Understanding the Effects of Cyclic Strain on Angiogenesis.

Abstract

The size of engineered tissues is currently limited by oxygen diffusion. Vascularization of these constructs could alleviate this problem if the cues that control blood vessel organization can be manipulated. Our laboratory has adapted a method for generating bona fide capillaries in fibrin gels to study how mechanical strain affects the growth and organization of engineered blood vessel networks and remodeling of the extracellular matrix. A PDMS multi-well platform was designed to allow cell culture in a mechanically labile environment that also allowed visualization of the extracellular matrix under strain. The platform was calibrated using a custom microscope stage mount composed of two clamps attached to a micrometer for strain application. Strains from 0 – 12% were applied to fibrin hydrogels and the corresponding principle strains were calculated, revealing a linear relationship between the two. Cell culture experiments were performed using a custom, automated linear stage able to apply cyclic or static strains to the PDMS platform under standard mammalian cell culture conditions. These experiments revealed that human umbilical vein endothelial cells, in the presence of supportive human aortic smooth muscle cells, formed vasculature parallel to the applied strain and that the alignment was reversible upon halting the strain. Examination of the fibrin matrix revealed no cell-mediated directional matrix remodeling and only modest alignment of the matrix at 10% strain, the maximum used in these studies. Contact guidance was thus ruled out as a primary source of capillary alignment, prompting an investigation of intracellular mechanotransduction as a mediator of the directional sprouting. The use of multiple genetic constructs to modulate RhoA activity and the application of drugs to inhibit Rho-associated kinase, myosin light chain kinase, and myosin ATPase showed that perturbation of this mechanosensitive pathway led to decreased capillary network lengths without diminishing the ability of the capillaries to align parallel to the direction of applied strain. These findings demonstrate that mechanical inputs can be used to control angiogenic patterning in engineered tissues and suggest that an alternative mechanosensitive pathway may be responsible for directional angiogenesis in response to strain.