The p.R482W hotspot mutation in A-type nuclear lamins causes familial partial lipodystrophy of Dunnigan-type (FPLD2), a lipodystrophic syndrome complicated by early onset atherosclerosis. Molecular mechanisms underlying endothelial cell dysfunction conferred by the lamin A mutation remain elusive. However, lamin A regulates epigenetic developmental pathways and mutations could perturb these functions. Here, we demonstrate that lamin A R482W elicits endothelial differentiation defects in a developmental model of FPLD2. Genome modeling in fibroblasts from patients with FPLD2 caused by the lamin A R482W mutation reveals repositioning of the mesodermal regulator T/Brachyury locus towards the nuclear center relative to normal fibroblasts, suggesting enhanced activation propensity of the locus in a developmental model of FPLD2. Addressing this issue, we report phenotypic and transcriptional alterations in mesodermal and endothelial differentiation of induced pluripotent stem cells we generated from a patient with R482W-associated FPLD2. Correction of the LMNA mutation ameliorates R482W-associated phenotypes and gene expression. Transcriptomics links endothelial differentiation defects to decreased Polycomb-mediated repression of the T/Brachyury locus and over-activation of T target genes. Binding of the Polycomb repressor complex 2 to T/Brachyury is impaired by the mutated lamin A network, which is unable to properly associate with the locus. This leads to a deregulation of vascular gene expression over time. By connecting a lipodystrophic hotspot lamin A mutation to a disruption of early mesodermal gene expression and defective endothelial differentiation, we propose that the mutation rewires the fate of several lineages, resulting in multi-tissue pathogenic phenotypes.

Ritonavir-boosted PIs differentially induced vascular endothelial cell dysfunction, reactive oxygen species production, inflammation and senescence with no effect or a mild effect of DRV/r, an intermediate effect of ATV/r, and a stronger effect of LPV/r. Statins could, in part, protect the cells from PI-induced endothelial dysfunction.

About 15% of colorectal cancers are called MSI because they demonstrate microsatellite instability. In most sporadic MSI cases, the DNA mismatch repair (MMR) defect is due to methylation of the MLH1 promoter. In hereditary MSI cases, it is the consequence of germline mutations of one of the MMR genes. We analysed the MLH1 promoter for methylation using the methylationspecific PCR technique. With a previously described and widely used primer set, a number of samples with an intact MMR system were found to have methylated MLH1 promoter, a finding normally associated with lack of MLH1 expression. Another primer set, specific for a more proximal region of the promoter, gave results that correlated more closely with loss of MLH1 expression. We then conducted a survey of the literature on the subject, and a total of 161 articles were examined. Although it was shown as early as 1999 that absence of MLH1 expression correlated with methylation of the proximal but not distal regions of the MLH1 promoter, 60% of published studies analysed nonspecific regions. Our findings suggest that these studies are likely to have wrongly estimated the association between methylation of the MLH1 gene and the lack of its protein expression.

The mechanisms underlying MMR deficiency in MSI-H IBD-Ns are different from those in sporadic MSI-H tumors and seem to be more related to those observed in hereditary MSI-H tumors. However, BRAF mutations were observed in MSI-H IBD-Ns, similar to sporadic MSI-H tumors, but unlike hereditary MSI-H tumors. Finally, the mutational events in target genes for instability are the same in MSI-H IBD-N tumors as in non-IBD sporadic and hereditary colorectal MSI-H cancers, indicating a colon-related repertoire of target gene alterations.

The various PIs differentially modified adipocyte functions. Darunavir alone did not affect adipocyte functions and only modestly altered differentiation and mitochondrial function when associated with ritonavir. Lopinavir/ritonavir adversely affected differentiation and lipid content, mitochondrial function, ROS production and insulin sensitivity, and the effect of atazanavir/ritonavir was intermediate. Thus, in vitro, darunavir/ritonavir presented a safer metabolic profile on adipocytes than atazanavir/ritonavir and lopinavir/ritonavir.

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Objective-Inactivating peroxisome proliferator-activated receptor-γ (PPARγ) mutations lead to a syndrome of familial partial lipodystrophy (FPLD3) associated with early-onset severe hypertension. PPARγ can repress the vascular reninangiotensin system (RAS) and angiotensin II receptor 1 expression. We evaluated the relationships between PPARγ inactivation and cellular RAS using FPLD3 patients' cells and human vascular smooth muscle cells expressing mutant or wild-type PPARγ. Approach and Results-We identified 2 novel PPARG mutations, R165T and L339X, located in the DNA and ligand-binding domains of PPARγ, respectively in 4 patients from 2 FPLD3 families. In cultured skin fibroblasts and peripheral blood mononuclear cells from the 4 patients and healthy controls, we compared markers of RAS activation, oxidative stress, and inflammation, and tested the effect of modulators of PPARγ and angiotensin II receptor 1. We studied the impact of the 2 mutations on the transcriptional activity of PPARγ and on the vascular RAS in transfected human vascular smooth muscle cells. Systemic RAS was not altered in patients. However, RAS markers were overexpressed in patients' fibroblasts and peripheral blood mononuclear cells, as in vascular cells expressing mutant PPARγ. Angiotensin II-mediated mitogenactivated protein kinase activity increased in patients' fibroblasts, consistent with RAS constitutive activation. Patients' cells also displayed oxidative stress and inflammation. PPARγ activation and angiotensin II receptor 1 mRNA silencing reversed RAS overactivation, oxidative stress, and inflammation, arguing for a role of angiotensin II receptor 1 in these processes. Conclusions-Two

Activation of thermogenic beige adipocytes has recently emerged as a promising therapeutic target in obesity and diabetes. Relevant human models for beige adipocyte differentiation are essential to implement such therapeutic strategies. We report a straightforward and efficient protocol to generate functional human beige adipocytes from human induced pluripotent stem cells (hiPSCs). Without overexpression of exogenous adipogenic genes, our method recapitulates an adipogenic developmental pathway through successive mesodermal and adipogenic progenitor stages. hiPSC-derived adipocytes are insulin sensitive and display beige-specific markers and functional properties, including upregulation of thermogenic genes, increased mitochondrial content, and increased oxygen consumption upon activation with cAMP analogs. Engraftment of hiPSC-derived adipocytes in mice produces well-organized and vascularized adipose tissue, capable of β-adrenergic–responsive glucose uptake. Our model of human beige adipocyte development provides a new and scalable tool for disease modeling and therapeutic screening.