Regulation of endothelial cell activation and angiogenesis by injectable peptide nanofibers

Abstract

RAD16-II peptide nanofibers are promising for vascular tissue engineering and were shown to enhance angiogenesis in vitro and in vivo, although the mechanism remains unknown. We hypothesized that the pro-angiogenic effect of RAD16-II results from low-affinity integrin-dependent interactions of microvascular endothelial cells (MVECs) with RAD motifs. Mouse MVECs were cultured on RAD16-II with or without integrin and MAPK/ERK pathway inhibitors, and angiogenic responses were quantified. Results were validated in vivo using mouse diabetic wound healing model with impaired neovascularization. RAD16-II stimulated spontaneous capillary morphogenesis, increased β3 integrin phosphorylation and VEGF expression in MVECs. These responses were abrogated in the presence of β3 and MEK inhibitors or on the control peptide without RAD motifs. Wide-spectrum integrin inhibitor echistatin completely abolished RAD16-II-mediated capillary morphogenesis in vitro and neovascularization and VEGF expression in the wound in vivo. Addition of the RGD motif to RAD16-II did not change nanofiber architecture or mechanical properties, but resulted in significant decrease in capillary morphogenesis. Overall, these results suggest that low-affinity non-specific interactions between cells and RAD motifs can trigger angiogenic responses via phosphorylation of β3 integrin and MAPK/ERK pathway, indicating that low-affinity sequences can be used to functionalize bio-compatible materials for the regulation of cell migration and angiogenesis, thus expanding the current pool of available motifs that can be used for such functionalization. Incorporation of RAD or similar motifs into protein engineered or hybrid peptide scaffolds may represent a novel strategy for vascular tissue engineering and will further enhance design opportunities for new scaffolds materials.

Angiogenic microenvironment augments impaired endothelial responses under diabetic conditions

“…Therefore, in our studies, diabetic and normal cell responses were quantified for two substrates, Matrigel and the proangiogenic nanofiber hydrogel. We have previously shown that these nanofibers support cellular function in multiple cardiac cell types including cardiomyocytes, endothelial cells, and fibroblasts (26,42), and that their proangiogenic mechanism of action involves sustained angiogenic activation of endothelial cells through ␣ v ␤ 3 -integrins (10). The results of the present study demonstrate that the stimulating effect of the nanofiber microenvironment on endothelial angiogenic and biomechanical responses is present under both normal and diabetic conditions.…”

“…The effect of diabetes on capillary morphogenesis was determined using Matrigel assay, and the quantitative analysis of the characteristic network sizes was performed as described previously (10,41). Under static conditions, capillary morphogenesis was significantly reduced in both db and acute HG groups, with a larger decrease in HG group compared with the db group (Fig.…”

“…A novel tissue engineering approach, which includes the use of a nanoscaffold composed of self-assembling peptide RAD16-II nanofibers, has been shown to promote angiogenesis in nondiabetic conditions (12,26,41) and in a mouse model of diabetic wound healing in vivo (5). Studies by our group and others have demonstrated that these nanofibers support cellular function in multiple cardiac cell types including cardiomyocytes, endothelial cells, and fibroblasts (26,42) and enhance angiogenic responses through engagement of specific cell surface integrins, namely ␣ v ␤ 3 (10,52).…”

Regulation of endothelial MAPK/ERK signalling and capillary morphogenesis by low-amplitude electric field

“…In the capillary morphogenesis assay used in this study, endothelial cells undergo spontaneous formation of multicellular capillary structures with clearly identifiable lumens by 12 h of cell seeding on the nanofibre hydrogel [41,42]. High-frequency EF exposure resulted in significantly larger structures, when compared with low-frequency and no-EF groups ( p , 0.001, figure 2a,b), while no significant differences between low-frequency and no-EF groups were observed.…”

Nanofiber Microenvironment Effectively Restores Angiogenic Potential of Diabetic Endothelial Cells

“…In vitro studies have shown that RAD16-II nanofibers support multiple cell types (including cardiomyocytes, ECs, and fibroblasts), enhance capillary morphogenesis and angiogenic growth factor expression, and allow for long-term study of cell-cell interactions. 21,22 Our recent study 23 presented a novel mechanism for nanofiber-mediated activation of angiogenic signaling via relatively weak low-affinity interactions between ECs and RAD motifs in the RAD16-II nanofibers, which can trigger phosphorylation of b 3 integrin domain. In ECs, integrin a v b 3 is the most abundant and influential receptor regulating angiogenesis.…”