Stromal Vascular Fraction Reverses the Age-Related Impairment in Revascularization following Injury

Abstract

Adipose-derived stromal vascular fraction (SVF) has emerged as a potential regenerative therapy, but few studies utilize SVF in a setting of advanced age. Additionally, the specific cell population in SVF providing therapeutic benefit is unknown. We hypothesized that aging would alter the composition of cell populations present in SVF and its ability to promote angiogenesis following injury, a mechanism that is T cell-mediated. SVF isolated from young and old Fischer 344 rats was examined with flow cytometry for cell composition. Mesenteric windows from old rats were isolated following exteriorization-induced (EI) hypoxic injury and intravenous injection of one of four cell therapies: (1) SVF from young or (2) old donors, (3) SVF from old donors depleted of or (4) enriched for T cells. Advancing age increased the SVF T-cell population but reduced revascularization following injury. Both young and aged SVF incorporated throughout the host mesenteric microvessels, but only young SVF significantly increased vascular area following EI. This study highlights the effect of donor age on SVF angiogenic efficacy and demonstrates how the ex vivo mesenteric-window model can be used in conjunction with SVF therapy to investigate its contribution to angiogenesis.

Keywords: Aging; Angiogenesis; Cell therapy; Injury; Revascularization; T cells.

Figure 1.. Summary of methodology.

Combinations of freshly isolated young or aged SVF cells seeded onto young or aged mesenteric windows was utilized to assess SVF’s angiogenic properties influenced by age of donor and/or host (Group 1). Previously frozen young or aged SVF was administered to aged hosts via systemic administration to assess SVF’s incorporation into the host mesenteric microvascular network (Group 2). Exteriorization of the mesentery was utilized to induce injury in aged rats treated with no cells or previously frozen SVF from either young or aged donors. The microvascular networks of injured windows and non-injured control were imaged over time to quantify angiogenic growth (Group 3). Previously frozen SVF from aged donors was subjected to magnetic antibody cell sorting to generate CD3 depleted and CD3 enriched SVF fractions. The modified SVF was then systemically administered following exteriorization injury (Group 4). Angiogenic growth in the microvascular networks of injured and non-injured windows was analyzed. Adipose-derived stromal vascular fraction (SVF), CD3+ (T cell marker), cell membrane stain (Dil), exteriorization injury (EI), immunohistochemistry (IHC), magnetic antibody cell sorting (MACS), months old (mo), non-exteriorized/non-injured (NEI). Aged rats treated with SVF from young donors (O+Y SVF), aged rats treated with SVF from aged donors (O+O SVF), aged rats treated with SVF from aged donors depleted of CD3+ T cells (O+O SVF CD3-), aged rats treated with SVF from aged donors containing only CD3+ T cells (O+O SVF CD3+). Image created with BioRender.com.

Figure 2.. Cell composition of SVF from young or aged donors.

Flow cytometry analysis of cell composition for SVF isolated from young or aged donors (A-C). A) Lymphocytes (forward and side scatter), hematopoietic (CD45+/CD11b+), hematopoietic stem cell (CD45+/CD34+), endothelial (CD31+), MSC (CD90+/CD45-/CD34-), and stromal fraction (CD45-/CD31-/CD34+). B) All macrophages (CD45+/CD11b+/CD68+), immature (CD45+/CD11b+/CD68+/CD86-/CD163-), M1 (CD45+/CD11b+/CD68+/CD86+/CD163-), M2 (CD45+/CD11b+/CD68+/CD86-/CD163+) and transitional (CD45+/CD11b+/CD68+/CD86+/CD163+). C) All T cells (CD3+), thymocytes (CD45-/CD90+), helper (CD3+/CD4+), cytotoxic (CD3+/CD8+), activated (CD3+/CD25+), CD4 memory (CD3+/CD4+/CD44+), and CD8 memory (CD3+/CD8+/CD44+). Data are represented as percentage of cells in SVF mean+SEM, n=3/group. p≤.05 when vs young SVF (*).

Figure 3.. Aging effects between donor SVF and host tissue on vascularized area.

Vascular coverage is greatest in young SVF + young tissue pairing (A) compared to aged SVF on young tissue (B), young SVF on aged tissue (C) and aged SVF on aged tissue (D). Quantitative analysis of percent vascularized area (E). Green outline indicates vascularized area while red outline indicates avascular area. Scale bar = 2 mm. 8 mesenteric windows from 2 rats per group, p≤.05 (*); values are shown as box and whisker plots.

Figure 4.. GFP+ SVF incorporated into mesenteric microvasculature following intravenous injection.

GFP+ SVF (green) cells from young or aged donors were found in the perivascular space and microvasculature (GSL-1 - red) of young host mesenteric windows six days following i.v. injection (A). GFP is co-localized (arrows) with CD3+ (T cell marker, pink), and DAPI (blue). An old control animal not treated with SVF was used as a negative control for GFP signal and secondary antibody control for CD3 (B). Scale bar = 20 μm.

Figure 5.. SVF rescues age-related impairment in revascularization following injury.

Representative images of injured (EI) and non-injured (NEI) mesenteric windows from NCC, O+YSVF, and O+OSVF groups at days 0, 3, and 5 of culture. Vascular networks stained with GSL-1 and scale bar = 200 μm (A). Quantitative analysis of percent vascular area (B). Data are shown as box and whisker plots (n=4/group), brackets indicate p≤.05 when comparisons are between days or groups. p≤.05 when EI vs NEI within group comparison (*).

Figure 6.. Modification of T cell population in SVF does not alter angiogenic potential following injury.

Representative images of injured (EI) and non-injured (NEI) mesenteric windows from O+OSVF animals that received CD3 depleted (CD3-) SVF or CD3 enriched (CD3+) SVF at days 0, 3, and 5 of culture. Vascular networks stained with GSL-1 and scale bar = 200 μm (A). Quantitative analysis of percent vascular area (B). Data are shown as box and whisker plots (n=4/group), brackets indicate p≤.05 when comparisons are between days or groups.