Identification of a physiologic vasculogenic fibroblast state to achieve tissue repair

Abstract

Tissue injury to skin diminishes miR-200b in dermal fibroblasts. Fibroblasts are widely reported to directly reprogram into endothelial-like cells and we hypothesized that miR-200b inhibition may cause such changes. We transfected human dermal fibroblasts with anti-miR-200b oligonucleotide, then using single cell RNA sequencing, identified emergence of a vasculogenic subset with a distinct fibroblast transcriptome and demonstrated blood vessel forming function in vivo. Anti-miR-200b delivery to murine injury sites likewise enhanced tissue perfusion, wound closure, and vasculogenic fibroblast contribution to perfused vessels in a FLI1 dependent manner. Vasculogenic fibroblast subset emergence was blunted in delayed healing wounds of diabetic animals but, topical tissue nanotransfection of a single anti-miR-200b oligonucleotide was sufficient to restore FLI1 expression, vasculogenic fibroblast emergence, tissue perfusion, and wound healing. Augmenting a physiologic tissue injury adaptive response mechanism that produces a vasculogenic fibroblast state change opens new avenues for therapeutic tissue vascularization of ischemic wounds.

Acoustofluidic Engineering of Functional Vessel-on-a-Chip

“…Figure B shows the confocal scanning of the fibroblast-supported vessel structure. The fibroblasts were labeled with red fluorescence conjugated α-smooth muscle actin antibody …”

“…The fibroblasts were labeled with red fluorescence conjugated α-smooth muscle actin antibody. 88 Then, the vascular barrier function was quantified by visualizing the diffusion of 70 kDa FITC-dextran solute. 89 The dextran solution was perfused into the parallel patterned vessel tubes through the anastomosis zone (Figure 2Eii) and permeated into the surrounding matrix.…”

Tissue Nanotransfection Silicon Chip and Related Electroporation-Based Technologies for In Vivo Tissue Reprogramming

“…TNT successfully demonstrated transdermal gene therapy for skin repair [ 18 ], tumor regression [ 19 ], ischemic stroke recovery [ 27 ], and extreme chronic wound healing [ 20 ]. The first generation of the TNT 1.0 chip utilizes the mechanism of nanoelectroporation via nanochannels as illustrated in Figure 1 E and Figure 2 C. Fibroblast cells of skin in vivo have been successfully reprogrammed into neuronal and endothelial cells by delivering specific gene cocktails in mice models [ 7 , 10 , 21 ]. The second generation of TNT 2.0 chips that feature a hollow microneedle array is shown in Figure 2 D. This modification is aimed at enhancing the physical contact between the TNT chip and skin to accommodate the nonuniform topography across the skin, thereby improving gene delivery efficiency.…”

“…This deficiency significantly affects the treatment outcomes and prevents the healing process. To improve this situation, in vivo tissue reprogramming via TNT presents a groundbreaking method for directly converting a type of cell (such as fibroblast) into desired functional cells in vivo [ 21 ]. This innovative approach bypasses the intermediate stem cell stage to increase efficiency and reduce the risk of tumorigenesis [ 10 ].…”

“…This technique involves applying short, high-voltage pulses to create temporary nanopores in cell membranes, allowing the introduction of genetic molecules into the target cells. It is a suitable gene therapy strategy for more invasive treatments such as cancer treatments and management of diabetic complications [ 7 , 10 , 15 , 18 , 19 , 20 , 21 , 22 ].…”