Matrix-guided 3D lumenized vessel network formation and stabilization by endothelial colony forming cells: Role of collagen intermolecular cross-links

Catherine Faye Whittington, Purdue University

Abstract

Compromised vascular perfusion is a major factor contributing to non-healing wounds and the progression of disease states such as ischemic heart disease and peripheral vascular disease due to inadequate blood flow throughout the damaged tissue region in vivo. It also becomes a problem in the design and success of engineered tissue constructs because of the lack of vascularization within the constructs. In both of these situations, the diffusion limitation of tissues has been exceeded and can only be remedied by creating a new vasculature with the ability to integrate with the host circulation and restore the blood flow to the affected area. This restoration can be achieved by recreating the process of vasculogenesis in which vessels are formed de novo undifferentiated endothelial precursor cell populations. Endothelial colony forming cells (ECFC) are promising therapeutic cells largely owing to their high proliferative capacity and ability to form vessel networks in vitro and in vivo. Combining ECFCs with a type I collagen-based delivery vehicle will allow for localization of ECFCs at the implantation site and guidance of ECFC vessel formation and stabilization. Type I collagen is an ideal biopolymer to serve as a 3D matrix for ECFCs because it determines in-vivo tissue form and function through its natural intermolecular cross-links and has a role in vascular development and maintaining vascular integrity. We have developed an uncommon set of collagen building blocks that differ in intermolecular cross-link composition and can be used to create 3D collagen matrices that differ in their biophysical properties. Using collagen oligomers, two or more covalently cross-linked collagen monomers, we can independently control collagen fibril density and interfibril branching to specify matrix stiffness in the current study, to guide vessel morphogenesis. Other studies in the laboratory have also shown that oligomers can be used to control matrix permeability and diffusivity, as well as proteolytic degradation. Increasing the oligomer/monomer ratio, while maintaining a constant collagen concentration, results in an increase in interfibril branching and subsequently an increase in matrix stiffness upon in-vitro neutralization and polymerization. Overall, oligomers show increased interfibril branching over monomer under matched stiffness and concentration conditions. Using a new 3D morphometric analysis method in which we quantified the microstructure of lumen-containing ECFC vessel networks in monomer and oligomer matrices, we observed that stable, mature intermolecular cross-links and associated oligomers support more complex vessel networks with larger lumen diameters, vessel length, and total volume than monomers at the same concentration and matrix stiffness. These vessels, with lumen, persist beyond 7 days, unlike endothelial cells or endothelial precursors seeded within traditional monomeric matrices, and begin to undergo maturation and stabilizing through basement membrane deposition, which contributes to vessel persistence, without the addition of anti-apoptotic agents, phorbol esters, or accessory cells. We propose that matrix-guided vasculogenesis is driven by a balance between ECM physical properties and cell traction forces and hypothesize that this balance is well established in oligomer matrices that are able to appropriately resist ECFC traction forces. To begin to explore and test this hypothesis, we focused on the role of the matrix-integrin-cytoskeletal signaling pathway in early- and late-stage vessel formation events to explain the mechanisms through which the cell-ECM force balance guides and regulates vessel morphogenesis. Using chemical perturbation of the actin cytoskeleton and focal adhesions and immunostaining of intra- and extracellular elements of the signaling pathway involved in vessel morphogenesis, we have observed qualitative differences in the ECFC response in vessel formation and the organization and expression of several elements. F-actin formation, β1 integrin binding, and focal adhesion kinase (FAK) activation all appeared to increase with increased matrix interfibril branching or oligomer content. Furthermore, stimulation of the actin cytoskeleton promoted ECFC vessel formation in monomer more than oligomer, and matrix metalloproteinase activity (MMP) appeared to be up-regulated in oligomer, as well. These results, combined with our vessel microstructure findings provide additional evidence to support our theory that ECM biophysical properties are essential in regulating vessel morphogenesis through a cell-ECM force balance. This work documents, for the first time, that intermolecular cross-links constitute a new and important design parameter and important determinant of the biophysical properties and vascular-instructive capacity of polymerizable collagen matrices to be used for cell culture, regenerative medicine, and engineered tissue applications.

Degree

Ph.D.

Advisors

Voytik-Harbin, Purdue University.

Subject Area

Cellular biology|Biomedical engineering

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