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

Tina Rauchenwald; Florian Handle; Catherine E Connolly; Antonia Degen; Christof Seifarth; Martin Hermann; Christoph H Tripp; Doris Wilflingseder; Susanne Lobenwein; Dragana Savic; Leo Pölzl; Evi M Morandi; Dolores Wolfram; Ira-Ida Skvortsova; Patrizia Stoitzner; Johannes Haybaeck; Marko Konschake; Gerhard Pierer; Christian Ploner

doi:10.1186/s41232-023-00302-5

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

Inflamm Regen. 2023 Oct 31;43(1):53. doi: 10.1186/s41232-023-00302-5.

Authors

Tina Rauchenwald¹, Florian Handle², Catherine E Connolly¹, Antonia Degen¹, Christof Seifarth³, Martin Hermann⁴, Christoph H Tripp⁵, Doris Wilflingseder⁶, Susanne Lobenwein¹, Dragana Savic⁷, Leo Pölzl⁸, Evi M Morandi¹, Dolores Wolfram¹, Ira-Ida Skvortsova⁷, Patrizia Stoitzner⁵, Johannes Haybaeck^{2

9}, Marko Konschake³, Gerhard Pierer¹, Christian Ploner¹⁰

Affiliations

¹ Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University of Innsbruck, Innsbruck, Austria.
² Institute of Pathology, Neuropathology and Molecular Pathology, Medical University Innsbruck, Innsbruck, Austria.
³ Institute of Clinical and Functional Anatomy, Medical University of Innsbruck, Innsbruck, Austria.
⁴ Department of Anesthesiology and Critical Care Medicine, Medical University of Innsbruck, Innsbruck, Austria.
⁵ Department of Dermatology, Venereology and Allergology, Medical University of Innsbruck, Innsbruck, Austria.
⁶ Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria.
⁷ Department of Therapeutic Radiology and Oncology, Medical University of Innsbruck, EXTRO-Lab, Tyrolean Cancer Research Institute, Innsbruck, Austria.
⁸ Department of Cardiac Surgery, Medical University of Innsbruck, Innsbruck, Austria.
⁹ Diagnostic and Research Center for Molecular Biomedicine, Institute of Pathology, Medical University of Graz, Graz, Austria.
¹⁰ Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University of Innsbruck, Innsbruck, Austria. christian.ploner@i-med.ac.at.

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.