Faculty Bibliography

The key regulators of endothelial differentiation that is induced by shear stress are mostly unclear. Human atonal homolog 6 (Hath6 or ATOH8) is an endothelial-selective and shear-stress-responsive transcription factor. In this study, we sought to elucidate the role of Hath6 in the endothelial specification of embryonic stem cells. In a stepwise human embryonic stem cell to endothelial cell (hESC-EC) induction system, Hath6 mRNA was upregulated synchronously with endothelial determination. Subsequently, gain-of-function and loss-of-function studies of Hath6 were performed using the hESC-EC induction model and endothelial cell lines. The overexpression of Hath6, which mimics shear stress treatment, resulted in an increased CD45(-)CD31(+)KDR(+) population, a higher tubular-structure-formation capacity and increased endothelial-specific gene expression. By contrast, the knockdown of Hath6 mRNA markedly decreased endothelial differentiation. Hath6 also facilitated the maturation of endothelial cells in terms of endothelial gene expression, tubular-structure formation and cell migration. We further demonstrated that the gene encoding eNOS is a direct target of Hath6 through a reporter system assay and western blot analysis, and that the inhibition of eNOS diminishes hESC-EC differentiation. These results suggest that eNOS plays a key role in linking Hath6 to the endothelial phenotype. Further in situ hybridization studies in zebrafish and mouse embryos indicated that homologs of Hath6 are involved in vasculogenesis and angiogenesis. This study provides the first confirmation of the positive impact of Hath6 on human embryonic endothelial differentiation and function. Moreover, we present a potential signaling pathway through which shear stress stimulates endothelial differentiation.

Telomeres protect and cap linear chromosome ends, yet these genomic buffers erode over an organism's lifespan. Short telomeres have been associated with many age-related conditions in humans, and genetic mutations resulting in short telomeres in humans manifest as syndromes of precocious aging. In women, telomere length limits a fertilized egg's capacity to develop into a healthy embryo. Thus, telomere length must be reset with each subsequent generation. Although telomerase is purportedly responsible for restoring telomere DNA, recent studies have elucidated the role of alternative telomeres lengthening mechanisms in the reprogramming of early embryos and stem cells, which we review here.

We reported previously that apolipoprotein A-I (apoA-I) is oxidatively modified in the artery wall at tyrosine 166 (Tyr(166)), serving as a preferred site for post-translational modification through nitration. Recent studies, however, question the extent and functional importance of apoA-I Tyr(166) nitration based upon studies of HDL-like particles recovered from atherosclerotic lesions. We developed a monoclonal antibody (mAb 4G11.2) that recognizes, in both free and HDL-bound forms, apoA-I harboring a 3-nitrotyrosine at position 166 apoA-I (NO2-Tyr(166)-apoA-I) to investigate the presence, distribution, and function of this modified apoA-I form in atherosclerotic and normal artery wall. We also developed recombinant apoA-I with site-specific 3-nitrotyrosine incorporation only at position 166 using an evolved orthogonal nitro-Tyr-aminoacyl-tRNA synthetase/tRNACUA pair for functional studies. Studies with mAb 4G11.2 showed that NO2-Tyr(166)-apoA-I was easily detected in atherosclerotic human coronary arteries and accounted for approximately 8% of total apoA-I within the artery wall but was nearly undetectable (>100-fold less) in normal coronary arteries. Buoyant density ultracentrifugation analyses showed that NO2-Tyr(166)-apoA-I existed as a lipid-poor lipoprotein with <3% recovered within the HDL-like fraction (d = 1.063-1.21). NO2-Tyr(166)-apoA-I in plasma showed a similar distribution. Recovery of NO2-Tyr(166)-apoA-I using immobilized mAb 4G11.2 showed an apoA-I form with 88.1 +/- 8.5% reduction in lecithin-cholesterol acyltransferase activity, a finding corroborated using a recombinant apoA-I specifically designed to include the unnatural amino acid exclusively at position 166. Thus, site-specific nitration of apoA-I at Tyr(166) is an abundant modification within the artery wall that results in selective functional impairments. Plasma levels of this modified apoA-I form may provide insights into a pathophysiological process within the diseased artery wall.

Introduction: The main function of connexins is to form gap junctions; yet, recent studies show that Cx43 is not only a gap junction protein. In fact, Cx43 is a part of a protein interacting network (the connexome), likely to regulate other functions in a gap junction-independent manner. Recently, it was reported that loss of the last five amino acids of Cx43 (Cx43D378stop) leads to lethal ventricular arrhythmias in mice. Localization of Cx43 at the membrane and electrical coupling between cells was normal. Interestingly, there was a significant loss of sodium current amplitude. These observations linked two fundamental steps in action potential propagation, excitability and electrical coupling, through a common molecular mechanism. Here, we explore the hypothesis that the microtubular network at the cell end is part of the common link. Methods: N/A Results: Functional assays: Macropatch, and super-resolution scanning patch clamp in ventricular myocytes isolated from Cx43D378stop and Cre-negative (control) mice revealed a reduction in the amplitude of sodium current exclusively at the intercalated disc (ID), without a change in channel unitary conductance. Super-resolution fluorescence microscopy: direct stochastic optical reconstruction microscopy (20 nm resolution) showed Nav1.5 clusters in close proximity (or overlapping) with N-cadherin plaques. The distance between NaV1.5 clusters and the cell end increased from 57.2+12nm, n=365 in control to 111.7+11nm, n=446 in Cx43D378stop myocytes (p<0.001), indicating that mutation Cx43D378stop reduced NaV1.5 surface expression. This coincided with separation of the microtubule plus-end protein EB1 from N-cadherin-rich cell ends, from 23.7+31.9nm, n=665 in control, to 123.5+13.5nm, n=502 in Cx43D378stop cells (p<0.05). Conclusions: Functional surface expression of NaV1.5 at the ID depends on preservation of the Cx43 C-end. Cx43 is part of a molecular complex that anchors the microtubule plus-end to the cell end, thus allowing proper delivery of its ca!

