Hypoxia-inducible hydrogels facilitate study of cluster-based neovascularization in vitro

Michael R. Blatchley1,2, Songnan Wang2, Franklyn Hall3, Sharon Gerecht2,4

1Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA;

2Department of Chemical and Biomolecular Engineering and Institute for NanoBioTechnology , Johns Hopkins University Whiting School of Engineering, Baltimore, MD, USA, 3Department of Chemical Engineering, Mississippi State University, Starkville, MS, USA, 4Department of Materials Science and Engineering, Johns Hopkins University Whiting School of Engineering, Baltimore, MD, USA

The mechanisms governing postnatal vasculogenesis have begun to be uncovered in the last several decades. These mechanisms have informed the design of numerous therapeutics, seeking to regenerate or control the formation of new vasculature following loss-of-function diseases, such as peripheral and coronary artery diseases, as well as provide an understanding of tumor angiogenesis. The processes which govern vascular regeneration in such clinical pathologies involve complex spatiotemporal interplay between physical and biochemical cues. The plenary work of several groups has described one mechanism by which blood vessels may form de novo. In this body of work, new blood vessels are formed through intracellular changes in a population wide phenomenon, that subsequently leads to sprouting and branching through coalescence of single cells to form multicellular, luminal vessels. In addition to these works, observational reports have indicated the presence of a secondary, potentially parallel mechanism. In particu lar, endothelial progenitor cell (EPC) cluster formation has been reported in ischemic and hypoxic tissues. This mechanism commences with the formation of multicellular clusters that then sprout to form luminal structures, which can anastomose with existing vasculature. However, an understanding of the regulatory mechanism governing these processes is limited, due to a shortage in model systems that recapitulate the pathological microenvironmental cues.
The advent of biomaterials technologies has enabled precise control over in vitro microenvironments. Such biomaterials have facilitated studies which have uncovered a variety of biomechanical and biochemical cues that influence tissue morphogenesis. Specifically, we have developed hydrogel materials with precise control over two potent regulators of vascular regeneration: oxygen tension and matrix viscoelasticity. Our previous works established hydrogels with O2 gradients ranging from <1% to 21%. However, cellular responses to hypoxia vary widely based on specific and precise changes in O2 tension. Here, by generating layered hydrogels that discretize O2 gradients, we have studied cellular behavior and examined the effects of varying O2 tensions in 3D. Additionally, we can predictably tune matrix mechanics within each layer by the addition or omission of a secondary crosslinker, microbial transglutaminase (mTG).
Our initial observations indicate that hypoxic cluster-based vasculogenesis comprises a two-step mechanism: (1) cluster formation, stabilization and expansion, and (2) cluster sprouting and branching. Interestingly, we have consistently observed that low O2 tension promotes clustering of EPCs. We have also shown that this process is mediated by matrix metalloproteinase (MMP) production, which leads to matrix degradation that enables cell clustering. Upon concurrent encapsulation of EPCs with DQ-gelatin, which fluoresces in the presence of MMPs, we have confirmed the role of MMP in cluster formation. Additionally, we have analyzed junctional and cytoskeletal protein expression including intercellular adhesion molecule 1 (ICAM-1), integrin β2, vascular endothelial cadherin (VE-cad), and phalloidin, to delineate how cell-cell interactions mediate cluster formation, maintenance, and expansion. To determine the important factors regulating the second step of the proposed process, we have specifically and dynamically controlled matrix viscoelasticity. Dynamic regulation of matrix mechanics, specifically increases in matrix stiffness and crosslinking density by addition of mTG, increased sprouting from EPC clusters by coaxing cell-ECM interactions.