Atherosclerosis, a progressive chronic inflammatory disease of mid-sized to large arteries, is the major underlying cause of clinical cardiovascular events, myocardial infarction and stroke. As such, atherosclerosis is a major cause of mortality and morbidity worldwide [1]. Atherosclerosis is characterised by the excessive accumulation of lipid and fibrotic material in the arterial intima to form complex atherosclerotic lesions or plaques, the development of which are orchestrated by intricate interactions of vascular cells with innate and adaptive immune cells [2]. A critical early event initiating atherosclerosis development is endothelial dysfunction, which occurs at sites of disturbed blood flow and characterised by the impaired vasodilatory and anti-inflammatory activity of endothelial-derived nitric oxide (NO) and increased endothelial permeability [3,4]. The activated and dysfunctional endothelium increasingly express leukocyte adhesion molecules (e.g., VCAM-1, ICAM-1, E-Selectin) that facilitate the infiltration of circulating immune cells, including monocytes, T cells and dendritic cells (DCs) into the arterial intima [3].

The dysfunctional endothelium also exhibits increased permeability to low-density lipoprotein (LDL), resulting in the lipoprotein's accumulation into the intima and subsequent binding to the sub-endothelial extracellular matrix (ECM) that increases its susceptibility to modification by leukocyte-derived oxidants or lipolytic and proteolytic enzymes [5,6]. Modified LDL is pro-inflammatory and recognised by scavenger receptors, resulting in excessive lipid accumulation by monocyte-derived macrophages to form foam cells, the hallmark of early lesions. Macrophages and DCs can process and present epitopes from modified LDL to T cells to promote a Th1-mediated inflammatory response [6], which progresses the formation of more complex plaques characterised by a fibrous cap, composed of collagen and smooth muscle cells (SMC), and a necrotic core, derived from dying foam cells and extracellular release of cholesterol crystals [7]. Persistent inflammation at plaque shoulder regions signals for SMC apoptosis and replacement by macrophages and T cells, increased production of proteolytic matrix metalloproteinases (MMPs), ECM degradation and fibrous cap thinning, events that can precipitate plaque rupture, thrombosis and a myocardial infarction or stroke [7].

Inflammation is central to all stages of atherosclerosis and the recent CANTOS or COLCOT clinical trials established that broad-spectrum inhibition of inflammation with a neutralizing antibody for interleukin-1β or colchicine reduced clinical cardiovascular events in coronary artery disease (CAD) patients [8,9]. However, patients receiving these treatments exhibited higher risk of infection. This highlights the importance of identifying the inflammatory mediators driving atherosclerotic cardiovascular disease that can be more selectively and safely targeted therapeutically.

The current study focuses on the role of the pro-inflammatory enzyme Hpse, an endo-β-d-glucuronidase that constitutes the only mammalian enzyme known to selectively cleave heparan sulfate (HS), a key component of the vascular ECM and basement membrane. By degrading HS, Hpse is involved in various physiological functions associated with ECM remodelling such as wound healing and tissue remodelling [10], hair growth [11], angiogenesis [10], embryo development [12], and leukocyte trafficking [13,14]. Hpse also plays an important role in several disease settings including cancer metastasis [14,15], inflammation [13,16] and renal diseases [17].

HS is a major constituent of the glycocalyx layer on the endothelial luminal surface and in the sub-endothelial ECM of arteries, with increased HS degradation at these sites a characteristic of heightened vascular inflammation and development of atherosclerotic cardiovascular disease [18,19]. Importantly, increasing evidence suggests the involvement of Hpse in several key steps of atherosclerotic lesion development. For example, Hpse-mediated HS-degradation can impair flow-mediated activation of NO synthesis by the endothelium [20], promote the binding of LDL to sub-endothelial ECM in the arterial intima [21], facilitate the liberation of HS-bound cytokines and degrade HS-structural barriers to promote the adhesion of circulating monocytes and other leukocytes to the activated endothelium and subsequent infiltration into the arterial intima, which are all important steps in the initiation of atherosclerosis [22,23]. Consistent with this, the formation of nascent fatty streak lesions are increased in Hpse overexpressing mice on a non-atherosclerosis prone background [24]. Notably, circulating Hpse levels are significantly increased in CAD patients and Hpse protein expression is elevated in human and experimental animal atherosclerotic lesions, where it is detected in endothelial cells (ECs), macrophages and T cells [25,26]. Furthermore, enhanced expression of Hpse mRNA, protein and activity is observed in advanced atherosclerotic lesions of hyperlipidemic swine [26].

However, despite this evidence, the role of Hpse in atherosclerosis and the mechanism(s) by which the enzyme impacts on disease development are not well defined. This study investigates the role of Hpse in atherosclerosis by generating novel Hpse gene-deficient mice on the atherosclerosis prone, ApoE−/− background and studying the impact of Hpse gene-deficiency of disease initiation and progression in response to a Western-style high-fat diet (HFD). This study demonstrates for the first time an essential role for Hpse in promoting the initiation and progression of atherosclerotic disease in HFD-fed ApoE−/− mice.