Fig. 3: rhPRG4 induces VEGF expression in vitro.

In primary MEFs, rhPRG4 induced VEGF protein expression in a dose dependent manner with a plateau at 100 µg/ml a, b. This effect was confirmed at the mRNA level c. Vegf and Hif1α mRNA expression was quantified under normoxic and hypoxic conditions in the presence or absence of rhPGR4 and/or Hif1α inhibition (BAY 87-2243) to demonstrate that rhPRG4 induction of Vegf was independent of Hif1α d, e. To determine if Vegf activation was downstream of Toll-like receptors (TLRs), HEK cells lacking all TLRs or expressing a single TLR (2, 4 or 5) were treated with rhPRG4 or their specific agonist f. TLR4 expressing cells demonstrated the maximal Vegf activation (f) and this activation was independent of MYD88 activity since the MYD88 inhibitor (ST2825) had no effect on rhPRG4 Vegf activation g. All experiments were undertaken on at least 3 biological and 3 technical replicates unless otherwise stated. n.s. = not significant. Error bars equal mean ± SD (a, c, d – 1 way ANNOVA, f, g – t-test).