The interplay of cyclic stretch and vascular endothelial growth factor in regulating the initial steps for angiogenesis.

Angiogenesis is regulated by chemical and mechanical factors in vivo. The regulatory role of mechanical factors and how chemical and mechanical angiogenic regulators work in concert remains to be explored. We investigated the effect of cyclic uniaxial stretch (20%, 1 Hz), with and without the stimulation of vascular endothelial growth factor (VEGF), on sprouting angiogenesis by employing a stretchable three-dimensional cell culture model. When compared to static controls, stretch alone significantly increased the density of endothelial sprouts, and these sprouts aligned perpendicular to the direction of stretch. The Rho-associated kinase (ROCK) inhibitor Y27632 suppressed stretch-induced sprouting angiogenesis and associated sprout alignment. While VEGF is a potent angiogenic stimulus through ROCK-dependent pathways, the combination of VEGF and stretch did not have an additive effect on angiogenesis. In the presence of VEGF stimulation, the ROCK inhibitor suppressed stretch-induced sprout alignment but did not affect stretch-induced sprout density; in contrast, the receptor tyrosine kinase (RTK) inhibitor sunitinib had no effect on stretch-induced alignment but trended toward suppressed stretch-induced sprout density. Our results suggest that the formation of sprouts and their directionality do not have completely identical regulatory pathways, and thus it is possible to separately manipulate the number and pattern of new sprouts.

Differential effects of cyclic stretch on bFGF- and VEGF-induced sprouting angiogenesis.

How mechanical factors affect angiogenesis and how they and chemical angiogenic factors work in concert remain not yet well-understood. This study investigated the interactive effects of cyclic uniaxial stretch and two potent proangiogenic molecules [basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)] on angiogenesis using a stretchable three-dimensional (3-D) cell culture model. Endothelial cells seeded atop a 3-D collagen gel underwent sprouting angiogenesis while being subjected to either 10 or 20% cyclic uniaxial stretch at a frequency of either 1/12 or 1 Hz, in conjunction with an elevated concentration of bFGF or VEGF. Without the presence of additional growth factors, 10 and 20% stretch at 1 Hz induced angiogenesis and the perpendicular alignment of new sprouts, and both inductive effects were abolished by cytochalasin D (an actin polymerization inhibitor). While "10% stretch at 1 Hz," "20% stretch at 1 Hz," bFGF, and VEGF were strong angiogenesis stimulants individually, only the combination of "20% stretch at 1 Hz" and bFGF had an additive effect on inducing new sprouts. Interestingly, the combination of "20% stretch at a lower frequency (1/12 Hz)" and bFGF decreased sprouting angiogenesis, even though the level of perpendicular alignment of new sprouts was the same for both stretch frequencies. Taken together, these results demonstrate that both stretch frequency and magnitude, along with interactions with various growth factors, are essential in mediating formation of endothelial sprouts and vascular patterning. Furthermore, work in this area is warranted to elucidate synergistic or competitive signaling mechanisms.