Mark P. White

miR-145 and miR-143 regulate smooth muscle cell fate and plasticity

MicroRNAs (miRNAs) are regulators of myriad cellular events, but evidence for a single miRNA that can efficiently differentiate multipotent stem cells into a specific lineage or regulate direct reprogramming of cells into an alternative cell fate has been elusive. Here we show that miR-145 and miR-143 are co-transcribed in multipotent murine cardiac progenitors before becoming localized to smooth muscle cells, including neural crest stem-cell-derived vascular smooth muscle cells. miR-145 and miR-143 were direct transcriptional targets of serum response factor, myocardin and Nkx2-5 (NK2 transcription factor related, locus 5) and were downregulated in injured or atherosclerotic vessels containing proliferating, less differentiated smooth muscle cells. miR-145 was necessary for myocardin-induced reprogramming of adult fibroblasts into smooth muscle cells and sufficient to induce differentiation of multipotent neural crest stem cells into vascular smooth muscle. Furthermore, miR-145 and miR-143 cooperatively targeted a network of transcription factors, including Klf4 (Kruppel-like factor 4), myocardin and Elk-1 (ELK1, member of ETS oncogene family), to promote differentiation and repress proliferation of smooth muscle cells. These findings demonstrate that miR-145 can direct the smooth muscle fate and that miR-145 and miR-143 function to regulate the quiescent versus proliferative phenotype of smooth muscle cells.

Relationship between plasma levels of lipopolysaccharide (LPS) and LPS- binding protein in patients with severe sepsis and septic shock

Plasma endotoxin and lipopolysaccharide-binding protein (LBP) levels were measured in a group of 253 patients at the onset of severe sepsis and/or septic shock. Endotoxin levels were significantly greater than control levels (n=33; mean +/- SD, 5.1+/-7.3 pg/mL) in 78.3% of patients. Median endotoxin levels in patients with sepsis were 300 pg/mL (25%-75% interquartile range, 110-726 pg/mL). LBP levels were elevated in 97% of patients compared with normal control values of 4.1+/-1.65 microgram/mL. Median LBP levels in patients with sepsis were 31.2 microgram/mL (interquartile range, 22.5-47.7 microgram/mL). Median endotoxin levels at study entry were more highly elevated (515 vs. 230 pg/mL; P<.01), and LBP levels were less highly elevated (28.0 vs. 33.2 microgram/mL; P<.05) in nonsurvivors than survivors over the 28-day study period. No correlation was found between endotoxin and LBP levels. The quantitative level of both endotoxin and LBP may have prognostic significance in patients with severe sepsis.

Limited Gene Expression Variation in Human Embryonic Stem Cell and Induced Pluripotent Stem Cell‐Derived Endothelial Cells

Recent evidence suggests human embryonic stem cell (hESC) and induced pluripotent stem (iPS) cell lines have differences in their epigenetic marks and transcriptomes, yet the impact of these differences on subsequent terminally differentiated cells is less well understood. Comparison of purified, homogeneous populations of somatic cells derived from multiple independent human iPS and ES lines will be required to address this critical question. Here, we report a differentiation protocol based on embryonic development that consistently yields large numbers of endothelial cells (ECs) derived from multiple hESCs or iPS cells. Mesoderm differentiation of embryoid bodies was maximized, and defined growth factors were used to generate KDR+ EC progenitors. Magnetic purification of a KDR+ progenitor subpopulation resulted in an expanding, homogeneous pool of ECs that expressed EC markers and had functional properties of ECs. Comparison of the transcriptomes revealed limited gene expression variability between multiple lines of human iPS‐derived ECs or between lines of ES‐ and iPS‐derived ECs. These results demonstrate a method to generate large numbers of pure human EC progenitors and differentiated ECs from pluripotent stem cells and suggest individual lineages derived from human iPS cells may have significantly less variance than their pluripotent founders. STEM Cells2013;31:92–103

Human Disease Modeling Reveals Integrated Transcriptional and Epigenetic Mechanisms of NOTCH1 Haploinsufficiency

The mechanisms by which transcription factor haploinsufficiency alters the epigenetic and transcriptional landscape in human cells to cause disease are unknown. Here, we utilized human induced pluripotent stem cell (iPSC)-derived endothelial cells (ECs) to show that heterozygous nonsense mutations in NOTCH1 that cause aortic valve calcification disrupt the epigenetic architecture, resulting in derepression of latent pro-osteogenic and -inflammatory gene networks. Hemodynamic shear stress, which protects valves from calcification in vivo, activated anti-osteogenic and anti-inflammatory networks in NOTCH1(+/+), but not NOTCH1(+/-), iPSC-derived ECs. NOTCH1 haploinsufficiency altered H3K27ac at NOTCH1-bound enhancers, dysregulating downstream transcription of more than 1,000 genes involved in osteogenesis, inflammation, and oxidative stress. Computational predictions of the disrupted NOTCH1-dependent gene network revealed regulatory nodes that, when modulated, restored the network toward the NOTCH1(+/+) state. Our results highlight how alterations in transcription factor dosage affect gene networks leading to human disease and reveal nodes for potential therapeutic intervention.

