Expression, regulation and function of heparanase in the vasculature and in atherosclerosis

The cleavage of heparan sulfate (HS) by the {u03B2}-D glucuronidase heparanase (HPSE) fundamental event in many physiological processes such as wound healing and embryogenesis. Elevated HPSE activity has also been shown to contribute to cell migration and invasion in tumour angiogenesis, metastasis and inflammatory disease. More recently, HPSE has been implicated in vascular pathologies including atherosclerosis and post-angioplasty restenosis, where it may play a role in leukocyte infiltration and smooth muscle cell (SMC) proliferation and migration. While HPSE expression and regulation have been well characterised in tumour cells, platelets and T lymphocytes, relatively little is known of its expression in endothelial cells and SMCs. The aims of this thesis were to examine the expression and regulation of HPSE in cells of the vasculature and to investigate the function of the enzyme in atherosclerosis. The investigation of vascular cells revealed that both endothelial cells and SMCs expressed a high basal level of HPSE mRNA relative to Jurkat and MM170 tumour cells. HPSE gene expression was induced in endothelial cells derived from coronary artery and the cardiac microvasculature upon stimulation with the pro-inflammatory cytokines TNF{u03B1} and IL-1{u03B2}. In contrast, HPSE was not induced in cells isolated from the umbilical vein and artery, highlighting the heterogeneity in gene expression between endothelial cells of different types. In SMCs, HPSE was not induced upon treatment with pro-inflammatory stimuli. A putative ETS response element, one of four identified in the minimal promoter, was shown to be absolutely required for basal HPSE promoter activity in SMCs and the transcription factor ETS1 was found to trans-activate HPSE gene expression. Additionally, EGR1, previously shown to regulate HPSE gene transcription in T lymphocytes and tumour cells, was found to trans-activate the HPSE promoter in SMCs. The capacity of SMCs but not endothelial cells to bind exogenous HPSE was increased upon activation through binding to cell surface HS and possibly the calcium-independent mannose-6 phosphate receptor (CIMPR), representing a novel mechanism by which SMCs may be able to acquire HPSE and enhance migration. Immunohistochemical staining of human coronary arteries displaying atherosclerosis revealed high HPSE expression in macrophage foam cells of atherosclerotic lesions. Human monocytes were purified from peripheral blood leukocytes and also exhibited a high level of HPSE mRNA expression and activity. Monocytes differentiated to macrophages (HMDM) under specific culture conditions were treated with modified LDL to mimic atherosclerotic foam cell formation. HPSE expression was inducible in HMDM upon treatment with oxidised or acetylated LDL, suggesting a mechanism by which macrophages may be induced to express high levels of HPSE within atherosclerotic lesions. Additionally, the level of HPSE mRNA was induced upon treatment with LPS and down-regulated by IL-4, suggesting that HPSE expression may be associated with M1 polarised macrophages, and reduced in M2 macrophages. Hpse constitutive gene knockout mice were generated, characterised and used to directly assess the functions of HPSE in vivo in physiological processes and disease. Surprisingly, these mice were viable and fertile. Elevated expression of several matrix metalloproteinases and the endosulfatase Sulf2 was detected in Hpse{u207B}/{u207B} heart, suggesting that compensation by other ECM-degrading enzymes may result from Hpse deficiency in these mice. Hpse{u207B}/{u207B} mice were crossed with the ApoE{u207B}/{u207B} mouse, an established murine model of atherosclerosis. Age-matched ApoE{u207B}/{u207B} Hpse{u207B}/{u207B} double knockout (DKO) and ApoE{u207B}/{u207B} mice were fed a high fat diet and after 14 weeks, atherosclerotic lesion formation in the aorta was assessed. Lesion size was significantly reduced at the aortic arch in DKO compared with ApoE{u207B}/{u207B} mice, suggesting that HPSE contributes to the severity of disease. Although there was no difference in lesion macrophage content between the two groups, expression of the pro-inflammatory marker Ccl2 was decreased in the hearts of DKO mice, implicating HPSE in inflammatory events during atherogenesis. These studies provide the first direct evidence that HPSE is involved in the pathogenesis of atherosclerosis and establish a foundation to examine the precise roles of the enzyme in the initiation, progression and rupture of atherosclerotic plaques