Sprouting endothelial cells (ECs) use soluble and insoluble cues to guide migration and expand the existing vascular network to meet changing trophic needs of the tissue during angiogenesis. A noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique was used to optically image endothelial sprouting morphogenesis in three dimensional (3D) collagen matrices with simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Sprout advancement and lumen expansion companying with ECM alteration were the synergistic results of membrane-associated matrix metalloproteinase and cell traction evidenced by proteinase inhibition and Rho-associated kinase (p160ROCK) inhibition experiments. These physical EC-ECM interactions suggest that ECM mechanical properties may influence angiogenic responses. In a 3D angiogenesis model, we measure angiogenic responses as a function of collagen matrix stiffness by inducing collagen cross-linking with microbial transglutaminase (mTG). Collagen matrices stiffen with both mTG treatment and incubation time as evidenced with biaxial mechanical test results and collagen TPF intensity increases with mTG treatment and that the ratio of TPF/SHG correlates with biaxial tested mechanical stiffness. SHG and optical coherence microscopy (OCM) are further used to show that other physical properties of the matrix do not change with mTG treatment, thus providing the same density but different stiffness with which to measure angiogenic responses. Stiffer matrices promote angiogenesis with more invading sprouts that invade deeper. No differences in lumen size were observed between control and mTG stiffened 3D cultures, but there was evidence of greater matrix remodeling in stiffer gels using NLOM. Results of this study show angiogenic responses are enhanced with increasing stiffness and suggest that these properties may be used in tissue engineering and regenerative medicine applications to engineer angiogenesis.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/148244 |
| Date | 14 March 2013 |
| Creators | Lee, Po-Feng 1976- |
| Contributors | Yeh, Alvin T |
| Source Sets | Texas A and M University |
| Detected Language | English |
| Type | Thesis, text |
| Format | application/pdf |
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