Vascular Smooth Muscle Cells in the Presence of Fibronectin Functionalized Collagen Scaffold Increases the Size of Endothelial Cell-based Vascular Aggregates

PURPOSE: Fibronectin-functionalized collagen scaffolds can promote induced-pluripotent-stem-cell-derived-vascular smooth muscle cell (iPSC-VSMC) to enhance their pro-angiogenic paracrine profiles. However, the influence of fibronectin collagen on human umbilical vein endothelial cells (HUVEC), a key component of angiogenesis, is still unknown. In this study, our objectives were to evaluate the behaviors of HUVEC within fibronectin-functionalized scaffolds and evaluate the integrity of iPSC-VSMC:HUVEC combination vascular formation in the setting of fibronectin-functionalized collagen scaffolds. METHODS: Fibronectin was added to type I collagen to obtain a final scaffold density of 4 mg per ml. HUVECs were incubated within the scaffold for a total of 7 days, and after the first 24 hours Echistatin, an integrin inhibitor of Alpha-v Beta-3 was added to scaffolds. The resultant scaffolds were evaluated for cellular viability via AlamarBlue assay. The scaffolds were immunofluorescence stained with CD144. Confocal microscope was used to count the total number of endothelial vascular aggregates, and then categorized based on sizes, greater or less than 50 µm. Next, we combined iPSC-VSMC and HUVEC at a ratio of 1:4 and embedded them into fibronectin-functionalized collagen for 7 days. The resultant scaffolds were then immunofluorescence stained with Sm-22alpha, NG2 and CD144. The number and sizes of the vascular aggregates were also evaluated. Result: iPSC-VSMCs embedded in fibronectin-functionalized collagen scaffold demonstrated no significant cellular viability from control collagen scaffold. However, the addition of Echistatin, an fibronectin inhibitor, to fibronectin scaffolds resulted in significant decrease in HUVEC viability when compared with control and fibronectin scaffold groups (P = 0.0001). The total number of vascular aggregates was significantly higher in fibronectin scaffolds than in control and Echistatin scaffolds (P value = 0.03). There was no significance between the number of large or small vascular aggregates amount the three groups. iPSC-VSMC:HUVEC combination scaffolds that were functionalized fibronectin scaffolds demonstrated increased number of vascular aggregates when compared with control and Echistatin scaffolds (P value = 0.003 and 0.002, respectively). In addition, the number of large vascular aggregates was increased in the fibronectin scaffolds containing iPSC-VSMC and HUVEC combination when compared with control and Echistatin scaffolds (P value = 0.0001, both). The number of large vascular aggregates was significantly decreased in the scaffolds treated with Echistatin (P value = 0.0001). However, the number of small vascular aggregate remained constant among the 3 groups. CONCLUSIONS: Fibronectin plays a key role in maintaining the HUVEC’s viability, since the addition of Echistatin, a fibronectin inhibitor, dramatically decreased the HUVEC’s viability. This suggested fibronectin to have an agonistic effect on HUVEC via interaction of Alpha-v Beta-3 integrin expressed on the cells. The higher number of large vascular aggregates in iPSC-VSMC and HUVEC combination in fibronectin scaffolds suggested that fibronectin was promoting iPSC-VSMC interaction with HUVEC to promote formation and maintenance of larger and complex vascular aggregates. Echistatin scaffolds as the inhibitor caused the number large aggregates to diminish down close to none. These findings resulted in a better understanding of potentially an underlying mechanism of how to optimize iPSC-VSMC and HUVEC interaction to improve vascular formations by specifically targeting sites such as Alpha-v Beta-3 integrin, which may eventually translate to more effective cell therapy-based wound healing treatments.