Extracellular lactic acidosis drives reprogramming of human subcutaneous adipocytes toward pathogenic myofibroblasts

Emerging evidence underlines that adipocytic progenitor cells/adipocytes may give rise to pathogenic myofibroblast-like cells both in solid tumors and in multiple forms of fibrosis. Lactic acidosis is commonly observed in the tumor microenvironment, as cancer cells and cancer-associated fibroblasts generate their energy through glycolysis with production of large amounts of lactic acid even in the presence of oxygen, a phenomenon known as aerobic glycolysis. A close association between aerobic glycolytic metabolism and myofibroblastic activation has also been reported in different fibrotic diseases. Based on these premises, the aim of our in vitro study was to investigate whether glycolysis-derived acidic microenvironment could be implicated in the adipocyte-to-myofibroblast transition (AMT) process. Human subcutaneous adipose tissue-derived stem cells (ADSCs) at low passage (passages 2–4) were cultured in adipocyte differentiation medium for 10 days to obtain cells displaying characteristic adipocyte morphology with accumulation of prominent intracytoplasmic lipid droplets by phase-contrast microscopy. Cells were subsequently cultured for 3 days in i) ADSC growth medium (pH 7.4), ii) a chemically induced acidic extracellular microenvironment obtained by 1N HCl administration directly in ADSC growth medium to reach pH 6.7 in the presence of 10mM lactate, or iii) ADSC growth medium with 10 ng/ml of recombinant human transforming growth factor β1 as positive control of AMT. Cell viability and proliferation were determined by annexin V/propidium iodide flow cytometry and MTT assay, respectively. The expression of adipogenic/adipocytic and myofibroblastic markers was assessed by quantitative real-time PCR, immunoblotting and immunofluorescence. Acidic conditioning did not alter cell viability and proliferation. The acidic microenvironment was found to induce the loss of the expression of the adipogenic/adipocytic markers FABP4, C/EBPα, PPARγ, adiponectin and perilipin in parallel with a significant upregulation of the mesenchymal/myofibroblastic markers α-smooth muscle actin (α-SMA), COL1A1 and COL1A2. Immunofluorescence analysis further revealed the loss of perilipin-coated cytoplasmic lipid droplets and the accumulation of intracellular type I collagen and expression of α-SMA–positive stress fibers characteristic of myofibroblasts in acidic cell cultures. This study reveals a previously unrecognized relationship of extracellular lactic acidosis and generation of pathogenic myofibroblast-like cells from adipocytic precursors that may have pathophysiologic implications in a variety of diseases characterized by metabolic reprogramming toward glycolysis.