Role of Heparanase on Hepatic Uptake of Intestinal Derived Lipoprotein and Fatty Streak Formation in Mice

BACKGROUND: Heparanase modulates the level of heparan sulfate proteoglycans (HSPGs) which have an important role in multiple cellular processes. Recent studies indicate that HSPGs have an important function in hepatic lipoprotein handling and processes involving removal of lipoprotein particles. PRINCIPAL FINDINGS: To determine the effects of decreased HSPGs chain length on lipoprotein metabolism and atherosclerosis, transgenic mice over-expressing the human heparanase gene were studied. Hepatic lipid uptake in hpa-Tg mice were evaluated by giving transgenic mice oral fat loads and labeled retinol. Sections of aorta from mice over-expressing heparanase (hpa-Tg) and controls (C57/BL6) fed an atherogenic diet were examined for evidence of atherosclerosis. Heparanase over-expression results in reduced hepatic clearance of postprandial lipoproteins and higher levels of fasting and postprandial serum triglycerides. Heparanase over-expression also induces formation of fatty streaks in the aorta. The mean lesion cross-sectional area in heparanase over-expressing mice was almost 6 times higher when compared to control mice (23,984 µm(2)±5,922 vs. 4,189 µm(2)±1,130, p<0.001). CONCLUSIONS: Over-expression of heparanase demonstrates the importance of HSPGs for the uptake of intestinal derived lipoproteins and its role in the formation of fatty streaks

Dissociation between Mature Phenotype and Impaired Transmigration in Dendritic Cells from Heparanase-Deficient Mice

To reach the lymphatics, migrating dendritic cells (DCs) need to interact with the extracellular matrix (ECM). Heparanase, a mammalian endo-β-D-glucuronidase, specifically degrades heparan sulfate proteoglycans ubiquitously associated with the cell surface and ECM. The role of heparanase in the physiology of bone marrow-derived DCs was studied in mutant heparanase knock-out (Hpse-KO) mice. Immature DCs from Hpse-KO mice exhibited a more mature phenotype; however their transmigration was significantly delayed, but not completely abolished, most probably due to the observed upregulation of MMP-14 and CCR7. Despite their mature phenotype, uptake of beads was comparable and uptake of apoptotic cells was more efficient in DCs from Hpse-KO mice. Heparanase is an important enzyme for DC transmigration. Together with CCR7 and its ligands, and probably MMP-14, heparanase controls DC trafficking

Matrix Metalloproteinases in Cytotoxic Lymphocytes Impact on Tumour Infiltration and Immunomodulation

To efficiently combat solid tumours, endogenously or adoptively transferred cytotoxic T cells and natural killer (NK) cells, need to leave the vasculature, traverse the interstitium and ultimately infiltrate the tumour mass. During this locomotion and migration in the three dimensional environment many obstacles need to be overcome, one of which is the possible impediment of the extracellular matrix. The first and obvious one is the sub-endothelial basement membrane but the infiltrating cells will also meet other, both loose and tight, matrix structures that need to be overridden. Matrix metalloproteinases (MMPs) are believed to be one of the most important endoprotease families, with more than 25 members, which together have function on all known matrix components. This review summarizes what is known on synthesis, expression patterns and regulation of MMPs in cytotoxic lymphocytes and their possible role in the process of tumour infiltration. We also discuss different functions of MMPs as well as the possible use of other lymphocyte proteases for matrix degradation

Eosinophil major basic protein: first identified natural heparanase-inhibiting protein

BACKGROUND: Heparanase and eosinophils are involved in several diseases, including inflammation, cancer, and angiogenesis. OBJECTIVE: We sought to determine whether eosinophils produce active heparanase. METHODS: Human peripheral blood eosinophils were isolated by immunoselection and tested for heparanase protein (immunocytochemistry, Western blot), mRNA (RT-PCR) and activity (Na(2)[(35)S]O(4)-labeled extracellular matrix degradation) before and after activation. Heparanase intracellular localization (confocal laser microscopy) and ability to bind to eosinophil major basic protein (MBP) were also evaluated (immunoprecipitation). A model of allergic peritonitis resulting in eosinophilia was induced in TNF knockout and wild-type mice for in vivo studies. RESULTS: Eosinophils synthesized heparanase mRNA and contained heparanase in the active (50-kd) and latent (65-kd) forms. Heparanase partially co-localized with and was bound to MBP. No heparanase enzymatic activity was detected in eosinophils resting or activated with various agonists, including GM-CSF/C5a. Eosinophil lysates and MBP inhibited recombinant heparanase activity in a concentration-dependent manner (100%, 2 x 10(-7) mol/L). Eosinophil peroxidase and eosinophil cationic protein, but not myelin basic protein or compound 48/80, partially inhibited heparanase activity. Poly-l-arginine at very high concentrations caused an almost complete inhibition. In allergic peritonitis, heparanase activity in the peritoneal fluid inversely correlated with eosinophil number. CONCLUSIONS: MBP is the first identified natural heparanase-inhibiting protein. Its presence in the eosinophil granules might indicate a protective function of these cells in diseases associated with inflammation and cancer progression

In vivo fragmentation of heparan sulfate by heparanase overexpression renders mice resistant to amyloid protein A amyloidosis

