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1.  ZO-1 controls endothelial adherens junctions, cell–cell tension, angiogenesis, and barrier formation 
The Journal of Cell Biology  2015;208(6):821-838.
ZO-1 regulates VE-cadherin–dependent endothelial junctions and actomyosin organization, thereby influencing cell–cell tension, migration, angiogenesis, and barrier formation
Intercellular junctions are crucial for mechanotransduction, but whether tight junctions contribute to the regulation of cell–cell tension and adherens junctions is unknown. Here, we demonstrate that the tight junction protein ZO-1 regulates tension acting on VE-cadherin–based adherens junctions, cell migration, and barrier formation of primary endothelial cells, as well as angiogenesis in vitro and in vivo. ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the α-catenin–VE-cadherin complex. Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation. ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF. ZO-1 is thus a central regulator of VE-cadherin–dependent endothelial junctions that orchestrates the spatial actomyosin organization, tuning cell–cell tension, migration, angiogenesis, and barrier formation.
PMCID: PMC4362456  PMID: 25753039
2.  A multifaceted approach to prevention of delirium on intensive care 
BMJ Quality Improvement Reports  2015;4(1):u209656.w4000.
Delirium is a frequent complication of critical illness with an increased risk of death which is difficult to treat effectively. We describe a seven year quality improvement programme that aimed to reduce rates of delirium on intensive care (ICU). We completed two PDSA programmes with a number of changes including alterations to sedation practice and environmental changes to the ICU itself.
Rates of delirium reduced from 70% to 44% after the first cycle of change. Rates of delirium were further reduced to 29% after the second cycle of change. The rates are now lower than predicted by the validated prediction tool PRE-DELIRIC.
This project demonstrates that a multifaceted approach to prevention of delirium on ICU can deliver sustained reductions in rates of delirium. The impact of the second cycle of change was less than the first, suggesting that further reductions may prove more challenging.
PMCID: PMC4693040  PMID: 26734390
3.  Rac1 functions as a reversible tension modulator to stabilize VE-cadherin trans-interaction 
The Journal of Cell Biology  2015;208(1):23-32.
In endothelial cells, the RhoGTPase Rac1 stabilizes VE-cadherin trans-dimers in mature adherens junctions by counteracting actomyosin tension.
The role of the RhoGTPase Rac1 in stabilizing mature endothelial adherens junctions (AJs) is not well understood. In this paper, using a photoactivatable probe to control Rac1 activity at AJs, we addressed the relationship between Rac1 and the dynamics of vascular endothelial cadherin (VE-cadherin). We demonstrated that Rac1 activation reduced the rate of VE-cadherin dissociation, leading to increased density of VE-cadherin at AJs. This response was coupled to a reduction in actomyosin-dependent tension across VE-cadherin adhesion sites. We observed that inhibiting myosin II directly or through photo-release of the caged Rho kinase inhibitor also reduced the rate of VE-cadherin dissociation. Thus, Rac1 functions by stabilizing VE-cadherin trans-dimers in mature AJs by counteracting the actomyosin tension. The results suggest a new model of VE-cadherin adhesive interaction mediated by Rac1-induced reduction of mechanical tension at AJs, resulting in the stabilization of VE-cadherin adhesions.
PMCID: PMC4284224  PMID: 25559184
5.  Fluid shear stress on endothelial cells modulates mechanical tension across VE-cadherin and PECAM-1 
Current biology : CB  2013;23(11):1024-1030.
Fluid shear stress (FSS) from blood flow acting on the endothelium critically regulates vascular morphogenesis, blood pressure and atherosclerosis [1]. FSS applied to endothelial cells (EC) triggers signaling events including opening of ion channels, activation of signaling pathways and changes in gene expression. Elucidating how ECs sense flow important for understanding both normal vascular function and disease. EC responses to FSS are mediated in part by a junctional mechanosensory complex consisting of VE-cadherin, PECAM-1, and VEGFR2 [2]. Previous work suggested that flow increases force on PECAM-1, which initiates signaling [2–4]. Deletion of PECAM-1 blocks responses to flow in vitro and flow-dependent vascular remodeling in vivo [2, 5]. To understand this process, we developed and validated FRET-based tension sensors for VE-cadherin and PECAM-1 using our previously developed FRET tension biosensor [6]. FRET measurements showed that in static culture, VE-cadherin in cell-cell junctions bears significant myosin-dependent tension, whereas there was no detectable tension on VE-cadherin outside of junctions. Onset of shear stress triggered a rapid (<30 sec) decrease in tension across VE-cadherin, which paralleled a decrease in total cell-cell junctional tension. Flow triggered a simultaneous increase in tension across junctional PECAM-1, while non-junctional PECAM-1 was unaffected. Tension on PECAM-1 was mediated by flow-stimulated association with vimentin. These data confirm the prediction that shear increases force on PECAM-1. However, they also argue against the current model of passive transfer of force through the cytoskeleton to the junctions [7], showing instead that flow triggers cytoskeletal remodeling, which alters forces across the junctional receptors.