The discovery that TNF receptors (TNFR) serve as the binding receptors for progranulin (PGRN) reveals the significant role of PGRN in inflammatory and autoimmune diseases, including inflammatory arthritis. Herein we describe a simple, antibody-free analytical assay, i.e., a biotin-based solid-phase binding assay, to examine the direct interaction of PGRN/TNFR and the PGRN inhibition of TNF/TNFR interactions. Briefly, a 96-well high-binding microplate is first coated with the first protein (protein A), and after blocking, the coated microplate is incubated with the biotin-labeled second protein (protein B) in the absence or presence of the third protein (protein C). Finally the streptavidin conjugated with a detecting enzyme is added, followed by a signal measurement. Also discussed in this chapter are the advantages of the strategy, key elements to obtain reliable results, and discrepancies among various PGRN proteins in view of the binding activity with TNFR.

BACKGROUND: The recent availability of sequenced genomes from a broad array of chordates (cephalochordates, urochordates and vertebrates) has allowed us to systematically analyze the evolution of uroplakins: tetraspanins (UPK1a and UPK1b families) and their respective partner proteins (UPK2 and UPK3 families). RESULTS: We report here: (1) the origin of uroplakins in the common ancestor of vertebrates, (2) the appearance of several residues that have statistically significantly positive dN/dS ratios in the duplicated paralogs of uroplakin genes, and (3) the existence of strong coevolutionary relationships between UPK1a/1b tetraspanins and their respective UPK2/UPK3-related partner proteins. Moreover, we report the existence of three new UPK2/3 family members we named UPK2b, 3c and 3d, which will help clarify the evolutionary relationships between fish, amphibian and mammalian uroplakins that may perform divergent functions specific to these different and physiologically distinct groups of vertebrates. CONCLUSIONS: Since our analyses cover species of all major chordate groups this work provides an extremely clear overall picture of how the uroplakin families and their partner proteins have evolved in parallel. We also highlight several novel features of uroplakin evolution including the appearance of UPK2b and 3d in fish and UPK3c in the common ancestor of reptiles and mammals. Additional studies of these novel uroplakins should lead to new insights into uroplakin structure and function.

The oligodendrocyte (OL), the myelinating cell of the central nervous system, undergoes dramatic changes in the organization of its cytoskeleton as it differentiates from a precursor (oligodendrocyte precursor cells) to a myelin-forming cell. These changes include an increase in its branching cell processes, a phenomenon necessary for OL to myelinate multiple axon segments. We have previously shown that levels and activity of non-muscle myosin II (NMII), a regulator of cytoskeletal contractility, decrease as a function of differentiation and that inhibition of NMII increases branching and myelination of OL in coculture with neurons. We have also found that mixed glial cell cultures derived from NMIIB knockout mice display an increase in mature myelin basic protein-expressing OL compared with wild-type cultures. We have now extended our studies to investigate the role of NMIIB ablation on myelin repair following focal demyelination by lysolecithin. To this end, we generated an oligodendrocyte-specific inducible knockout model using a Plp-driven promoter in combination with a temporally activated CRE-ER fusion protein. Our data indicate that conditional ablation of NMII in adult mouse brain, expedites lesion resolution and remyelination by Plp+ oligodendrocyte-lineage cells when compared with that observed in control brains. Taken together, these data validate the function of NMII as that of a negative regulator of OL myelination in vivo and provide a novel target for promoting myelin repair in conditions such as multiple sclerosis.

Since it has been recognized that mitochondria are crucial not only for energy metabolism but also for other cellular functions, there has been a growing interest in cardiolipin, the specific phospholipid of mitochondrial membranes. Indeed, cardiolipin is a universal component of mitochondria in all eukaryotes. It has a unique dimeric structure comprised of two phosphatidic acid residues linked by a glycerol bridge, which gives rise to unique physicochemical properties. Cardiolipin plays an important role in the structural organization and the function of mitochondrial membranes. In this article, we review the literature on cardiolipin biology, focusing on the most important discoveries of the past decade. Specifically, we describe the formation, the migration, and the degradation of cardiolipin and we discuss how cardiolipin affects mitochondrial function. We also give an overview of the various phenotypes of cardiolipin deficiency in different organisms.

The yellow fever vaccine YF-17D is one of the most successful vaccines ever developed in humans. Despite its efficacy and widespread use in more than 600 million people, the mechanisms by which it stimulates protective immunity remain poorly understood. Recent studies using systems biology approaches in humans have revealed that YF-17D-induced early expression of general control nonderepressible 2 kinase (GCN2) in the blood strongly correlates with the magnitude of the later CD8(+) T cell response. We demonstrate a key role for virus-induced GCN2 activation in programming dendritic cells to initiate autophagy and enhanced antigen presentation to both CD4(+) and CD8(+) T cells. These results reveal an unappreciated link between virus-induced integrated stress response in dendritic cells and the adaptive immune response.