Hypoplastic left heart syndrome (HLHS) is a severe cardiac malformation characterized by left ventricle (LV) hypoplasia and abnormal LV perfusion and oxygenation. We studied hypoxia-associated injury in fetal HLHS and human pluripotent stem cells during cardiac differentiation to assess the effect of microenvironmental perturbations on fetal cardiac reprogramming. We studied LV myocardial samples from 32 HLHS and 17 structurally normal midgestation fetuses. Compared with controls, the LV in fetal HLHS samples had higher nuclear expression of hypoxia-inducible factor-1α but lower angiogenic growth factor expression, higher expression of oncogenes and transforming growth factor (TGF)-β1, more DNA damage and senescence with cell cycle arrest, fewer cardiac progenitors, myocytes and endothelial lineages, and increased myofibroblast population (P < 0.05 versus controls). Smooth muscle cells (SMCs) had less DNA damage compared with endothelial cells and myocytes. We recapitulated the fetal phenotype by subjecting human pluripotent stem cells to hypoxia during cardiac differentiation. DNA damage was prevented by treatment with a TGF-β1 inhibitor (P < 0.05 versus nonhypoxic cells). The hypoplastic LV in fetal HLHS samples demonstrates hypoxia-inducible factor-1α up-regulation, oncogene-associated cellular senescence, TGF-β1-associated fibrosis and impaired vasculogenesis. The phenotype is recapitulated by subjecting human pluripotent stem cells to hypoxia during cardiac differentiation and rescued by inhibition of TGF-β1. This finding suggests that hypoxia may reprogram the immature heart and affect differentiation and development.

Elastin haploinsufficiency in Williams-Beuren syndrome (WBS) leads to increased vascular smooth muscle cell (SMC) proliferation and stenoses. Our objective was to generate a human induced pluripotent stem (hiPS) cell model for in vitro assessment of the WBS phenotype and to test the ability of candidate agents to rescue the phenotype. hiPS cells were reprogrammed from skin fibroblasts of a WBS patient with aortic and pulmonary stenosis and healthy control BJ fibroblasts using four-factor retrovirus reprogramming and were differentiated into SMCs. Differentiated SMCs were treated with synthetic elastin-binding protein ligand 2 (EBPL2) (20 g/ml) or the antiproliferative drug rapamycin (100 nM) for 5 days. We generated four WBS induced pluripotent stem (iPS) cell lines that expressed pluripotency genes and differentiated into all three germ layers. Directed differentiation of BJ iPS cells yielded an 85%-92% pure SMC population that expressed differentiated SMC markers, were functionally contractile, and formed tube-like structures on three-dimensional gel assay. Unlike BJ iPS cells, WBS iPS cells generated immature SMCs that were highly proliferative, showed lower expression of differentiated SMC markers, reduced response to the vasoactive agonists, carbachol and endothelin-1, impaired vascular tube formation, and reduced calcium flux. EBPL2 partially rescued and rapamycin fully rescued the abnormal SMC phenotype by decreasing the smooth muscle proliferation rate and enhancing differentiation and tube formation. WBS iPS cellderived SMCs demonstrate an immature proliferative phenotype with reduced functional and contractile properties, thereby recapitulating the human disease phenotype. The ability of rapamycin to rescue the phenotype provides an attractive therapeutic candidate for patients with WBS and vascular stenoses. STEM CELLS TRANSLATIONAL MEDICINE 2013;2:2-15

