Involvement of heparanase in the pathogenesis of acute kidney injury: Nephroprotective effect of PG545

Despite the high prevalence of acute kidney injury (AKI) and its association with increased morbidity and mortality, therapeutic approaches for AKI are disappointing. This is largely attributed to poor understanding of the pathogenesis of AKI. Heparanase, an endoglycosidase that cleaves heparan sulfate, is involved in extracellular matrix turnover, inflammation, kidney dysfunction, diabetes, fibrosis, angiogenesis and cancer progression. The current study examined the involvement of heparanase in the pathogenesis of ischemic reperfusion (I/R) AKI in a mouse model and the protective effect of PG545, a potent heparanase inhibitor. I/R induced tubular damage and elevation in serum creatinine and blood urea nitrogen to a higher extent in heparanase over-expressing transgenic mice vs. wild type mice. Moreover, TGF-\u3b2, vimentin, fibronectin and \u3b1-smooth muscle actin, biomarkers of fibrosis, and TNF\u3b1, IL6 and endothelin-1, biomarkers of inflammation, were upregulated in I/R induced AKI, primarily in heparanase transgenic mice, suggesting an adverse role of heparanase in the pathogenesis of AKI. Remarkably, pretreatment of mice with PG545 abolished kidney dysfunction and the up-regulation of heparanase, pro-inflammatory (i.e., IL-6) and pro-fibrotic (i.e., TGF-\u3b2) genes induced by I/R. The present study provides new insights into the involvement of heparanase in the pathogenesis of ischemic AKI.Our results demonstrate that heparanase plays a deleterious role in the development of renal injury and kidney dysfunction,attesting heparanase inhibition as a promising therapeutic approach for AKI

Heparanase and macrophage interplay in the onset of liver fibrosis

Abstract The heparan sulfate endoglycosidase heparanase (HPSE) is involved in tumor growth, chronic inflammation and fibrosis. Since a role for HPSE in chronic liver disease has not been demonstrated to date, the current study was aimed at investigating the involvement of HPSE in the pathogenesis of chronic liver injury. Herein, we revealed that HPSE expression increased in mouse livers after carbon tetrachloride (CCl4)-mediated chronic induction of fibrosis, but with a trend to decline during progression of the disease. In mouse fibrotic liver tissues HPSE immunostaining was restricted in necro-inflammatory areas, co-localizing with F4/80 macrophage marker and TNF-α. TNF-α treatment induced HPSE expression as well as HPSE secretion in U937 macrophages. Moreover, macrophage-secreted HPSE regulated the expression of α-SMA and fibronectin in hepatic stellate LX-2 cells. Finally, HPSE activity increased in the plasma of patients with liver fibrosis but it inversely correlated with liver stiffness. Our results suggest the involvement of HPSE in early phases of reaction to liver damage and inflammatory macrophages as an important source of HPSE. HPSE seems to play a key role in the macrophage-mediated activation of hepatic stellate cells (HSCs), thus suggesting that HPSE targeting could be a new therapeutic option in the treatment of liver fibrosis

Glycosaminoglycans and Glycosaminoglycan Mimetics in Cancer and Inflammation

Glycosaminoglycans (GAGs) are a class of biomolecules expressed virtually on all mammalian cells and usually covalently attached to proteins, forming proteoglycans. They are present not only on the cell surface, but also in the intracellular milieu and extracellular matrix. GAGs interact with multiple ligands, both soluble and insoluble, and modulate an important role in various physiological and pathological processes including cancer, bacterial and viral infections, inflammation, Alzheimer’s disease, and many more. Considering their involvement in multiple diseases, their use in the development of drugs has been of significant interest in both academia and industry. Many GAG-based drugs are being developed with encouraging results in animal models and clinical trials, showcasing their potential for development as therapeutics. In this review, the role GAGs play in both the development and inhibition of cancer and inflammation is presented. Further, advancements in the development of GAGs and their mimetics as anti-cancer and anti-inflammatory agents are discussed

Heparanase isoform expression and extracellular matrix remodeling in intervertebral disc degenerative disease

OBJECTIVE: To determine the molecules involved in extracellular matrix remodeling and to identify and quantify heparanase isoforms present in herniated and degenerative discs. INTRODUCTION: Heparanase is an endo-beta-glucuronidase that specifically acts upon the heparan sulfate chains of proteoglycans. However, heparanase expression in degenerative intervertebral discs has not yet been evaluated. Notably, previous studies demonstrated a correlation between changes in the heparan sulfate proteoglycan pattern and the degenerative process associated with intervertebral discs. METHODS: Twenty-nine samples of intervertebral degenerative discs, 23 samples of herniated discs and 12 samples of non-degenerative discs were analyzed. The expression of both heparanase isoforms (heparanase-1 and heparanase-2) was evaluated using immunohistochemistry and real-time RT-PCR analysis. RESULTS: Heparanase-1 and heparanase-2 expression levels were significantly higher in the herniated and degenerative discs in comparison to the control tissues, suggesting a possible role of these proteins in the intervertebral degenerative process. CONCLUSION: The overexpression of heparanase isoforms in the degenerative intervertebral discs and the herniated discs suggests a potential role of both proteins in the mediation of inflammatory processes and in extracellular matrix remodeling. The heparanase-2 isoform may be involved in normal metabolic processes, as evidenced by its higher expression in the control intervertebral discs relative to the expression of heparanase-1

