Angiogenic endothelial cell invasion into fibrin is stimulated by proliferating smooth muscle cells

Abstract

These studies aimed to determine the effect of smooth muscle cells (SMCs) on angiogenic behavior of endothelial cells (ECs) within fibrin hydrogels, an extracellular matrix (ECM) commonly used in tissue engineering. We developed a 3-D, fibrin-based co-culture assay of angiogenesis consisting of aggregates of SMCs with ECs seeded onto the aggregates’ surface. Using digital fluorescence micrography, EC matrix invasion was quantified by average length of sprouts (ALS) and density of sprout formation (DSF). We demonstrated that ECs and SMCs co-invade into the ECM in close proximity to one another. ECs that were co-cultured with SMCs demonstrated increased invasion compared to ECs that were cultured alone at all time points. At Day 19, the ALS of ECs in co-culture was 327 +/− 58 µm versus 70 +/− 11 µm of ECs cultured alone (p=.01). The DSF of co-cultured ECs was also significantly greater than that of ECs cultured alone (p=.007 on Day 19). This appeared to be a function of both increased EC invasion and well as improved persistence of EC sprout networks. At 7 days, ECs in co-culture with proliferation-inhibited SMCs previously treated with Mitomycin-C (MMC) demonstrated significantly attenuated sprouting compared to ECs co-cultured with SMCs that were untreated with MMC (82 +/− 14 µm versus 205 +/− 32 µm; p < .05). In assays in which multiple co-culture aggregates were cultured within a single hydrogel, we observed directional invasion of sprouts preferentially towards the other aggregates within the hydrogel. In co-culture assays without early EC/SMC contact, the ALS of ECs cultured in the presence of SMCs was significantly greater than those cultured in the absence of SMCs by Day 3 (320 +/− 21 µm versus 187 +/− 16 µm; p < .005). We conclude that SMCs augment EC matrix invasion into 3-D fibrin hydrogels, at least in part resulting from SMC proliferative and invasive activity. Directed invasion between co-culture aggregates and augmented angiogenesis in the absence of early contact suggests a paracrine mechanism for the observed results.

Beyond organoids: In vitro vasculogenesis and angiogenesis using cells from mammals and zebrafish

“…Other endothelial cell types such as bovine aortic endothelial cells [111] and rat aortic endothelial cells [112] have also been used to culture vascular networks. mesenchymal stem cells [109], fibroblasts [107], hepatocytes [114], smooth muscle cells [115] and adipose-derived stem cells (ASCs) [106]. The importance of fibroblasts in enhancing angiogenesis has been shown in co-culture with HUVECs [107,116].…”

Porcine arterial ECM hydrogel: Designing an in vitro angiogenesis model for long‐term high‐throughput research

“…To address the use of the newly developed paECM hydrogel as an angiogenesis model, we tested the co‐culture of endothelial cells with two types of supporting cells. SMCs and MSCs, often used for supporting angiogenesis function, 24,34 were addressed in different ratios with endothelial cells, and the cell growth, survival, and the formation of a net‐like structure were examined. When cultivating HUVECs with MSCs at a 2:1 ratio, the cells had formed a complex network structure, which stably persisted for 14 days.…”

Multilineage Constructs for Scaffold‐Based Tissue Engineering: A Review of Tissue‐Specific Challenges

“…The application of mechanical solicitations in situ (tensile strain, compression) is another way to tune the environment to trigger specific cell behaviors in coculture environment. In addition, the outcomes of the procedure will be different in direct cell–cell contact and indirect coculture systems, where the two cell types are seeded in different layers of a multiphasic scaffold and communicate by paracrine signaling . Paracrine signaling in coculture is very important, as it can eliminate the need for the addition of growth factors in the culture medium, thus leading to a cheaper and safer process .…”

“…This finding is very interesting, since it can be used for surface endothelialization of implants or scaffolds prior to their implantation . The effects of coculture on endothelial cell functions were investigated by seeding ECs with SMCs on fibrin hydrogel . SMCs strongly enhanced the invasion of the 3D fibrin matrix by the ECs, in both indirect and direct contact conditions, thus indicating their mutually beneficial effects in the development of in vitro rebuilt constructs.…”