PMCID: PMC3676707  PMID: 23684974
6.  Prevalence of Congenital Anomalies in Infants with in Utero Exposure to Antiretrovirals 
While use of efficacious interventions, including antiretrovirals (ARVs), has reduced dramatically the rate of mother-to-child transmission (MTCT) of HIV, the safety of in utero ARV exposure remains of concern.
Data regarding 1112 infants enrolled in the International Maternal Pediatric Adolescent AIDS Clinical Trials Group (IMPAACT) protocol P1025 born between 2002 and 2007 were analyzed for this study. Congenital anomalies were classified based on the Metropolitan Atlanta Congenital Defects Program (MACDP) guidelines. Associations between congenital anomalies and timing of first in utero exposure to ARVs were evaluated by logistic regression analysis.
61 of the 1112 infants had congenital anomalies identified and confirmed, resulting in a prevalence of 5.49/100 live births (95%CI: 4.22–6.99). Among the 80 anomalies identified, the organ systems involved included: cardiovascular (n=33), musculoskeletal (n=15), renal (n=9), genitourinary (n=6), craniofacial (n=4), and central nervous system (n=2). First trimester exposure to efavirenz was associated with a significantly increased risk of congenital anomalies (OR 2.84, 95%CI: 1.13–7.16). No significant associations were observed between exposure to other individual ARVs or classes of ARVs started at any time during pregnancy and infant congenital anomalies.
The observed rate of congenital anomalies in this cohort is higher than previously reported for the general population, but is consistent with rates observed in other recent studies of children born to HIV-infected women. Cardiovascular anomalies occurred most frequently. With the exception of a known teratogen (efavirenz), no statistically significant associations between in utero exposure to ARVs and congenital anomalies were identified.
PMCID: PMC3261302  PMID: 21983213
congenital anomalies; in utero exposure; HIV; antiretroviral
7.  Lessons from the endothelial junctional mechanosensory complex 
Mechanotransduction plays a key role in both normal physiology and in diseases such as cancer, atherosclerosis and hypertension. Nowhere is this more evident than in the vascular system, where fluid shear stress from blood flow plays a critical role in shaping the blood vessels and in determining their function and dysfunction. Responses to flow are mediated in part by a complex of proteins comprised of PECAM-1, VE-cadherin and VEGFR2 at endothelial cell-cell junctions; all proteins that clearly have other, non-mechanical functions. We review recent progress toward understanding the functions and mechanisms of mechanotransduction by this complex and suggest some principles that may apply more broadly.
PMCID: PMC3251317  PMID: 22238515
8.  Birth defects among children born to HIV-infected women: Pediatric AIDS Clinical Trials Protocols 219 and 219C 
Some studies have detected associations between in utero antiretroviral therapy (ARV) exposure and birth defects but evidence is inconclusive.
2,202 HIV-exposed children enrolled in the Pediatric AIDS Clinical Trials Group 219 and 219C protocols before one year of age were included. Birth defects were classified using the Metropolitan Atlanta Congenital Defects Program (MACDP) coding. Logistic regression models were used to evaluate associations between first trimester in utero ARV exposure and birth defects.
117 live-born children had birth defects for a prevalence of 5.3% (95% CI: 4.4, 6.3). Prevalence did not differ by HIV infection status or overall ARV exposure; rates were 4.8% (95% CI: 3.7, 6.1) and 5.8% (95% CI: 4.2, 7.8) in children without and with first trimester ARV exposure, respectively. The defect rate was higher among children with first trimester efavirenz exposure (5/32, 15.6%) versus children without first trimester efavirenz exposure [adjusted odds ratio (aOR)=4.31 (95% CI: 1.56, 11.86)]. Protective effects of first trimester zidovudine exposure on musculoskeletal defects were detected [aOR=0.24 (95% CI: 0.08, 0.69)], while a higher risk of heart defects was found [aOR=2.04 (95% CI: 1.03, 4.05)].
