Fig 2: Schematic of the mechanisms underlying the therapeutic effects of HMW HA on injured human lung microvascular endothelial cells. Cytomix, a mixture of IL-1ß, TNFa, and IFN? often used as a surrogate for acute respiratory distress syndrome pulmonary edema fluid, caused an increase in protein permeability across HLMVEC which was associated with an increase in apoptosis/LDH release and actin stress fiber and gap formations between cells. Cytomix was also capable of breaking down HMW HA into LMW HA further exacerbating the injury. In response to cytomix, AKT phosphorylation and HAS2 expression were increased in HLMVECs as an inherent reparative effect although there was no restoration of intracellular HMW HA levels or protein permeability. Surprisingly, exogenous HMW HA administration increased HAS2 expression further after cytomix through CD44, leading to an increase in intracellular HMW HA synthesis and restoration of protein permeability. This finding was unexpected given normal feedback inhibition seen with receptors and end-products and may result from an increase in AKT phosphorylation from HMW HA leading to increased HAS2 expression. Although exogenous LMW HA administration suppressed Caspase-3 activation in the current study, LMW HA is known to increase inflammation, apoptosis, and permeability in endothelial cells whether through a direct effect or through increased release of cytokines or through inhibition of HMW HA binding to CD44 as a competitive antagonist

Fig 3: Expression of predicted target genes in RCC models (A) Relative to Actinß expression of Hif-1a in Renca pten/WT ptenKO cells cultured in normoxia and hypoxia (B) Relative to GusB expression of Hif-1a in Caki-1 and 786-O cells cultured in normoxia and hypoxia (C) Relative to Actinß expression of Vegfa in Renca pten/WT ptenKO cells cultured in normoxia and hypoxia (D) Relative to GusB expression of Vegfa in Caki-1 and 786-O cells cultured in normoxia and hypoxia (E) Relative to Actinß expression of Bcl2 in Renca pten/WT ptenKO cells cultured in normoxia and hypoxia (F) Fold change of number of live cells collected form normoxic and hypoxic culture: Renca pten/WT pten/KO (G) and Caki-1, 786-O (H) Relative to Actinß expression of Akt1 (I) mTOR (J) Tgfßr3 (K) p53 in Renca pten/WT ptenKO cells cultured in normoxia and hypoxia, Student’s t-test *** p-value < 0.001; ** p-value < 0.01; * p-value < 0.05.

Fig 4: Effect of 4-MU, a HAS inhibitor, with or without hyaluronic acid on HLMVEC Injured With an Inflammatory Insult. (A) The addition of 4-MU significantly suppressed the expression of HAS2 mRNA in HLMVEC in a dose-dependent manner over 24 h. Only 4-MU at a concentration of 1.0 mM led to an increase in protein permeability, whereas there were no significant differences with a 4-MU concentration of 0.1–0.5 mM on permeability compared with vehicle control. Data are presented as mean ± SD. ****p < 0.0001; N = 3–6 for PCR and N = 9 for permeability experiments. (B) Addition of 4-MU 0.5 mM with cytomix further decreased HAS2 mRNA expression and increased protein permeability across HLMVEC. There was no restorative effect of HMW HA on the decrease in HAS2 mRNA or increase in protein permeability. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; N = 3–6 for PCR and N = 9 for Permeability experiments. (C–E) However, by fluorescence microscopy and Western blot analyses, the addition of HMW HA partially decreased F-actin stress fiber and increased both ZO-1 and VE-Cadherin total protein levels and distribution to cell–cell junctions in HLMVEC injured with both cytomix and 4-MU 0.5 mM. Yellow Arrows = Gap formations between cells. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; N = 3. (F) 4-MU further suppressed CD44 protein expression and the phosphorylation of AKT after the addition of cytomix in HLMVEC. Only the addition of HMW HA partially increased the expression of CD44 at 24 h but not the phosphorylation of AKT. Data are presented as mean ± SD. *p < 0.05, **p < 0.01; N = 3. Individual p values are the statistical comparison between two groups by Student’s t test. Graphs were created using GraphPad Prism 8.4.3 for OS X (GraphPad Software, www.graphpad.com)

Fig 5: OGD-induced vascular leakage in vitro is prevented by scCM. Treatment with scCM during reoxygenation (24 h) abolished the OGD-induced increase in permeability on CMECs (a). WB analysis revealed that scCM increased occludin, ZO-1, and tricellulin expression (b) and favoured their localization at cell junctions (c). scCM increased VEGFR2 activation, while it did not affect ERK1/2 or AKT activation (d). Besides, scCM upregulated VCAM-1, ICAM-1, and COX2 expression (d). Data represent median (interquartile range) [b and d] or mean ± SD [a] (n = 3–5). *P < 0.05 vs scEBM; $P < 0.05 vs R-OGD scEBM. Scale bar: 10 µm