FIGURE 1.

Characterization of LMW-HA and its effects on EC proliferation. Panel A, to evaluate the molecular weight (MW) and purity of our LMW-HA preparation, a 4–20% Tris borate-EDTA (TBE) gel was run with HA molecular weight standards (Select-HA^TM 75–150 kDa (Sigma), Select-HA^TM 15–30 kDa (Sigma), small HA 1.5–3.0 kDa (Enzo Life Sciences), and OligoHA^TM10 ∼1.9 kDa (Hyalose, L.L.C.)) and our purified LMW-HA (see “Experimental Procedures”). Development of the resulting gel was achieved with Stains-All (Sigma). Panel B, determination of the minimal molar concentration of our purified LMW-HA to induce maximal HLMVEC proliferation. Control, VEGF (200 pg/ml), or LMW-HA (0.1–1000 nm) pretreated EC (5 × 10³ cells/well) were incubated with 0.2 ml of serum-free media for 72 h at 37 °C in 5%CO₂, 95% air in 96-well culture plates. The in vitro cell proliferation assay was analyzed by measuring increases in cell number using the CellTiter96^TM MTS assay (Promega) and read at 492 nm. Each assay was set up in triplicate and repeated at least five times. An asterisk indicates a statistically significant difference (p < 0.05) from Control. Panel C, to further test the purity of our purified LMW-HA, it was subjected to boiling and proteinase K digestion (50 μg/ml), which did not affect the ability to promote EC proliferation compared with untreated 100 nm LMW-HA. LMW-HA was also fully digested with hyaluronidase SD (100 milliunits/ml), and there was virtually no increase in EC proliferation, a process that was reversed with boiling (inactivating) hyaluronidase SD before HA digestion. Each assay was set up in triplicate and repeated at least five times.