Angiogenic properties and intercellular communication of differentiated porcine endothelial cells in vascular therapy

Abstract

Endothelial cell dysfunction can lead to various vascular diseases. Blood flow disorder is a common symptom of vascular diseases. Regenerative angiogenesis, which involves transplanting vascular cells or stem cells into the body to shape new vasculature, can be a good therapeutic strategy. However, there are several limitations to using autologous cells from the patients themselves. We sought to investigate the new vascular cells that can play a role in the formation of angiogenesis in vivo using stem cells from alternative animals suitable for cellular therapy. Porcine is an optimal animal model for xenotransplantation owing to its physiological similarity to humans. We used differentiated porcine endothelial cells (pECs) as a therapeutic strategy to restore vessel function. Differentiated pECs formed vessel-like structures in mice, distinguishing them from stem cells. MMPs activity and migration assays indicated that differentiated pECs possessed angiogenic potential. Tube formation and 3D spheroid sprouting assays further confirmed the angiogenic phenotype of the differentiated pECs. Immunofluorescence and immunoprecipitation analyses revealed claudin-mediated tight junctions and connexin 43-mediated gap junctions between human ECs and differentiated pECs. Additionally, the movement of small RNA from human ECs to differentiated pECs was observed under co-culture conditions. Our findings demonstrated the in vivo viability and angiogenetic potential of differentiated pECs and highlighted the potential for intercellular communication between human and porcine ECs. These results suggest that transplanted cells in vascular regeneration completed after cell therapy have the potential to achieve intercellular communication within the body.

Keywords: Angiogenesis; Cellular therapy; Endothelial cells; Porcine; Xenotransplantation.

Fig. 1

Formation of vascular structures in vivo by differentiated pECs. (A) schematic outline of porcine cells transplantation in Balb/c nude mouse. Created with BioRender.com (B) Immunofluorescence images depicting CD-31 expression (green) in injected cells, including porcine epiblast stem cells (pEpiSCs), non-purified differentiated pECs (unsorted EC), and purified differentiated pECs (sorted EC). All cells were labeled with PKH 26 (red) prior to injection. Nuclei was counterstained with DAPI (blue). (C) Fluorescence intensity analysis of representative immunofluorescence images measured in RGB profile of ImageJ. (D) Hematoxylin and eosin (H&E) staining images illustrating the morphological characteristics of injected cells, including pEpiSCs, non-purified differentiated pECs, and purified differentiated pECs. Scale bar = 50 µm.

Fig. 2

Differentiated pECs MMP activity and migration ability. (A) Zymography analysis illustrating MMP activity in HUVECs, SUVECs, and differentiated pECs. (B) Assessing migration ability through a transwell migration assay for HUVECs, SUVECs, and differentiated pECs. Scale bar = 20 µm. (C) Migration cell number was counted by using ImageJ.

Fig. 3

Differentiated pECs have angiogenesis function. (A) Tube formation images in HUVECs, SUVECs, and differentiated pECs on Matrigel for 4 h. Scale bar = 500 µm. (B) Branch points of HUVECs, SUVECs, and differentiated pECs. (n = 3) (C) Spheroid sprouting images of HUVECs, SUVECs, and differentiated pECs embedded in collagen gels. Phalloidin (red) stained cytoskeleton for sprouting measurement. The nucleus was stained with DAPI (blue). Scale bar = 275 µm. (D) Sprouts length was measured by ImageJ. (n = 4) Values presented as mean SEM. * P < 0.05, ** P < 0.01.

Fig. 4

Tight junction formation between porcine and human ECs. (A) Immunofluorescence image depicting claudin (green) in HUVECs and SUVECs stained with PKH 26 (red). Nuclei were stained with DAPI (blue). (B) Immunofluorescence image illustrating claudin (green) in HUVECs and differentiated pECs stained with PKH 26 (red). Nuclei was stained with DAPI (blue). Scale bar = 20 µm.

Fig. 5

GAP junction formation between porcine and human ECs. (A) Immunofluorescence image illustrating connexin 43 (green) in HUVECs and SUVECs, with SUVECs stained using PKH 26 (red). Nuclei was stained with DAPI (blue). (B) Immunofluorescence depicting connexin 43 (green) in HUVECs and differentiated pECs, with differentiated pECs stained using PKH 26 (red). Nuclei was stained with DAPI (blue). (C) Western blotting data for co-IP showing the interaction between Myc-pCx43 and HA-hCx43 in HEK293T cells transfected with Myc-pCx43 and HA-hCx43. (D) Fluorescence image displaying small nucleotide tagged Cy3 (red) in SUVECs, transported by connexin channels from HUVECs. (E) Fluorescence image showing small nucleotide tagged Cy3 (red) in differentiated pECs, transported by connexin channels from HUVECs. Scale bar = 20 µm.