Prolonged cyclic strain inhibits human endothelial cell growth

Abstract

The vascular endothelium is continuously exposed to cyclic mechanical strain due to the periodic change in vessel diameter as a result of pulsatile blood flow. Since emerging evidence indicates the cyclic strain plays an integral role in regulating endothelial cell function, the present study determined whether application of a physiologic regimen of cyclic strain (6% at 1 hertz) influences the proliferation of human arterial endothelial cells. Prolonged exposure of human dermal microvascular or human aortic endothelial cells to cyclic strain for up to 7 days resulted in a marked decrease in cell growth. The strain-mediated anti-proliferative effect was associated with the arrest of endothelial cells in the G2/M phase of the cell cycle, did not involve cell detachment or cytotoxicity, and was due to the induction of p21. Interestingly, the inhibition in endothelial cell growth was independent of the strain regimen since prolonged application of constant or intermittent 6% strain was also able to block endothelial cell proliferation. The ability of chronic physiologic cyclic strain to inhibit endothelial cell growth represents a previously unrecognized mechanism by which hemodynamic forces maintain these cells in a quiescent, non-proliferative state.

1.

Physiologic cyclic strain inhibits endothelial cell proliferation. HMECs (A) or HAECs (B) were treated with serum (5%) and subjected to strain-free control or cyclic strain (6% at 1 Hz) conditions for up to 7 days. Results are means ±SEM (n=3–5). *Statistically significant effect of cyclic strain.

2.

Intermittent or constant strain inhibits endothelial cell proliferation. HMECs were treated with serum (5%) and subjected to strain-free control or intermittent strain (6% strain for 5 minutes followed by a two minute strain-free interval) (A), or constant strain (6%) (B) for up to 6 days. Results are means ±SEM (n=3–4). *Statistically significant effect of strain.

3.

Physiologic cyclic strain inhibits cell cycle progression in endothelial cells. Representative histograms of DNA content (in arbitrary units) in HMECs treated with serum (5%) and subjected to strain-free control or cyclic strain (6% at 1 Hz) conditions for 3 days (A). Distribution of cells in the cell cycle in HMECs treated with serum (5%) and subjected to strain-free control or cyclic strain (6% at 1 Hz) conditions for 3 days (B). Results are means ±SEM (n=4). *Statistically significant effect of cyclic strain.

4.

Physiologic cyclic strain inhibits endothelial cell proliferation via the induction of p21. Western blotting demonstrating the expression of cell cycle regulatory proteins in HMECs treated with serum (5%) and subjected to strain-free control or cyclic strain (6% at 1 Hz) conditions for 3 days (A) or 1 day (B). Northern blotting demonstrating the expression of p21 mRNA in HMECs treated with serum (5%) and subjected to strain-free control or cyclic strain (6% at 1 Hz) conditions for 3 days (C). Expression of p21 protein in cells transfected with p21 siRNA (0.1μM) or non-targeting (NT) siRNA (0.1μM) and subjected to strain-free control or cyclic strain (6% at 1 Hz) conditions for 3 days (D). HMEC proliferation in cells transfected with p21 siRNA (0.1μM) or NT siRNA (0.1μM) and subjected to strain-free control or cyclic strain (6% at 1 Hz) conditions for up to 3 days (E). Protein and mRNA expression was quantified by scanning densitometry, normalized with respect to β-actin or GAPDH, respectively, and expressed relative to that of control cells or in arbitrary units. Results are means ±SEM (n=3–5). *Statistically significant effect of cyclic strain.