TGFβ counteracts LYVE-1-mediated induction of lymphangiogenesis by small hyaluronan oligosaccharides

Abstract

During tissue injury, inflammation, and tumor growth, enhanced production and degradation of the extracellular matrix glycosaminoglycan hyaluronan (HA) can lead to the accumulation of small HA (sHA) oligosaccharides. We have previously reported that accumulation of sHA in colorectal tumors correlates with lymphatic invasion and lymph node metastasis, and therefore, investigated here are the effects of sHA on the lymphatic endothelium. Using cultured primary lymphatic endothelial cells (LECs) and ex vivo and in vivo lymphangiogenesis assays, we found that in contrast to high-molecular-weight HA (HMW-HA), sHA of 4-25 disaccharides in length can promote the proliferation of LECs and lymphangiogenesis in a manner that is dependent on their size and concentration. At pathophysiologically relevant concentrations found in tumor interstitial fluid, sHA is pro-proliferative, acts synergistically with VEGF-C and FGF-2, and stimulates the outgrowth of lymphatic capillaries in ex vivo lymphangiogenesis assays. In vivo, intradermally injected sHA acts together with VEGF-C to increase lymphatic vessel density. Higher concentrations of sHA were found to induce expression of the anti-lymphangiogenic cytokine TGFβ in LECs, which serves to counter-regulate sHA-induced LEC proliferation and lymphangiogenesis. Using appropriate knockout mice and blocking antibodies, we found that the effects of sHA are mediated by the sialylated form of the lymphatic HA receptor LYVE-1, but not by CD44 or TLR-4. These data are consistent with the notion that accumulation of sHA in tumors may contribute to tumor-induced lymphangiogenesis, leading to increased dissemination to regional lymph nodes. KEY MESSAGES : sHA promotes lymphangiogenesis primarily through increased LEC proliferation sHA induces proliferation in a narrow concentration window due to upregulated TGFβ Smaller HA oligosaccharides more potently induce proliferation than larger ones VEGF-C and FGF-2-induced LEC proliferation and lymphangiogenesis is augmented by sHA Sialylated LYVE-1, but not CD44 or TLR-4, mediate the effects of sHA on LEC.

Effect of Co-presentation of Adhesive Ligands and Short Hyaluronan on Lymphendothelial Cells

“…Thus, the application of sHA with 4–20 repeating units on blood vessel cells stimulates proliferation in a concentration range of 3–20 µg/mL showing a maximum response at 10 µg/mL (Mo et al, 2011 ). This biphasic effect on the proliferation of primary human dermal lymphatic endothelial cells (LECs) was also found for sHA with 4–13 disaccharide units in solution, with maximal effect on proliferation observed at a concentration of 5 µg/mL (Bauer et al, 2018 ).…”

“…Thus, effects due to the sHA modification as well as the cleavage mechanism on the metabolic activity of LECs can be excluded. We have previously reported a biphasic effect on the proliferation of primary human LEC upon incubation with sHA (4, 8, and 13 disaccharide units) in an LYVE-1-dependent manner (Bauer et al, 2018 ). In these experiments, we observed a maximal proliferation-promoting effect for 5.0 µg/mL of sHA in solution.…”

“…Short HA with a weight range from 10 to 20 kDa (25- to 50-mer) produced by a heat fragmentation of high molecular weight HA was obtained from Lifecore Biomedical. Enzymatically digested sHA with an average weight <10 kDa (25-mer) was produced from Healon 5 (Abbott Medical Optics), a highly purified high molecular weight HA with an average weight of 4 MDa, as previously described (Bauer et al, 2018 ). Briefly, Healon 5 was dissolved at 5 mg/mL in 0.3 M sodium phosphate, pH 5.3, sonified, and subsequently enzymatically digested with 200 U/mL bovine testes hyaluronidase (Sigma-Aldrich) for 6 h at 37°C.…”

The Role of the Tumor Microenvironment in the Development and Progression of Hepatocellular Carcinoma

“…The low molecular weight form is mostly present in pathological conditions such as inflammation and cancer (22,23). HA acts by inducing intracellular signals through several receptors: toll-like receptor 4, lymphatic vessel endothelial hyaluronan receptor 1, and receptor for hyaluronanmediated motility (24,25). The main receptor, CD44, is also considered a marker of cancer stem cells (CSC).…”

TGFBIp mediates lymphatic sprouting in corneal lymphangiogenesis

“…Thus, recent studies on the molecular regulation of lymphangiogenesis have focused on the interaction between lymphatic endothelial cells (LECs) and the ECM microenvironment. ECM molecules including hyaluronan, integrins, galectin‐8, matrix metalloproteinases and fibronectin (FN) play a pivotal role in lymphangiogenesis process by regulating LECs' proliferation, migration and tube formation …”