An Overview of the Pharmacokinetics and Pharmacodynamics of Efalizumab: A Monoclonal Antibody Approved for Use in Psoriasis

Efalizumab is a recombinant humanized monoclonal IgG1 antibody shown to be efficacious for the treatment of moderate to severe chronic plaque psoriasis. Efalizumab, a targeted inhibitor of T cell interactions, binds to the CD11a subunit of lymphocyte function—associated antigen 1 (LFA‐1), thereby preventing LFA‐1 binding to intercellular adhesion molecule 1 (ICAM‐1). The authors review the pharmacokinetic and pharmacodynamic data from the efalizumab clinical development program and discuss how these data led to selection of the optimal weekly subcutaneous (SC) dose of efalizumab (1.0 mg/kg) in adults. Efalizumab SC dosages of 1.0 mg/kg/wk or greater exerted maximal pharmacodynamic effects for CD11a expression and available CD11a binding sites on T lymphocytes. Dosages greater than 1.0 mg/kg/wk SC did not provide additional benefits; moreover, higher doses did not alter the safety profile. During long‐term administration of efalizumab, serum levels were generally stable and pharmacodynamic markers remained maximally affected.

MicroRNA-10 Regulates the Angiogenic Behavior of Zebrafish and Human Endothelial Cells by Promoting Vascular Endothelial Growth Factor Signaling

Rationale: Formation and remodeling of the vasculature during development and disease involve a highly conserved and precisely regulated network of attractants and repellants. Various signaling pathways control the behavior of endothelial cells, but their posttranscriptional dose titration by microRNAs is poorly understood. Objective: To identify microRNAs that regulate angiogenesis. Methods and Results: We show that the highly conserved microRNA family encoding miR-10 regulates the behavior of endothelial cells during angiogenesis by positively titrating proangiogenic signaling. Knockdown of miR-10 led to premature truncation of intersegmental vessel growth in the trunk of zebrafish larvae, whereas overexpression of miR-10 promoted angiogenic behavior in zebrafish and cultured human umbilical venous endothelial cells. We found that miR-10 functions, in part, by directly regulating the level of fms-related tyrosine kinase 1 (FLT1), a cell-surface protein that sequesters vascular endothelial growth factor, and its soluble splice variant sFLT1. The increase in FLT1/sFLT1 protein levels upon miR-10 knockdown in zebrafish and in human umbilical venous endothelial cells inhibited the angiogenic behavior of endothelial cells largely by antagonizing vascular endothelial growth factor receptor 2 signaling. Conclusions: Our study provides insights into how FLT1 and vascular endothelial growth factor receptor 2 signaling is titrated in a microRNA-mediated manner and establishes miR-10 as a potential new target for the selective modulation of angiogenesis.

The neural crest-enriched microRNA miR-452 regulates epithelial-mesenchymal signaling in the first pharyngeal arch

Neural crest cells (NCCs) are a subset of multipotent, migratory stem cells that populate a large number of tissues during development and are important for craniofacial and cardiac morphogenesis. Although microRNAs (miRNAs) have emerged as important regulators of development and disease, little is known about their role in NCC development. Here, we show that loss of miRNA biogenesis by NCC-specific disruption of murine Dicer results in embryos lacking craniofacial cartilaginous structures, cardiac outflow tract septation and thymic and dorsal root ganglia development. Dicer mutant embryos had reduced expression of Dlx2, a transcriptional regulator of pharyngeal arch development, in the first pharyngeal arch (PA1). miR-452 was enriched in NCCs, was sufficient to rescue Dlx2 expression in Dicer mutant pharyngeal arches, and regulated non-cell-autonomous signaling involving Wnt5a, Shh and Fgf8 that converged on Dlx2 regulation in PA1. Correspondingly, knockdown of miR-452 in vivo decreased Dlx2 expression in the mandibular component of PA1, leading to craniofacial defects. These results suggest that post-transcriptional regulation by miRNAs is required for differentiation of NCC-derived tissues and that miR-452 is involved in epithelial-mesenchymal signaling in the pharyngeal arch.

Plasma bactericidal/permeability-increasing protein concentrations in critically ill children with the sepsis syndrome.

