Preadipocytes in human granulation tissue: role in wound healing and response to macrophage polarization

Abstract

Background: Chronic non-healing wounds pose a global health challenge. Under optimized conditions, skin wounds heal by the formation of scar tissue. However, deregulated cell activation leads to persistent inflammation and the formation of granulation tissue, a type of premature scar tissue without epithelialization. Regenerative cells from the wound periphery contribute to the healing process, but little is known about their cellular fate in an inflammatory, macrophage-dominated wound microenvironment.

Methods: We examined CD45^-/CD31^-/CD34⁺ preadipocytes and CD68⁺ macrophages in human granulation tissue from pressure ulcers (n=6) using immunofluorescence, immunohistochemistry, and flow cytometry. In vitro, we studied macrophage-preadipocyte interactions using primary human adipose-derived stem cells (ASCs) exposed to conditioned medium harvested from IFNG/LPS (M1)- or IL4/IL13 (M2)-activated macrophages. Macrophages were derived from THP1 cells or CD14⁺ monocytes. In addition to confocal microscopy and flow cytometry, ASCs were analyzed for metabolic (OXPHOS, glycolysis), morphological (cytoskeleton), and mitochondrial (ATP production, membrane potential) changes. Angiogenic properties of ASCs were determined by HUVEC-based angiogenesis assay. Protein and mRNA levels were assessed by immunoblotting and quantitative RT-PCR.

Results: CD45^-/CD31^-/CD34⁺ preadipocytes were observed with a prevalence of up to 1.5% of total viable cells in human granulation tissue. Immunofluorescence staining suggested a spatial proximity of these cells to CD68⁺ macrophages in vivo. In vitro, ASCs exposed to M1, but not to M2 macrophage secretome showed a pro-fibrotic response characterized by stress fiber formation, elevated alpha smooth muscle actin (SMA), and increased expression of integrins ITGA5 and ITGAV. Macrophage-secreted IL1B and TGFB1 mediated this response via the PI3K/AKT and p38-MAPK pathways. In addition, ASCs exposed to M1-inflammatory stress demonstrated reduced migration, switched to a glycolysis-dominated metabolism with reduced ATP production, and increased levels of inflammatory cytokines such as IL1B, IL8, and MCP1. Notably, M1 but not M2 macrophages enhanced the angiogenic potential of ASCs.

Conclusion: Preadipocyte fate in wound tissue is influenced by macrophage polarization. Pro-inflammatory M1 macrophages induce a pro-fibrotic response in ASCs through IL1B and TGFB1 signaling, while anti-inflammatory M2 macrophages have limited effects. These findings shed light on cellular interactions in chronic wounds and provide important information for the potential therapeutic use of ASCs in human wound healing.

Keywords: Adipose-derived stem cells; Granulation tissue; Inflammation; Macrophage polarization; Myofibroblasts; Preadipocytes; Tissue fibrosis; Wound healing.

Fig. 1

CD34⁺/CD31⁻ cells are present in human wound granulation tissue. A Overview of human granulation tissue cellularity and identification of CD34-expressing cells shown as uniform manifold approximation and projection (UMAP) plots. B Unsupervised clustering of fibroblast subtypes and expression of CD34. C Immunofluorescence staining of a representative granulation tissue section from a chronic lower limb wound in a 53-year-old male patient. The higher magnification detail (right image) indicates the presence of CD34⁺/CD31⁻ single and CD34⁺/CD31⁺ double positive cells. D Immunofluorescence staining of the same specimen. The higher magnification detail (right image) shows the proximity of CD68⁺ cells to CD34⁺ cells. E, F Flow cytometry analysis of single-cell suspensions from human granulation tissue samples (n = 6). E Left panel: Frequency of CD45⁺ and CD45⁻ cells shown as a percentage of viable granulation tissue cells. Right panel: Frequency of CD31⁺ cells shown as a percentage of CD45⁻ cells. F Left panel: frequency of CD34⁺ endothelial progenitor cells shown as a percentage of CD45⁻/CD31⁺ endothelial cells. Right panel: frequency of CD34⁺ cells shown as a percentage of CD45⁻/CD31⁻/CD90⁺ cells. Details of patients are described in Supplement Table 1