Heparinase selectively sheds heparan sulphate from the endothelial glycocalyx

A healthy vascular endothelium is coated by the endothelial glycocalyx. Its main constituents are transmembrane syndecans and bound heparan sulphates. This structure maintains the physiological endothelial permeability barrier and prevents leukocyte and platelet adhesion, thereby mitigating inflammation and tissue oedema. Heparinase, a bacteria] analogue to heparanase, is known to attack the glycocalyx. However, the exact extent and specificity of degradation is unresolved. We show by electron microscopy, immunohistological staining and quantitative measurements of the constituent parts, that heparinase selectively sheds heparan sulphate from the glycocalyx, but not the synclecans

Heparanase Levels Are Elevated in the Urine and Plasma of Type 2 Diabetes Patients and Associate with Blood Glucose Levels

Heparanase is an endoglycosidase that specifically cleaves heparan sulfate side chains of heparan sulfate proteoglycans. Utilizing an ELISA method capable of detection and quantification of heparanase, we examined heparanase levels in the plasma and urine of a cohort of 29 patients diagnosed with type 2 diabetes mellitus (T2DM), 14 T2DM patients who underwent kidney transplantation, and 47 healthy volunteers. We provide evidence that heparanase levels in the urine of T2DM patients are markedly elevated compared to healthy controls (1162±181 vs. 156±29.6 pg/ml for T2DM and healthy controls, respectively), increase that is statistically highly significant (P<0.0001). Notably, heparanase levels were appreciably decreased in the urine of T2DM patients who underwent kidney transplantation, albeit remained still higher than healthy individuals (P<0.0001). Increased heparanase levels were also found in the plasma of T2DM patients. Importantly, urine heparanase was associated with elevated blood glucose levels, implying that glucose mediates heparanase upregulation and secretion into the urine and blood. Utilizing an in vitro system, we show that insulin stimulates heparanase secretion by kidney 293 cells, and even higher secretion is observed when insulin is added to cells maintained under high glucose conditions. These results provide evidence for a significant involvement of heparanase in diabetic complications

Small RNA interference-mediated gene silencing of heparanase abolishes the invasion, metastasis and angiogenesis of gastric cancer cells

<p>Abstract</p> <p>Background</p> <p>Heparanase facilitates the invasion and metastasis of cancer cells, and is over-expressed in many kinds of malignancies. Our studies indicated that heparanase was frequently expressed in advanced gastric cancers. The aim of this study is to determine whether silencing of heparanase expression can abolish the malignant characteristics of gastric cancer cells.</p> <p>Methods</p> <p>Three heparanase-specific small interfering RNA (siRNAs) were designed, synthesized, and transfected into cultured gastric cancer cell line SGC-7901. Heparanase expression was measured by RT-PCR, real-time quantitative PCR and Western blot. Cell proliferation was detected by MTT colorimetry and colony formation assay. The <it>in vitro </it>invasion and metastasis of cancer cells were measured by cell adhesion assay, scratch assay and matrigel invasion assay. The angiogenesis capabilities of cancer cells were measured by tube formation of endothelial cells.</p> <p>Results</p> <p>Transfection of siRNA against 1496-1514 bp of encoding regions resulted in reduced expression of heparanase, which started at 24 hrs and lasted for 120 hrs post-transfection. The siRNA-mediated silencing of heparanase suppressed the cellular proliferation of SGC-7901 cells. In addition, the <it>in vitro </it>invasion and metastasis of cancer cells were attenuated after knock-down of heparanase. Moreover, transfection of heparanase-specific siRNA attenuated the <it>in vitro </it>angiogenesis of cancer cells in a dose-dependent manner.</p> <p>Conclusions</p> <p>These results demonstrated that gene silencing of heparanase can efficiently abolish the proliferation, invasion, metastasis and angiogenesis of human gastric cancer cells <it>in vitro</it>, suggesting that heparanase-specific siRNA is of potential values as a novel therapeutic agent for human gastric cancer.</p

HEPARANASE EXPRESSION IN RENAL INTERSTITIAL MAY CONTRIBUTE TO EPITHELIAL AND ENDOTHELIAL CELLS INJURIES AFTER KIDNEY ISCHEMIC/ REPERFUSION EPISODE IN MICE

Kidney ischemia/reperfusion injury (I/R) is the most frequent cause of acute kidney injury (AKI). It had been reported that epithelial and endothelial injuries occurred during kidney I/R injury. Heparanase is an enzyme that degrades glycocalyx and contributes to I/R injury in the heart and liver. This study is to elucidate the association between heparanase expression and cell injuries in kidney I/R injury. We performed kidney I/R injury model in mice using renal pedicle clamping for 30 minutes and sacrificed the mice in day 1 (n=6) after operation. Sham-operation procedure (SO, n=5) was used as control. PAS staining was used to quantify tubular injury score. Serum creatinine was measured from orbital venous. Heparanase expression was quantified using western blot and real-time PCR. Heparanase localization and endothelial injury were shown by immunostaining of heparanase and double glycocalyx-von Willebrand factor. Kidney I/R induced an increase of serum creatinine level that was accompanied by elevation of tubular injury score and glycocalyx damage. Glycocalyx damage was identified using immunofluorescent staining that revealed a disruption of glycocalyx or lectin layer in the endothelial cells of intra-renal artery. This finding was associated with significant elevation of heparanase mRNA and protein level expression. We found that heparanase was expressed in the renal epithelial and interstitial cells. In conclusion, heparanase may induce endothelial and epithelial injury in the kidney I/R episode. Using heparanase expression as a early marker of AKI may possibly promising. Keywords: kidney I/R, epithelial injury, endothelial injury, heparanase, glycocalyx