Mechanical Intercellular Communication via Matrix-Borne Cell Force Transmission During Vascular Network Formation.

Intercellular communication is critical to the formation and homeostatic function of all tissues. Previous work has shown that cells can communicate mechanically via the transmission of cell-generated forces through their surrounding extracellular matrix, but this process is not well understood. Here, mechanically defined, synthetic electrospun fibrous matrices are utilized in conjunction with a microfabrication-based cell patterning approach to examine mechanical intercellular communication (MIC) between endothelial cells (ECs) during their assembly into interconnected multicellular networks. It is found that cell force-mediated matrix displacements in deformable fibrous matrices underly directional extension and migration of neighboring ECs toward each other prior to the formation of stable cell-cell connections enriched with vascular endothelial cadherin (VE-cadherin). A critical role is also identified for calcium signaling mediated by focal adhesion kinase and mechanosensitive ion channels in MIC that extends to multicellular assembly of 3D vessel-like networks when ECs are embedded within fibrin hydrogels. These results illustrate a role for cell-generated forces and ECM mechanical properties in multicellular assembly of capillary-like EC networks and motivates the design of biomaterials that promote MIC for vascular tissue engineering.

Fiber Crimp Confers Matrix Mechanical Nonlinearity, Regulates Endothelial Cell Mechanosensing, and Promotes Microvascular Network Formation.

Mechanical interactions between cells and their surrounding extracellular matrix (ECM) guide many fundamental cell behaviors. Native connective tissue consists of highly organized, 3D networks of ECM fibers with complex, nonlinear mechanical properties. The most abundant stromal matrix component is fibrillar type I collagen, which often possesses a wavy, crimped morphology that confers strain- and load-dependent nonlinear mechanical behavior. Here, we established a new and simple method for engineering electrospun fibrous matrices composed of dextran vinyl sulfone (DexVS) with controllable crimped structure. A hydrophilic peptide was functionalized to DexVS matrices to trigger swelling of individual hydrogel fibers, resulting in crimped microstructure due to the fixed anchorage of fibers. Mechanical characterization of these matrices under tension confirmed orthogonal control over nonlinear stress-strain responses and matrix stiffness. We next examined ECM mechanosensing of individual endothelial cells (ECs) and found that fiber crimp promoted physical matrix remodeling alongside decreases in cell spreading, focal adhesion area, and nuclear localization of Yes-associated protein (YAP). These changes corresponded to an increase in migration speed, along with evidence for long-range interactions between neighboring cells in crimped matrices. Interestingly, when ECs were seeded at high density in crimped matrices, capillary-like networks rapidly assembled and contained tube-like cellular structures wrapped around bundles of synthetic matrix fibers due to increased physical reorganization of matrix fibers. Our work provides an additional level of mechanical and architectural tunability to synthetic fibrous matrices and implicates a critical role for mechanical nonlinearity in EC mechanosensing and network formation.

Myofibroblast activation in synthetic fibrous matrices composed of dextran vinyl sulfone.

Mechanical interactions between fibroblasts and their surrounding extracellular matrix (ECM) guide fundamental behaviors such as spreading, migration, and proliferation that underlie disease pathogenesis. The challenges of studying ECM mechanics in vivo have motivated the development of in vitro models of the fibrous ECM in which fibroblasts reside. Natural materials such as collagen hydrogels bear structural and biochemical resemblance to stromal ECM, but mechanistic studies in these settings are often confounded by cell-mediated material degradation and the lack of structural and mechanical tunability. Here, we established a new material system composed of electrospun dextran vinyl sulfone (DexVS) polymeric fibers. These fibrous matrices exhibit mechanical tunability at both the single fiber (80-340 MPa) and bulk matrix (0.77-11.03 kPa) level, as well as long-term stability in mechanical properties over a two-week period. Cell adhesion to these matrices can be either user-defined by functionalizing synthetic fibers with thiolated adhesive peptides or methacrylated heparin to sequester cell-derived ECM proteins. We utilized DexVS fibrous matrices to investigate the role of matrix mechanics on the activation of fibroblasts into myofibroblasts, a key step of the fibrotic progression. In contrast to previous findings with non-fibrous hydrogel substrates, we find that fibroblasts in soft and deformable matrices exhibit increased spreading, focal adhesion formation, proliferation, and myofibroblast activation as compared to cells on stiffer matrices with equivalent starting architecture. STATEMENT OF SIGNIFICANCE: Cellular mechanosensing of fibrillar extracellular matrices plays a critical role in homeostasis and disease progression in stromal connective tissue. Here, we established a new material system composed of electrospun dextran vinyl sulfone polymeric fibers. These matrices exhibit architectural, mechanical, and biochemical tunability to accurately model diverse tissue microenvironments found in the body. In contrast to previous observations with non-fibrous hydrogels, we find that fibroblasts in soft and deformable fibrous matrices exhibit increased spreading and focal adhesion formation as compared to those in stiffer matrices with equivalent architecture. We also investigated the role of matrix stiffness on myofibroblast activation, a critical step in the fibrotic cascade, and find that low stiffness matrices promote increased myofibroblast activation.

Cell force-mediated matrix reorganization underlies multicellular network assembly.

Vasculogenesis is the de novo formation of a vascular network from individual endothelial progenitor cells occurring during embryonic development, organogenesis, and adult neovascularization. Vasculogenesis can be mimicked and studied in vitro using network formation assays, in which endothelial cells (ECs) spontaneously form capillary-like structures when seeded in the appropriate microenvironment. While the biochemical regulators of network formation have been well studied using these assays, the role of mechanical and topographical properties of the extracellular matrix (ECM) is less understood. Here, we utilized both natural and synthetic fibrous materials to better understand how physical attributes of the ECM influence the assembly of EC networks. Our results reveal that active cell-mediated matrix recruitment through actomyosin force generation occurs concurrently with network formation on Matrigel, a reconstituted basement membrane matrix regularly used to promote EC networks, and on synthetic matrices composed of electrospun dextran methacrylate (DexMA) fibers. Furthermore, modulating physical attributes of DexMA matrices that impair matrix recruitment consequently inhibited the formation of cellular networks. These results suggest an iterative process in which dynamic cell-induced changes to the physical microenvironment reciprocally modulate cell behavior to guide the formation and stabilization of multicellular networks.