Glycosaminoglycan-based hydrogels to modulate heterocellular communication in in vitro angiogenesis models

Abstract

Angiogenesis, the outgrowth of blood vessels, is crucial in development, disease and regeneration. Studying angiogenesis in vitro remains challenging because the capillary morphogenesis of endothelial cells (ECs) is controlled by multiple exogenous signals. Therefore, a set of in situ-forming starPEG-heparin hydrogels was used to identify matrix parameters and cellular interactions that best support EC morphogenesis. We showed that a particular type of soft, matrix metalloproteinase-degradable hydrogel containing covalently bound integrin ligands and reversibly conjugated pro-angiogenic growth factors could boost the development of highly branched, interconnected, and lumenized endothelial capillary networks. Using these effective matrix conditions, 3D heterocellular interactions of ECs with different mural cells were demonstrated that enabled EC network modulation and maintenance of stable vascular capillaries over periods of about one month in vitro. The approach was also shown to permit in vitro tumor vascularization experiments with unprecedented levels of control over both ECs and tumor cells. In total, the introduced 3D hydrogel co-culture system could offer unique options for dissecting and adjusting biochemical, biophysical, and cell-cell triggers in tissue-related vascularization models.A ngiogenesis describes the outgrowth of new blood vessels and is a research 'hot spot' as emerging therapeutic opportunities for various different pathologic conditions directly relate to this widespread phenomenon. The formation of vascular sprouts is a complex multistep process including: (1) growth factor (GF) gradient formation, (2) endothelial cell (EC) activation, migration, and proliferation, (3) capillary development, and (4) stabilization by mural cells 1 . Studying the process of vascular sprout formation in vitro requires 3D models, which recapitulate the consecutive steps of capillary formation. Most angiogenesis research focuses on the biology of ECs, which are the core component of vascular structures. However, their activity in vivo is tightly regulated by supporting cell types such as pericytes and smooth muscle cells (SMCs) 2 indicating the importance of the often-neglected heterocellular interactions in in vitro models of angiogenesis. Isolated ECs preserve their vasculogenic and angiogenic characteristics in culture. When embedded in a suitable 3D scaffold, ECs temporarily form inter-connected tubular networks resembling vascular capillaries 3 , thus enabling in vitro vascular sprouting studies. Tissue-derived biopolymer matrices such as collagen 4-6 and fibrin 7,8 were shown to induce EC vascular morphogenesis successfully, and significantly contributed to our knowledge about angiogenesis, but their poor stability limits the development of defined, long term, in vitro assays. In search for improvement, synthetic polymer materials were systematically tested and studies using poly(ethylene glycol) (PEG) 9 and hyaluronic acid (HA) 10,11 based hydrogels demonstrated that EC capillary formation is str...

Dual Role of Mesenchymal Stem Cells Allows for Microvascularized Bone Tissue‐Like Environments in PEG Hydrogels

“…This is in agreement with previous studies in semisynthetic hydrogels (PEG-Heparin, PEG-Collagen, GelMA) where the presence of mesenchymal cells including 10T1/2 cells, human fi broblasts, and MSCs [28][29][30] supported the formation of microvascular structures by HUVECs or late outgrowth endothelial cells (OECs). Similarly, in fully synthetic hydrogels, the presence of mesenchymal lineage cells could stabilize cord-like structures by HUVECs in vitro.…”

Perivascular signals alter global gene expression profile of glioblastoma and response to temozolomide in a gelatin hydrogel

“…A few studies have examined the interactions between GBM cells, perivascular niche cells, and macrophages within 3D in vitro platforms, but these models feature spatial segregation between the different cell types that is not reminiscent of the in vivo microenvironment [20,[31][32][33][34]. Mixed cultures of tumor cells, endothelial cells, and stromal cells have been achieved within biomaterials for breast, lung, and prostate cancers, and these models have been used to investigate how non-tumor cells impact tumor proliferation, phenotype, and therapeutic response [35][36][37][38][39]. Our lab has recently described a tri-culture biomaterial platform for the purpose of modeling the perivascular niche of GBM [40].…”

Hydrogels for therapeutic cardiovascular angiogenesis

“…Recently, a novel 3D in vitro model of heterocellular angiogenesis utilizing in situ-forming starPEG-heparin hydrogels as a scaffold was reported [94]. These scaffolds are composed of multi-armed PEG and heparin crosslinked via cytocompatible Michael type addition that enabled cells to be embedded in 3D.…”