The prevalence of birth defects was higher in this cohort of HIV-exposed children than in other pediatric cohorts. There was no association with overall ARV exposure, but there were some associations with specific agents including efavirenz. Additional studies are needed to rule out confounding and to evaluate newer ARV agents.
PMCID: PMC2948952  PMID: 20539252
9.  Expression of CYP1A1 and CYP1B1 in human endothelial cells: regulation by fluid shear stress 
Cardiovascular Research  2009;81(4):669-677.
CYP1A1 and CYP1B1, members of the cytochrome P450 protein family, are regulated by fluid shear stress. This study describes the effects of duration, magnitude and pattern of shear stress on CYP1A1 and CYP1B1 expressions in human endothelial cells, towards the goal of understanding the role(s) of these genes in pro-atherogenic or anti-atherogenic endothelial cell functions.
Methods and results
We investigated CYP1A1 and CYP1B1 expressions under different durations, levels, and patterns of shear stress. CYP1A1 and CYP1B1 mRNA, protein, and enzymatic activity were maximally up-regulated at ≥24 h of arterial levels of shear stress (15–25 dynes/cm2). Expression of both genes was significantly attenuated by reversing shear stress when compared with 15 dynes/cm2 steady shear stress. Small interfering RNA knockdown of CYP1A1 resulted in significantly reduced CYP1B1 and thrombospondin-1 expression, genes regulated by the aryl hydrocarbon receptor (AhR). Immunostaining of human coronary arteries showed constitutive CYP1A1 and CYP1B1 protein expressions in endothelial cells. Immunostaining of mouse aorta showed nuclear localization of AhR and increased expression of CYP1A1 in the descending thoracic aorta, whereas reduced nuclear localization of AhR and attenuated CYP1A1 expression were observed in the lesser curvature of the aortic arch.
CYP1A1 and CYP1B1 gene and protein expressions vary with time, magnitude, and pattern of shear stress. Increased CYP1A1 gene expression modulates AhR-regulated genes. Based on our in vitro reversing flow data and in vivo immunostained mouse aorta, we suggest that increased expression of both genes reflects an anti-atherogenic endothelial cell phenotype.
PMCID: PMC2642602  PMID: 19126602
CYP1A1; CYP1B1; Shear stress; Vascular endothelial cell; Aryl hydrocarbon receptor
10.  Behaviour sequelae following acute Kawasaki disease 
BMC Pediatrics  2005;5:14.
Kawasaki disease is a systemic vasculitis and may affect cerebral function acutely. The aim of the present study was to measure a number of behaviour and social parameters within a cohort of Kawasaki disease patients.
Parents of children with past diagnosis of Kawasaki disease were recruited to complete several behaviour screening questionnaires. Sixty five sets of questionnaires relating to the patient cohort received were eligible for inclusion. Two control groups were used, a hospital (HC) control and a sibling control (SC) group.
40% of the Kawasaki disease group showed elevated internalising scores in the clinical or borderline-clinical range. This compared with 18% of hospital controls and 13% of sibling controls. Additionally, the Kawasaki disease (KD) group were shown to be experiencing greater overall total difficulties when compared with the controls (KD 13.7, HC 8.6, SC 8.9). The KD group attained higher behavioural scores within the internalising sub-categories of somatic problems (KD 61, HC 57, SC 54) and withdrawn traits (KD 56, HC 53, SC 51). The KD group were also shown to be suffering more thought problems (KD 57, HC 53, SC 50) compared with the controls. Further difficulties relating to conduct (KD 3.3, HC 1.4) and social interactions (KD 6.7, HC 8.3) are also highlighted for the KD group compared with hospital controls. Positron emission tomograms were performed on nine patients to investigate severe behavioural problems. Three showed minor changes, possibly a resolving cerebral vasculopathy.
Kawasaki disease can be associated with significant behavioural sequelae. This is an important consideration in the long-term follow up and referral to a clinical psychologist may be necessary in selected patients.
PMCID: PMC1156909  PMID: 15916701
Kawasaki Disease; Cerebral Vasculitis; Psychological Difficulties; Long Term Management

Results 1-10 (10)