Fig. 2

M1 macrophage secretome effects on human ASCs metabolism. A Representative confocal images of human ASCs cultured in differentially activated MQ-CM for 72 h. Cells were stained with CellMask Orange to visualize cell morphology. Scale bars indicate 200 µm. B Quantification of relative cell numbers in 72 h treated ASC. C ATP levels measured by ATP-Red and mitochondrial membrane potential (MMP) determined by TMRM staining (n = 6). Asterisks indicate p values of p<0.05 (*) and p<0.01 (**). D Energy profiling of MQ-CM treated ASCs at 72 h determined by Agilent Seahorse technology. The graph shows the correlation of mitochondrial respiration determined by oxygen consumption rate (OCR) and glycolysis measured by extracellular acidification rate (ECAR). Open squares indicate levels under basal conditions; closed squares indicate levels under oligomycin-induced mitochondrial uncoupling (stressed conditions) (n = 4). E Metabolic potential indicates the ability of cells to meet an energy demand via mitochondrial respiration and glycolysis. This is shown as the percentage increase of stressed OCR over baseline OCR and stressed ECAR over baseline ECAR (n = 4). F Representative immunoblots of MQ-CM treated ASC at 72 h analyzed for the expression of key regulatory enzymes of glycolysis. G Representative immunoblots of MQ-CM-treated ASCs showing levels and activation of intracellular kinases over time. All data are shown as mean ± SEM

Fig. 3

M1 macrophage secretome induces stress fiber formation in ASCs. A Representative confocal images of MQ-CM-treated ASCs at 72 h showing increased actin stress fibers (phalloidin) and focal adhesions (vinculin) in MQ-CM- and TGFB1-treated ASCs. Scale bars indicate 20 µm. B Representative immunoblots of MQ-CM-treated ASCs after 72 h analyzing levels of ECM-cytoskeleton signaling enzymes including SMA, phosphorylation of FAK and RGD-recognizing integrins ITGA5 and ITGAV. C Flow cytometry analysis of cell surface expression of ITGA5 and ITGAV (n = 6). D Representative images of a conventional scratch assay showing the migration ability of MQ-CM-treated ASCs. The blue lines indicate the initial scratch lines; the yellow lines indicate the relative migration distance after 24 h. Scale bars indicate 200 µm. Quantification of the relative migration distance was calculated using INCUCYTE^TM software based on cell confluence within the wound region over time (n = 4). All data are shown as mean ± SEM. Asterisks indicate p values of p<0.05 (*), p<0.01 (**), and p<0.0001 (****). TGFB1 treatment served as a positive control for maximal stress fiber induction

Fig. 4

Monocyte-derived macrophages prove M1 effects on ASCs. A Representative confocal images of stress fibers (phalloidin) and focal adhesions (vinculin) of ASCs exposed to CM of CD14⁺ monocyte-derived pro-inflammatory (GMCSF and GMCSF (IFNG/LPS)) or anti-inflammatory (MCSF and MCSF (IL4/IL13)) macrophages. Scale bars indicate 20 µm. B–D Representative immunoblots analyzing the levels of B intracellular kinases, C key enzymes regulating glycolysis, and D ECM-cytoskeleton signaling

Fig. 5

M1 macrophage secretome induces a myofibroblast-like secretory phenotype in ASCs. A, B Gene profiling by quantitative qPCR mRNA analysis of MQ-CM-treated ASCs at 72 h. Genes analyzed were clustered for A potential inflammatory, angiogenic, and keratinocyte (KC) mobilizing function (n = 3) or B ECM remodeling properties (n = 5). Data are shown as -ΔCt ± SEM. C Representative images of HUVEC-based in vitro angiogenesis assay and analysis thereof at 6 h. D Quantification of master meshes, master segments, branch lengths, and junctions of identified HUVEC structures. Data are shown as mean ± SEM (n = 6). Asterisks indicate p values of p<0.05 (*), p<0.01 (**), and p<0.001(***)

Fig. 6

Stress fiber assembly in M1-CM-treated cells is mediated by IL1B, TGFB1, and AKT signaling. A Representative confocal microscopy images of MQ-CM-treated ASCs at 72 h. Supplementation of M1-CM with TGFB1 inhibitor SB431542 or IL1B inhibitor IL1RA reduced stress fiber assembly. B, C Representative immunoblots analyzing ECM-cytoskeletal signaling (B) and intracellular activated kinases (C). D Quantification of cell migration of MQ-CM-treated ASC with at least partially rescued by impaired IL1B and TGFB1 signaling. E Representative confocal microscopy images of MQ-CM-treated ASCs including co-treatment with either PI3K/AKT inhibitor LY294002 (25 µM), ERK1/2 inhibitor U0126 (10 µM), or p38-MAPK inhibitor Losmapimod (25 µM) during incubation with M1-CM treatment. F Representative immunoblots analyzing ECM-cytoskeleton signaling. G Cell migration of MQ-CM-treated ASCs with impaired AKT, ERK1/2, or p38-MAPK signaling. All scale bars indicate 20 µm. All data are shown as mean ± SEM. Asterisks indicate p values of <0.05 (*)