Role of Heparanase Inhibition in Atherosclerosis Prevention. A Potential Novel Therapeutic Strategy

Atherosclerosis (AS) is the process in which lipid particles,
mainly oxidized LDL (Ox-LDL) accumulate in the luminal side of the...

as well as in several pathologic processes [7,11,12,[17][18][19][20][21][22][23], and their normal structure and function are crucial for maintaining normal tissue structure, integrity and function [24]. Heparan sulfate endoglycosidase heparanase (Heparanse), the only enzyme in mammalians that degrades HS chains in the HSPGs [19,[25][26][27], is implicated in the tight regulation of HSPG turnover, through both intracellular and extracellular roles [28][29][30]. As HSPGs exist in all the body organs and systems, heparanase inhibition is under extensive investigation in various diseases. Recent studies implicate heparanase in AS development and progression. In their study, Blich, et al. demonstrated intense staining for heparanase in the intima of vulnerable atherosclerotic plaques in human coronary arteries compared to a weak staining in stable plaques [31].
Similarly, Osterholm, et al. demonstrated a 6.6 fold increased heparanase mRNA levels in atherosclerotic plaques in human carotid arteries in comparison to non-atherosclerotic iliac arteries [23]. Likewise, Baker, et al. documented elevated heparanase levels and activity associated with coronary atherosclerosis progression in diabetic hyperlipidemic swine [22]. In their review, Vlodavsky, et al. reported in detail the role of heparanase in atherosclerosis and other vessel wall pathologies [32]. In line with these studies, we demonstrated in apolipoprotein E deficient (E0) mice that heparanase inhibition by PG545 (Pixatimod) significantly decreased serum OS and lowered plasma lipid levels [33]. In addition, we demonstrated in E0 mice placed on high fat diet (HFD) that heparanase inhibition by PG545 significantly decreased serum OS, along decreasing aortic wall thickness and atherosclerotic plaque surface area. In the same study, we demonstrated that PG545 significantly diminished the development of liver steatosis, an issue under current investigation [34].
In biochemical staining and western blotting studies, we demonstrated that PG545 caused significant reduction of IL-1, TNF-α, and aKT, together with increasing FGF-2 and LC-III expression, reflecting the fact that heparanase inhibition resulted in anti-inflammatory effects, besides augmenting regeneration process and increased autophagy, in an attempt of the liver to repair injured liver tissue. All these effects, together with lowering serum OS and lipid levels, can be suggested as the pathogenetic mechanisms by which heparanase inhibition exerts the beneficial anti-atherosclerotic and anti-steatosis effects ( Figure 1). Moreover, we also studied the effect of Roneparstat (SST0001) on OS, AS, and liver steatosis in E0 mice placed on HFD for eight weeks. SST0001 showed metabolic effects similar to PG545 (decreased serum lipids levels and OS, and significantly attenuated the development of liver steatosis), but had no effect against the development of atherosclerosis in the aortas of the mice. Like PG545, also SST0001 demonstrated neither heptotoxicity nor renal toxicity, and did not affect blood pressure. In contrast to PG545, which caused prominent weight loss in mice despite minimal effect on food intake, SST0001 affected neither food intake nor mice body weight (data not published).

Heparanase Inhibition in Acute Kidney Injury
In ischemia-reperfusion acute kidney injury (AKI) rat model, pre-treatment with PG545 significantly attenuated the development of AKI, which was expressed by lower serum creatinine and blood urea nitrogen levels in the treated mice. Histologic studies of the kidneys in the sham-control mice revealed the development of acute tubular necrosis, with tubular lysis, loss of brush border and the accumulation of debris in the tubular lumen. Electron microscopy analyses revealed mitochondrial distortion in the sham-control mice, compared to normal ultrastructure of the mitochondria in mice pre-treated with the heparanase inhibitor [35].

Heparanase Inhibition in Malignant Diseases
The role of heparanase in the development of malignant diseases was well established, and the effect of heparanase inhibition in the treatment of different malignancies has been extensively studied. Recent studies demonstrated favorable effects of the heparanase inhibitor SST0001 (Roneparstat) when added to standard treatment protocols in patients with multiple myeloma [36][37][38] or in different kinds of human pediatric osteosarcoma and other sarcoma models [39,40], as well as in cases of mesothelioma [41] and different pediatric malignancies [42], and thus heparanase inhibitors are under consideration as an additional modality for treating malignant diseases [43][44][45].

Heparanase Inhibitors and the Coagulation System
Impaired endothelial dysfunction is an early feature of formation of the atherosclerotic lesion. Acute cardiac ischemic syndromes, such as unstable angina pectoris or myocardial infarctions, occur following the abrupt rupture of an atherosclerotic plaque, an event that leads to exposure of the highly thrombogenic lipid core constituents of the plaque to the intra-vascular content, resulting in activation of the coagulation system, which further accelerates the atherosclerotic plaque formation [46][47][48]. Heparan sulfate molecules play key roles in activation of the coagulation system.
In a book chapter, Nadir Y. had described in detail the role of heparanase in the activation of the coagulation system and reported increased heparanase level and activity in several clinical settings associated with hypercoagulability, such as in women using oral contraceptives, cases of diabetic foot, women at delivery, after orthopedic surgeries, and in patients with lung cancers and other malignancies [49]. Former studies have reported in detail the role of heparanase in increasing coagulation via three mechanisms, including enhancement of tissue factor activity, upregulation of tissue factor expression in endothelial cells, and by releasing the single chain polypeptide tissue factor pathway inhibitor (TFPI) from cell surface [50], and heparanase inhibition by peptides derived from TFPI-2 was proved to inhibit the procoagulant activity of heparanase and to attenuate sepsis in mouse model [51].

Conclusion
There is a large evidence regarding the involvement of heparanase in several disease processes, supported by the fact that higher heparanase levels and activity are associated with more advanced and complicated diseases, as was shown in However, lack of pathogenic mechanism and effect of long term use of heparanase inhibitors in humans limit the possibility to apply using heparanase inhibitors in humans for prevention or treating atherosclerosis, and further research is absolutely warranted. In the literature, additional heparanase inhibitors are available, where their effect was not examined neither on AS nor on liver steatosis. In face of the existing non-consistent results demonstrated so far, it is highly warranted that the effect of additional heparanase inhibitors on atherosclerosis prevention and treatment be studied.