Rationale The phenotypes of vascular smooth-muscle cells (vSMCs) comprise a continuum bounded by predominantly contractile and synthetic cells. Some evidence suggests that contractile vSMCs can assume a more synthetic phenotype in response to ischemic injury, but the mechanisms that activate this phenotypic switch are poorly understood. Objective To determine whether lactate, which increases in response to regional ischemia, may promote the synthetic phenotype in vSMCs. Methods and Results Experiments were performed with vSMCs that had been differentiated from human induced pluripotent stem cells and then cultured in glucose-free, lactate-enriched (L+) medium or in standard (L−) medium. Compared to the L− medium, the L+ medium was associated with significant increases in synthetic vSMC marker expression, proliferation, and migration, and with significant declines in contractile and apoptotic activity. Furthermore, these changes were accompanied by increases in the expression of monocarboxylic acid transporters (MCT) and were generally attenuated both by the blockade of MCT activity and by transfection with iRNA for N-myc downstream regulated gene (NDRG). Proteomics, biomarker, and pathway analyses suggested that the L+ medium tended to upregulate the expression of synthetic vSMC markers, the production of extracellular proteins that participate in tissue construction or repair, and the activity of pathways that regulate cell proliferation and migration. Observations in hypoxia-cultured vSMCs were similar to those in L+ cultured vSMCs, and assessments in a swine myocardial infarction model suggested that measurements of lactate levels, lactate dehydrogenase levels, vSMC proliferation, and MCT and NDRG expression were greater in the ischemic zone than in nonischemic tissues. Conclusions These results demonstrate for the first time that vSMCs assume a more synthetic phenotype in a microenvironment that is rich in lactate. Thus, mechanisms that link glucose metabolism to vSMC phenotypic switching could play a role in the pathogenesis and treatment of cardiovascular disease.

Conventional protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into smooth-muscle cells (SMCs) can be inefficient and generally fail to yield cells with a specific SMC phenotype (i.e., contractile or synthetic SMCs). Here, we present two novel hiPSC-SMC differentiation protocols that yield SMCs with predominantly contractile or synthetic phenotypes. Flow cytometry analyses of smooth-muscle actin (SMA) expression indicated that ~45% of the cells obtained with each protocol assumed an SMC phenotype, and that the populations could be purified to ~95% via metabolic selection. Assessments of cellular mRNA and/or protein levels indicated that SMA, myosin heavy chain II, collagen 1, calponin, transgelin, connexin 43, and vimentin expression in the SMCs obtained via the Contractile SMC protocol and in SMCs differentiated via a traditional protocol were similar, while SMCs produced via the Sythetic SMC protocol expressed less calponin, more collagen 1, and more connexin 43. Differences were also observed in functional assessments of the two SMC populations: the two-dimensional surface area of Contractile SMCs declined more extensively (to 12% versus 44% of original size) in response to carbachol treatment, while quantification of cell migration and proliferation were greater in Synthetic SMCs. Collectively, these data demonstrate that our novel differentiation protocols can efficiently generate SMCs from hiPSCs.

Elongating ketosynthases (KSs) catalyze carbon–carbon bond-forming reactions during the committed step for each round of chain extension in both fatty acid synthases (FASs) and polyketide synthases (PKSs). A small α-helical acyl carrier protein (ACP) shuttles fatty acyl intermediates between enzyme active sites. To accomplish this task, the ACP relies on a series of dynamic interactions with multiple partner enzymes of FAS and associated FAS-dependent pathways. Recent structures of the Escherichia coli FAS ACP, AcpP, in covalent complexes with its two cognate elongating KSs, FabF and FabB, provide high-resolution details of these interfaces, but a systematic analysis of specific interfacial interactions responsible for stabilizing these complexes has not yet been undertaken. Here, we use site-directed mutagenesis with both in vitro and in vivo activity analyses to quantitatively evaluate these contacting surfaces between AcpP and FabF. We delineate the FabF interface into three interacting regions and demonstrate the effects of point mutants, double mutants, and region deletion variants. Results from these analyses reveal a robust and modular FabF interface capable of tolerating seemingly critical interface mutations with only the deletion of an entire region significantly compromising activity. Structure and sequence analyses of FabF orthologs from related type II FAS pathways indicate significant conservation of type II FAS KS interface residues and, overall, support its delineation into interaction regions. These findings strengthen our mechanistic understanding of molecular recognition events between ACPs and FAS enzymes and provide a blueprint for engineering ACP-dependent biosynthetic pathways.