Bactericidal/permeability-increasing protein (BPI) is a neutrophil azurophilic granule component that is bactericidal towards Gram-negative bacteria and inhibits lipopolysaccharide-mediated inflammatory responses. We conducted a prospective study to measure plasma BPI concentrations in 36 critically ill children with and without the sepsis syndrome. Plasma BPI concentrations ranged from 0.5 to 452 ng/ml. Patients with the sepsis syndrome had higher median plasma BPI concentrations than critically ill controls (5.1 v8. 1.8 ng/ml, P = 0.006). Patients with organ system failure had higher median plasma BPI concentrations than those with no organ system failure (4.5 v8. 1.3 ng/ml, P = 0.001). Plasma BPI concentrations were positively associated with pediatric risk of mortality score (P = 0.03, r8 = 0.4). These data provide the first clinical insights regarding the role of endogenous BPI production in critically ill children and suggest that BPI may play an important role in host defenses.

NOTCH1 regulates matrix gla protein and calcification gene networks in human valve endothelium.

Valvular and vascular calcification are common causes of cardiovascular morbidity and mortality. Developing effective treatments requires understanding the molecular underpinnings of these processes. Shear stress is thought to play a role in inhibiting calcification. Furthermore, NOTCH1 regulates vascular and valvular endothelium, and human mutations in NOTCH1 can cause calcific aortic valve disease. Here, we determined the genome-wide impact of altering shear stress and NOTCH signaling on human aortic valve endothelium. mRNA-sequencing of primary human aortic valve endothelial cells (HAVECs) with or without knockdown of NOTCH1, in the presence or absence of shear stress, revealed NOTCH1-dependency of the atherosclerosis-related gene connexin 40 (GJA5), and numerous repressors of endochondral ossification. Among these, matrix gla protein (MGP) is highly expressed in aortic valve and vasculature, and inhibits soft tissue calcification by sequestering bone morphogenetic proteins (BMPs). Altering NOTCH1 levels affected MGP mRNA and protein in HAVECs. Furthermore, shear stress activated NOTCH signaling and MGP in a NOTCH1-dependent manner. NOTCH1 positively regulated endothelial MGP in vivo through specific binding motifs upstream of MGP. Our studies suggest that shear stress activates NOTCH1 in primary human aortic valve endothelial cells leading to downregulation of osteoblast-like gene networks that play a role in tissue calcification.

The ACVR1 R206H mutation found in fibrodysplasia ossificans progressiva increases human induced pluripotent stem cell-derived endothelial cell formation and collagen production through BMP-mediated SMAD1/5/8 signaling.

BackgroundThe Activin A and bone morphogenetic protein (BMP) pathways are critical regulators of the immune system and of bone formation. Inappropriate activation of these pathways, as in conditions of congenital heterotopic ossification, are thought to activate an osteogenic program in endothelial cells. However, if and how this occurs in human endothelial cells remains unclear.MethodsWe used a new directed differentiation protocol to create human induced pluripotent stem cell (hiPSC)-derived endothelial cells (iECs) from patients with fibrodysplasia ossificans progressiva (FOP), a congenital disease of heterotopic ossification caused by an activating R206H mutation in the Activin A type I receptor (ACVR1). This strategy allowed the direct assay of the cell-autonomous effects of ACVR1 R206H in the endogenous locus without the use of transgenic expression. These cells were challenged with BMP or Activin A ligand, and tested for their ability to activate osteogenesis, extracellular matrix production, and differential downstream signaling in the BMP/Activin A pathways.ResultsWe found that FOP iECs could form in conditions with low or absent BMP4. These conditions are not normally permissive in control cells. FOP iECs cultured in mineralization media showed increased alkaline phosphatase staining, suggesting formation of immature osteoblasts, but failed to show mature osteoblastic features. However, FOP iECs expressed more fibroblastic genes and Collagen 1/2 compared to control iECs, suggesting a mechanism for the tissue fibrosis seen in early heterotopic lesions. Finally, FOP iECs showed increased SMAD1/5/8 signaling upon BMP4 stimulation. Contrary to FOP hiPSCs, FOP iECs did not show a significant increase in SMAD1/5/8 phosphorylation upon Activin A stimulation, suggesting that the ACVR1 R206H mutation has a cell type-specific effect. In addition, we found that the expression of ACVR1 and type II receptors were different in hiPSCs and iECs, which could explain the cell type-specific SMAD signaling.ConclusionsOur results suggest that the ACVR1 R206H mutation may not directly increase the formation of mature chondrogenic or osteogenic cells by FOP iECs. Our results also show that BMP can induce endothelial cell dysfunction, increase expression of fibrogenic matrix proteins, and cause differential downstream signaling of the ACVR1 R206H mutation. This iPSC model provides new insight into how human endothelial cells may contribute to the pathogenesis of heterotopic ossification.