Amyloid diseases encompass >20 medical disorders that include amyloid protein A (AA) amyloidosis, Alzheimer's disease, and type 2 diabetes. A common feature of these conditions is the selective organ deposition of disease-specific fibrillar proteins, along with the sulfated glycosaminoglycan, heparan sulfate. We have generated transgenic mice that overexpress human heparanase and have tested their susceptibility to amyloid induction. Drastic shortening of heparan sulfate chains was observed in heparanase-overproducing organs, such as liver and kidney. These sites selectively escaped amyloid deposition on experimental induction of inflammation-associated AA amyloidosis, as verified by lack of material staining with Congo Red, as well as lack of associated polysaccharide, whereas the same tissues from control animals were heavily infiltrated with amyloid. By contrast, the spleens of transgenic mice that failed to significantly overexpress heparanase contained heparan sulfate chains similar in size to those of control spleen and remained susceptible to amyloid deposition. Our findings provide direct in vivo evidence that heparan sulfate is essential for the development of amyloid disease

Reduction of anionic sites in the glomerular basement membrane by heparanase does not lead to proteinuria.

Contains fulltext : 71200.pdf (publisher's version ) (Open Access)Heparan sulfate in the glomerular basement membrane has been considered crucial for charge-selective filtration. In many proteinuric diseases, increased glomerular expression of heparanase is associated with decreased heparan sulfate. Here, we used mice overexpressing heparanase and evaluated the expression of different heparan sulfate domains in the kidney and other tissues measured with anti-heparan sulfate antibodies. Glycosaminoglycan-associated anionic sites were visualized by the cationic dye cupromeronic blue. Transgenic mice showed a differential loss of heparan sulfate domains in several tissues. An unmodified and a sulfated heparan sulfate domain resisted heparanase action in vivo and in vitro. Glycosaminoglycan-associated anionic sites were reduced about fivefold in the glomerular basement membrane of transgenic mice, whereas glomerular ultrastructure and renal function remained normal. Heparanase-resistant heparan sulfate domains may represent remnant chains or chains not susceptible to cleavage. Importantly, the strong reduction of glycosaminoglycan-associated anionic sites in the glomerular basement membrane without development of a clear renal phenotype questions the primary role of heparan sulfate in charge-selective filtration. We cannot, however, exclude that overexpression of heparanase and heparan sulfate loss in the basement membrane in glomerular diseases contributes to proteinuria

Two Heparanase Splicing Variants with Distinct Properties Are Necessary in Early Xenopus Development*

Heparanase is an endoglycosidase that cleaves heparan sulfate (HS) side chains from heparan sulfate proteoglycans (HSPGs) present in extracellular matrix and cell membranes. Although HSPGs have many functions during development, little is known of the role of the enzyme that degrades HS, heparanase. We cloned and characterized the expression of two heparanase splicing variants from Xenopus laevis and studied their function in early embryonic development. The heparanase gene (termed xHpa) spans over 15 kb and consists of at least 12 exons. The long heparanase (XHpaL) cDNA encodes a 531-amino acid protein, whereas the short splicing variant (XHpaS) results in a protein with the same open reading frame but missing 58 amino acids as a consequence of a skipped exon 4. Comparative studies of both isoforms using heterologous expression systems showed: 1) XHpaL is enzymatically active, whereas XHpaS is not; 2) XHpaL and XHpaS interact with heparin and HS; 3) both proteins traffic through the endoplasmic reticulum and Golgi apparatus, but XHpaL is secreted into the medium, whereas XHpaS remains associated with the membrane as a consequence of the loss of three glycosylation sites; 4) overexpression of XHpaS but not XHpaL increases cell adhesion of glioma cells to HS-coated surfaces; 5) XHpaL and XHpaS mRNA and protein levels vary as development progresses; 6) specific antisense knock-down of both XHpaL and XHpaS, but not XHpaL alone, results in failure of embryogenesis to proceed. Interestingly, rescue experiments suggest that the two heparanases regulate the same developmental processes, but via different mechanisms

Alternatively spliced Spalax heparanase inhibits extracellular matrix degradation, tumor growth, and metastasis

Heparanase is an endoglycosidase that degrades heparan sulfate (HS) at the cell surface and in the extracellular matrix. Heparanase is expressed mainly by cancer cells, and its expression is correlated with increased tumor aggressiveness, metastasis, and angiogenesis. Here, we report the cloning of a unique splice variant (splice 36) of heparanase from the subterranean blind mole rat (Spalax). This splice variant results from skipping part of exon 3, exons 4 and 5, and part of exon 6 and functions as a dominant negative to the wild-type enzyme. It inhibits HS degradation, suppresses glioma tumor growth, and decreases experimental B16–BL6 lung colonization in a mouse model. Intriguingly, Spalax splice variant 7 of heparanase (which results from skipping of exon 7) is devoid of enzymatic activity, but unlike splice 36 it enhances tumor growth. Our results demonstrate that alternative splicing of heparanase regulates its enzymatic activity and might adapt the heparanase function to the fluctuating normoxic–hypoxic subterranean environment that Spalax experiences. Development of anticancer drugs designed to suppress tumor growth, angiogenesis, and metastasis is a major challenge, of which heparanase inhibition is a promising approach. We anticipate that the heparanase splicing model, evolved during 40 million years of Spalacid adaptation to underground life, would pave the way for the development of heparanase-based therapeutic modalities directed against angiogenesis, tumor growth, and metastasis