RESUMO
In obese adipose tissue (AT), hypertrophic expansion of adipocytes is not matched by new vessel formation, leading to AT hypoxia. As a result, hypoxia inducible factor-1⺠(HIF-1âº) accumulates in adipocytes inducing a transcriptional program that upregulates profibrotic genes and biosynthetic enzymes such as lysyl oxidase (LOX) synthesis. This excess synthesis and crosslinking of extracellular matrix (ECM) components cause AT fibrosis. Although fibrosis is a hallmark of obese AT, the role of fibroblasts, cells known to regulate fibrosis in other fibrosis-prone tissues, is not well studied. Here we have developed an in vitro model of AT to study adipocyte-fibroblast crosstalk in a hypoxic environment. Further, this in vitro model was used to investigate the effect of hypoxia on adipocyte mechanical properties via ras homolog gene family member A (RhoA)/Rho-associated coiled-coil kinases (ROCK) signaling pathways. We confirmed that hypoxia creates a diseased phenotype by inhibiting adipocyte maturation and inducing actin stress fiber formation facilitated by myocardin-related transcription factor A (MRTF-A/MKL1) nuclear translocation. This work presents new potential therapeutic targets for obesity by improving adipocyte maturation and limiting mechanical stress in obese AT.
Assuntos
Adipócitos/patologia , Fibroblastos/patologia , Fibrose/patologia , Hipóxia/fisiopatologia , Células-Tronco Mesenquimais/patologia , Obesidade/patologia , Fibras de Estresse/patologia , Adipócitos/metabolismo , Fibroblastos/metabolismo , Fibrose/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Obesidade/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Fibras de Estresse/metabolismo , Transativadores/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
INTRODUCTION: Adipose derived stem cells (ASCs) hold great promise for clinical applications such as soft tissue regeneration and for in vitro tissue models and are notably easy to derive in large numbers. Specifically, ASCs provide an advantage for in vitro models of adipose tissue, where they can be employed as tissue specific cells and for patient specific models. However, ASC in vitro expansion may unintentionally reduce adipogenic capacity due to the stiffness of tissue culture plastic (TCPS). METHODS: Here, we expanded freshly isolated ASCs on soft and stiff substrates for 4 passages before adipogenic differentiation. At the last passage we swapped the substrate from stiff to soft, or soft to stiff to determine if short term exposure to a different substrate altered adipogenic capacity. RESULTS: Expansion on stiff substrates reduced adipogenic capacity by 50% which was not rescued by swapping to a soft substrate for the last passage. Stiff substrates had greater nuclear area and gene expression of nesprin-2, a protein that mediates the tension of the nuclear envelope by tethering it to the actin cytoskeleton. Upon swapping to a soft substrate, the nuclear area was reduced but nesprin-2 levels did not fully recover, which differentially regulated cell commitment transcriptional factors. CONCLUSION: Therefore, in vitro expansion on stiff substrates must be carefully considered when the end-goal of the expansion is for adipose tissue or soft tissue applications.