Figure 1.
Biomechanical stress induces MEC proliferation.
Proliferation of Ben-Men-I cells (A) and primary porcine MECs (B) exposed to elevated and ambient pressure for 2 days. Proliferation of Ben-Men-I cells and PMECs increased in all cell concentration groups that were exposed to elevated pressure compared to control cells cultivated under ambient pressure conditions (shown is a representative result of three independent experiments; error bars represent SD; Student's t-test: p<0.01, marked with ** for highly significant).
Figure 2.
Endocytotic function of MECs is impacted by elevated pressure.
Ben-Men-I cells (A) or PMECs (B) were treated with elevated pressure for two days and fluorescent latex beads were added to assess endocytotic activity by fluorescence microscopy (>100 cells scored/condition). Ben-Men-I cells (Student's t-test: p<0.01, marked with ** for highly significant) as well as PMECs (Student's t-test: p<0.05, marked with * for significant) showed a significant decrease in endocytotic activity after pressure treatment compared to control treated cells (shown is a representative result of three independent experiments; error bars represent SD).
Figure 3.
Hypoxia does not impact MEC function.
Ben-Men-I cells and PMECs were incubated under limited oxygen conditions (1% O2) and compared to cells cultivated under normoxia. Cellular viability was only slightly impacted by this treatment in Ben-Men-I (A – representative result shown of three independent experiments; error bars represent SD; Student's t-test: p<0.05, marked with *) but not significantly in PMECs (C, marked with n.s. for not significant). Evaluation of endocytotic activity by scoring fluorescent latex bead uptake did not reveal a significant difference after hypoxia in Ben-Men-I cells (B, marked with n.s. for not significant) or PMECs (D, marked with n.s. for not significant).
Figure 4.
Oxidative stress impairs MEC function.
Ben-Men-I cells (A; error bars represent +SEM; ANOVA p<0.01, Tukey's HSD post-hoc test see Figure S1A) (5×104 cells/ml) or PMECs (C; error bars represent +SEM; ANOVA p<0.01, Tukey's HSD post-hoc test see Figure S1B) were treated with varying concentrations of the complex I inhibitor rotenone (0–20 µM) for 24 hours to induce oxidative stress. Oxidative damage resulting in decreased viability was measured using MTS. The impact of oxidative stress on MEC function in terms of endocytosis was analyzed by scoring fluorescent latex bead uptake following treatment with different concentrations (0, 0.05 and 0.75 µM) of rotenone. Intensity of latex beads engulfed by Ben-Men-I cells (B; >100 cells/condition; error bars represent SD of three independent counts; Student's t-test: p<0.05; marked with * for significant) or PMECs (D; >100 cells/condition; error bars represent SD of three independent counts; Student's t-test: p>0.05; marked with n.s. for not significant) decreases with increasing of rotenone concentration where Ben-Men-I cells display a high sensitivity towards rotenone with a reduction in endocytosis at low, non toxic rotenone concentrations while PMECs are more resistant to oxidative stress and maintain their rate of endocytosis.