Sulfated hyaluronic acid inhibits the hyaluronidase CEMIP and regulates the HA metabolism, proliferation and differentiation of fibroblasts.

Hyaluronan (HA) is an extracellular matrix component that regulates a variety of physiological and pathological processes. The function of HA depends both on its overall amount and on its size, properties that are controlled by HA synthesizing and degrading enzymes. The lack of inhibitors that can specifically block individual HA degrading enzymes has hampered attempts to understand the contribution of individual hyaluronidases to different physiological and pathological processes. CEMIP is a recently discovered hyaluronidase that cleaves HA through mechanisms and under conditions that are distinct from those of other hyaluronidases such as HYAL1 or HYAL2. The role of its hyaluronidase activity in physiology and disease is poorly understood. Here, we characterized a series of sulfated HA derivatives (sHA) with different sizes and degrees of sulfation for their ability to inhibit specific hyaluronidases. We found that highly sulfated sHA derivatives potently inhibited CEMIP hyaluronidase activity. One of these compounds, designated here as sHA3.7, was characterized further and shown to inhibit CEMIP with considerable selectivity over other hyaluronidases. Inhibition of CEMIP with sHA3.7 in fibroblasts, which are the main producers of HA in the interstitial matrix, increased the cellular levels of total and high molecular weight HA, while decreasing the fraction of low molecular weight HA fragments. Genetic deletion of CEMIP in mouse embryonic fibroblasts (MEFs) produced analogous results and confirmed that the effects of sHA3.7 on HA levels were mediated by CEMIP inhibition. Importantly, both CEMIP deletion and its inhibition by sHA3.7 suppressed fibroblast proliferation, while promoting differentiation into myofibroblasts, as reflected in a lack of CEMIP in myofibroblasts within skin wounds in experimental mice. By contrast, adipogenic and osteogenic differentiation were attenuated upon CEMIP loss or inhibition. Our results demonstrate the importance of CEMIP for the HA metabolism, proliferation and differentiation of fibroblasts, and suggest that inhibition of CEMIP with sulfated HA derivatives such as sHA3.7 has potential utility in pathological conditions that are dependent on CEMIP function.

Antitumor activity of sulfated hyaluronic acid fragments in pre-clinical models of bladder cancer.

Tumor cell-derived hyaluronidase HYAL-1 degrades hyaluronic acid (HA) into angiogenic fragments (AGF: 10-12 disaccharides). AGF support tumor growth and progression. Urine and tissue HAase/HYAL-1 levels are sensitive markers for high-grade bladder cancer (BCa) and its metastasis. In preclinical models of BCa, we evaluated whether o-sulfated AGF (sHA-F) inhibits HAase activity and has antitumor activity. At IC50 for HAase activity inhibition (5-20 µg/ml [0.4-1.7 µM]), sHA-F significantly inhibited proliferation, motility and invasion of HYAL-1 expressing BCa cells (253J-Lung, HT1376, UMUC-3), P<0.001. sHA-F did not affect the growth of HYAL-1 non-expressing BCa (5637, RT4, T24, TCCSUP) and normal urothelial (Urotsa, SV-HUC1) cells. sHA-F treatment induced apoptosis by death receptor pathway. sHA-F downregulated transcript and/or protein levels of HA receptors (CD44, RHAMM), p-AKT, ß-catenin, pß-Catenin(S552), Snail and Twist but increased levels of pß-Catenin(T41/S45), pGSK-3α/ß(S21/S9) and E-cadherin. sHA-F also inhibited CD44/Phosphoinositide 3-kinase (PI-3K) complex formation and PI-3K activity. AGF addition or myristoylated-AKT overexpression attenuated sHA-F effects. Contrarily, HYAL-1 expression sensitized RT4 cells to sHA-F treatment. In the 253J-L and HT1376 xenograft models, sHA-F treatment significantly inhibited tumor growth (P<0.001), plausibly by inhibiting angiogenesis and HA receptor-PI-3K/AKT signaling. This study delineates that sHA-F targets tumor-associated HA-HAase system and could be potentially useful in BCa treatment.

A new potential spreading factor: Streptomyces koganeiensis hyaluronidase. A comparative study with bovine testes hyaluronidase and recombinant human hyaluronidase of the HA degradation in ECM.

BACKGROUND: Recombinant human hyaluronidase has been used in the interstitial matrix to promote the dispersion of therapeutics. The production and isolation of an extracellular hyaluronidase from Streptomyces koganeiensis (rHyal_Sk) has recently been described. METHODS: The specificity of rHyal_Sk has been assessed against heparan sulfate, chondroitin sulfates and sulfated HAs. The oligomers generated by HA degradation have been investigated by MALDI-TOF MS analysis. rHyal_Sk has been compared with BTH and PH20 in vitro, against cross-linked HA (ACP) and HA-aggrecan complex, and in vivo, by means of a diffusion assay in nude mice. RESULTS: Depolymerization of HA by rHyal_Sk gave tetra-, hexa- and octasaccharides in high yields. The reaction mechanism and the high HA specificity were demonstrated. The in vivo diffusion assay, supported by the in vitro tests, evidenced an initially enhanced enzymatic activity of rHyal_Sk compared to BTH and PH20. CONCLUSIONS: rHyal_Sk, compared to BTH and PH20, showed higher substrate specificity and no inhibition from GAGs sulfate, together with a superior performance for HA depolymerization in ECM. As better predictive tests for the in vivo activity of hyaluronidase we developed two assays based on the degradation of ACP or of the HA-aggrecan complex. GENERAL SIGNIFICANCE: rHyal_Sk is a new potential spreading factor for intradermal drug administration. Hyaluronidases of distinct classes, that show equivalent activities in a common turbidimetric assay, could have different potencies and dose-efficacies in vivo which influences the therapeutic effect. The new proposed in vitro tests are designed to obtain a predictive characterization of the enzyme activity in vivo.