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1.  Dual Role of Endothelial Nitric Oxide Synthase in Oxidized LDL-Induced, p66Shc-Mediated Oxidative Stress in Cultured Human Endothelial Cells 
PLoS ONE  2014;9(9):e107787.
The aging gene p66Shc, is an important mediator of oxidative stress-induced vascular dysfunction and disease. In cultured human aortic endothelial cells (HAEC), p66Shc deletion increases endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability via protein kinase B. However, the putative role of the NO pathway on p66Shc activation remains unclear. This study was designed to elucidate the regulatory role of the eNOS/NO pathway on p66Shc activation.
Methods and Results
Incubation of HAEC with oxidized low density lipoprotein (oxLDL) led to phosphorylation of p66Shc at Ser-36, resulting in an enhanced production of superoxide anion (O2-). In the absence of oxLDL, inhibition of eNOS by small interfering RNA or L-NAME, induced p66Shc phosphorylation, suggesting that basal NO production inhibits O2- production. oxLDL-induced, p66Shc-mediated O2- was prevented by eNOS inhibition, suggesting that when cells are stimulated with oxLDL eNOS is a source of reactive oxygen species. Endogenous or exogenous NO donors, prevented p66Shc activation and reduced O2- production. Treatment with tetrahydrobiopterin, an eNOS cofactor, restored eNOS uncoupling, prevented p66Shc activation, and reduced O2- generation. However, late treatment with tetrahydropterin did not yield the same result suggesting that eNOS uncoupling is the primary source of reactive oxygen species.
The present study reports that in primary cultured HAEC treated with oxLDL, p66Shc-mediated oxidative stress is derived from eNOS uncoupling. This finding contributes novel information on the mechanisms of p66Shc activation and its dual interaction with eNOS underscoring the importance eNOS uncoupling as a putative antioxidant therapeutical target in endothelial dysfunction as observed in cardiovascular disease.
PMCID: PMC4172699  PMID: 25247687
2.  Alternatively Spliced Tissue Factor Is Not Sufficient for Embryonic Development 
PLoS ONE  2014;9(5):e97793.
Tissue factor (TF) triggers blood coagulation and is translated from two mRNA splice isoforms, encoding membrane-anchored full-length TF (flTF) and soluble alternatively-spliced TF (asTF). The complete knockout of TF in mice causes embryonic lethality associated with failure of the yolk sac vasculature. Although asTF plays roles in postnatal angiogenesis, it is unknown whether it activates coagulation sufficiently or makes previously unrecognized contributions to sustaining integrity of embryonic yolk sac vessels. Using gene knock-in into the mouse TF locus, homozygous asTF knock-in (asTFKI) mice, which express murine asTF in the absence of flTF, exhibited embryonic lethality between day 9.5 and 10.5. Day 9.5 homozygous asTFKI embryos expressed asTF protein, but no procoagulant activity was detectable in a plasma clotting assay. Although the α-smooth-muscle-actin positive mesodermal layer as well as blood islands developed similarly in day 8.5 wild-type or homozygous asTFKI embryos, erythrocytes were progressively lost from disintegrating yolk sac vessels of asTFKI embryos by day 10.5. These data show that in the absence of flTF, asTF expressed during embryonic development has no measurable procoagulant activity, does not support embryonic vessel stability by non-coagulant mechanisms, and fails to maintain a functional vasculature and embryonic survival.
PMCID: PMC4039448  PMID: 24879059
3.  Peripheral Blood Monocyte Sirt1 Expression Is Reduced in Patients with Coronary Artery Disease 
PLoS ONE  2013;8(1):e53106.
Inflammation plays a key role in atherosclerosis. Sirt1 regulates transcription factors involved in inflammatory processes and blunts atherosclerosis in mice. However, its role in humans remains to be defined. This study was therefore designed to investigate the role of Sirt1 in the development of atherosclerosis.
Methods and Results
48 male subjects admitted for cardiac catheterization were subdivided into healthy subjects, patients with stable coronary artery disease (CAD), and with acute coronary syndromes (ACS). Monocytes were isolated and Sirt1 mRNA levels were determined. Sirt1 gene expression was higher in healthy subjects as compared to patients with CAD or ACS (P<0.05), respectively. Interestingly, HDL levels correlated positively with Sirt1 expression. Thus, HDL from the three groups was isolated and incubated with THP-1 monocytes to determine the effects of HDL on Sirt1 protein in controlled experimental conditions. HDL from healthy subjects stimulated Sirt1 expression in THP-1 monocytes to a higher degree than HDL from CAD and ACS patients (P<0.05). Paraoxonase-1 (PON-1), a HDL-associated enzyme, showed a reduced activity in HDL isolated from CAD and ACS patients as compared to the controls (P<0.001).
Monocytic Sirt1 expression is reduced in patients with stable CAD and ACS. Experiments on THP-1 monocytes suggest that this effect is HDL-dependent and is mediated by a reduced activity of HDL-associated enzyme PON1.
PMCID: PMC3558418  PMID: 23382833
5.  Globotriaosylsphingosine Accumulation and Not Alpha-Galactosidase-A Deficiency Causes Endothelial Dysfunction in Fabry Disease 
PLoS ONE  2012;7(4):e36373.
Fabry disease (FD) is caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (GLA) resulting in the accumulation of globotriaosylsphingosine (Gb3) in a variety of tissues. While GLA deficiency was always considered as the fulcrum of the disease, recent attention shifted towards studying the mechanisms through which Gb3 accumulation in vascular cells leads to endothelial dysfunction and eventually multiorgan failure. In addition to the well-described macrovascular disease, FD is also characterized by abnormalities of microvascular function, which have been demonstrated by measurements of myocardial blood flow and coronary flow reserve. To date, the relative importance of Gb3 accumulation versus GLA deficiency in causing endothelial dysfunction is not fully understood; furthermore, its differential effects on cardiac micro- and macrovascular endothelial cells are not known.
Methods and Results
In order to assess the effects of Gb3 accumulation versus GLA deficiency, human macro- and microvascular cardiac endothelial cells (ECs) were incubated with Gb3 or silenced by siRNA to GLA. Gb3 loading caused deregulation of several key endothelial pathways such as eNOS, iNOS, COX-1 and COX-2, while GLA silencing showed no effects. Cardiac microvascular ECs showed a greater susceptibility to Gb3 loading as compared to macrovascular ECs.
Deregulation of key endothelial pathways as observed in FD vasculopathy is likely caused by intracellular Gb3 accumulation rather than deficiency of GLA. Human microvascular ECs, as opposed to macrovascular ECs, seem to be affected earlier and more severely by Gb3 accumulation and this notion may prove fundamental for future progresses in early diagnosis and management of FD patients.
PMCID: PMC3340376  PMID: 22558451

Results 1-6 (6)