation), and a weak activation of Akt and ERK1/2 in response to mitogenic stimuli. Accordingly, neointimal thickening induced by carotid artery ligation was attenuated in TgLOX mice that also displayed a reduction in PCNA-immunopositive VSMC. Conclusions: Our results evidence that LOX plays a critical role in vascular remodelling. We have developed a new animal model to study the role of LOX in vascular biology.