Mechanism of Matrix Gla Protein (MGP); Adipose Fibrosis

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PROJECT SUMMARY: Adipose fibrosis is characterized by progressive stromal fibrosis that causes adipose dysfunction in obesity, diabetes, and inflammatory conditions. The sustained activation of unwanted myofibroblasts promotes progressive tissue changes with excessive amounts of altered fibrotic matrix. Although PDGFRα+ and CD9+ progenitor cells have been implicated, the cellular mechanisms remain unclear. Matrix Gla Protein (MGP) is an extracellular inhibitor of bone morphogenetic protein (BMP) 4, a mediator of endothelial cell (EC) inflammation. Loss of MGP triggers endothelial-mesenchymal transitions (EndMTs), a source of multipotent cells. We discovered that MGP is expressed in PDGFRα+ and CD9+ progenitor cells, and plays a limiting role in myofibroblast lineage in mice. By characterizing the adipose fibrosis in Mgp-knockout (KO) mice, integrating profiles from single-cell RNA sequencing (scRNA-seq) combined with cell sorting and culture, we propose to dissect the mechanism of MGP in adipose fibrosis. In preliminary studies, global Mgp deletion caused extensive adipose fibrosis in white adipose tissue (WAT) in mice. ScRNA-seq uncovered MGP expression in ECs and progenitors with projected trajectories towards myofibroblasts. Loss of Mgp enhanced abnormal ECs and cell populations with myofibrogenic potential, isolated by FACS. Mice with mutated Mgp lacking BMP- binding displayed fibrosis limited to perivascular areas, suggesting a structural role for MGP. EC-specific Mgp deletion largely mimicked the global fibrotic phenotype. We also found robust changes in brown adipose tissue (BAT) with excessive ECs, myofibroblasts, and abnormal thermogenesis. Potential mechanisms of differentiation involved BMP4/7, the BMP receptor ALK1, the TGFβ receptor ALK5. Our hypothesis is that loss of MGP causes transition and expansion of ECs and specific progenitors, followed by enhanced myofibroblast differentiation, in a two-step model. Aim 1 will elucidate the mechanism by which MGP regulates the transition of ECs and progenitors to myofibroblasts in WAT. We will characterize the Mgp- KO mouse as a new model of adipose fibrosis, using scRNA-seq, FACS and adipogenic cultures. We will assess EC contribution to myofibroblasts by lineage tracing using tdTomato-labeled ECs. In vitro, we will test profibrotic effects of BMP3b and BMP4/ALK1/ALK5 signaling. Aim 2 will determine whether loss of BMP- binding in MGP or endothelial deletion of MGP is sufficient to cause myofibroblasts transitions or a shift in the pattern of fibrosis. Comparisons of myofibroblast transition without BMP binding and endothelial deletion of Mgp allow us to deduce the anti-fibrotic of MGP and the relative importance of endothelial MGP. Aim 3 will characterize BAT after Mgp deletion. We will examine fibrotic and vascular phenotypes in Mgp-KO mice. We will identify MGP-expressing cell populations, their transcriptional profiles and cell trajectories, and determine how MGP helps direct brown adipogenesis by early BMP7. Our results may impact the field of obesity and obesity-related complications and lead to new therapeutic strategies that limit fibrosis in chronic disease.