Objective-Elevated apolipoprotein D (apoD) levels are associated with reduced proliferation of cancer cells. We therefore investigated whether apoD, which occurs free or associated with HDL, suppresses vascular smooth muscle cell (VSMC) proliferation, which is related to the pathobiology of disease. Methods and Results-Intense immunoreactivity for apoD was observed in human atherosclerotic plaque but not in normal coronary artery. However, an increase in apoD mRNA was seen in quiescent relative to proliferating fetal lamb aortic VSMCs, and in the rat aortic VSMC line (A10), we demonstrated uptake of apoD from serum. Stable transfection of apoD in A10 cells in the absence of serum did not influence VSMC proliferation assessed by [ 3 H]-thymidine incorporation. ApoD, administered at a dose of 100 ng/mL, completely inhibited basal as well as platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation (PϽ0.01) but had no effect on fibroblast growth factor-induced VSMC proliferation. ApoD did not suppress PDGF-BB or fibroblast growth factor-2-induced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 but selectively inhibited PDGF-BB-mediated ERK1/2 nuclear translocation. Conclusions-Our data suggest that apoD selectively modulates the proliferative response of VSMC to growth factors by a mechanism related to nuclear translocation of ERK1/2. Key Words: apolipoprotein D Ⅲ vascular smooth muscle cells Ⅲ platelet-derived growth factor-BB Ⅲ fibroblast growth factor-2 Ⅲ extracellular signal-regulated kinase phosphorylation and nuclear translocation Ⅲ proliferation A bnormal proliferation of vascular smooth muscle cells (VSMCs) is a critical component of atherosclerosis and arterial restenosis after angioplasty. 1 The mechanism has been related to a response to injury in which growth factors such as basic fibroblast growth factor (FGF-2) and plateletderived growth factor (PDGF) are released, stimulating proliferation and migration of VSMCs, leading to the formation of a neointima. Binding of PDGF to its receptor leads to the activation of several cell-signaling pathways associated with both VSMC proliferation and migration, such as those related to mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2, phosphatidylinositol 3-kinase (PI3-kinase), and phospholipase C-␥ (PLC-␥). 2 Many epidemiological studies have demonstrated that elevated levels of LDL and reduced levels of HDL are risk factors in the development of atherosclerosis. 3 The apolipoprotein (apo) portion of HDL consists mainly of apoA-I (70%) and A-II (20%), in addition to A-IV, C, E, J, and D. There is an inverse relationship, both in human subjects and in experimental animals, between apoA-I, apoE, and atherosclerosis. 3,4 ApoE is induced by growth arrest in human and mouse fibroblasts, 5 and in rat VSMC, apoE significantly inhibits PDGF-BB-induced VSMC proliferation by partially suppressing MAPK activity and by preventing the increase in cyclin D1, which is necessary for cells to enter the G1 phase o...

Obstructive arterial diseases including supravalvular aortic stenosis (SVAS), atherosclerosis and restenosis share two important features: an abnormal or disrupted elastic lamellae structure and excessive smooth muscle cells (SMCs). However, the relationship between these pathological features is poorly delineated. SVAS is caused by heterozygous lossof-function, hypomorphic or deletion mutations in the elastin gene ELN, and SVAS patients and elastin mutant mice display increased arterial wall cellularity and luminal obstructions.Pharmacological treatments for SVAS are lacking as underlying pathobiology is inadequately defined. Herein, using human aortic vascular cells, mouse models as well as aortic samples and SMCs derived from induced pluripotent stem cells of ELN-deficient patients, we demonstrated that elastin insufficiency induced epigenetic changes, upregulating the Notch pathway in SMCs.Specifically, reduced elastin increased levels of γ-secretase, activated NOTCH3 intracellular domain and downstream genes. Notch3 deletion or pharmacological inhibition of γ-secretase attenuated aortic hypermuscularization and stenosis in Eln (-/-) mutants. Eln (-/-) mice expressed higher levels of Notch ligand JAGGED1 (JAG1) in aortic SMCs and endothelial cells (ECs).Finally, Jag1 deletion in SMCs, but not ECs, mitigated the hypermuscular and stenotic phenotype in the aorta of Eln (-/-) mice. Our findings reveal that NOTCH3 pathway upregulation induced pathological aortic SMC accumulation during elastin insufficiency and provide potential therapeutic targets for SVAS. Dave et al., p3

Induced pluripotent stem cells (iPSC) derived from healthy individuals are important controls for disease-modeling studies. Here we apply precision health to create a high-quality resource of control iPSCs. Footprint-free lines were reprogrammed from four volunteers of the Personal Genome Project Canada (PGPC). Multilineage-directed differentiation efficiently produced functional cortical neurons, cardiomyocytes and hepatocytes. Pilot users demonstrated versatility by generating kidney organoids, T lymphocytes, and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited the expected hypertrophic phenotype. Whole-genome sequencing-based annotation of PGPC lines revealed on average 20 coding variants. Importantly, nearly all annotated PGPC and HipSci lines harbored at least one pre-existing or acquired variant with cardiac, neurological, or other disease associations. Overall, PGPC lines were efficiently differentiated by multiple users into cells from six tissues for disease modeling, and variant-preferred healthy control lines were identified for specific disease settings.