KLF4 (Kruppel-Like Factor 4)-Dependent Perivascular Plasticity Contributes to Adipose Tissue inflammation

Objective: Smooth muscle cells and pericytes display remarkable plasticity during injury and disease progression. Here, we tested the hypothesis that perivascular cells give rise to Klf4-dependent macrophage-like cells that augment adipose tissue (AT) inflammation and metabolic dysfunction associated with diet-induced obesity (DIO). Approach and Results: Using Myh11-Cre(ERT2) eYFP (enhanced yellow fluorescent protein) mice and flow cytometry of the stromovascular fraction of epididymal AT, we observed a large fraction of smooth muscle cells and pericytes lineage traced eYFP(+) cells expressing macrophage markers. Subsequent single-cell RNA sequencing, however, showed that the majority of these cells had no detectable eYFP transcript. Further exploration revealed that intraperitoneal injection of tamoxifen in peanut oil, used for generating conditional knockout or reporter mice in thousands of previous studies, resulted in large increase in the autofluorescence and false identification of macrophages within epididymal AT as being eYFP(+); and unintended proinflammatory consequences. Using newly generated Myh11-Dre(ERT2)tdTomato mice given oral tamoxifen, we virtually eliminated the problem with autofluorescence and identified 8 perivascular cell dominated clusters, half of which were altered upon DIO. Given that perivascular cell KLF4 (kruppel-like factor 4) can have beneficial or detrimental effects, we tested its role in obesity-associated AT inflammation. While smooth muscle cells and pericytes-specific Klf4 knockout (smooth muscle cells and pericytes Klf4(Delta/Delta)) mice were not protected from DIO, they displayed improved glucose tolerance upon DIO, and showed marked decreases in proinflammatory macrophages and increases in LYVE1(+) lymphatic endothelial cells in the epididymal AT. Conclusions: Perivascular cells within the AT microvasculature dynamically respond to DIO and modulate tissue inflammation and metabolism in a KLF4-dependent manner.