Thuan Thai

Tumor Necrosis Factor–Related Apoptosis‐Inducing Ligand (TRAIL) Promotes Angiogenesis and Ischemia‐Induced Neovascularization Via NADPH Oxidase 4 (NOX4) and Nitric Oxide–Dependent Mechanisms

Background Tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) has the ability to inhibit angiogenesis by inducing endothelial cell death, as well as being able to promote pro‐angiogenic activity in vitro. These seemingly opposite effects make its role in ischemic disease unclear. Using Trail −/− and wildtype mice, we sought to determine the role of TRAIL in angiogenesis and neovascularization following hindlimb ischemia. Methods and Results Reduced vascularization assessed by real‐time 3‐dimensional Vevo ultrasound imaging and CD31 staining was evident in Trail −/− mice after ischemia, and associated with reduced capillary formation and increased apoptosis. Notably, adenoviral TRAIL administration significantly improved limb perfusion, capillary density, and vascular smooth‐muscle cell content in both Trail −/− and wildtype mice. Fibroblast growth factor‐2, a potent angiogenic factor, increased TRAIL expression in human microvascular endothelial cell‐1, with fibroblast growth factor‐2‐mediated proliferation, migration, and tubule formation inhibited with TRAIL siRNA. Both fibroblast growth factor‐2 and TRAIL significantly increased NADPH oxidase 4 (NOX4) expression. TRAIL‐inducible angiogenic activity in vitro was inhibited with siRNAs targeting NOX4, and consistent with this, NOX4 mRNA was reduced in 3‐day ischemic hindlimbs of Trail −/− mice. Furthermore, TRAIL‐induced proliferation, migration, and tubule formation was blocked by scavenging H2O2, or by inhibiting nitric oxide synthase activity. Importantly, TRAIL‐inducible endothelial nitric oxide synthase phosphorylation at Ser‐1177 and intracellular human microvascular endothelial cell‐1 cell nitric oxide levels were NOX4 dependent. Conclusions This is the first report demonstrating that TRAIL can promote angiogenesis following hindlimb ischemia in vivo. The angiogenic effect of TRAIL on human microvascular endothelial cell‐1 cells is downstream of fibroblast growth factor‐2, involving NOX4 and nitric oxide signaling. These data have significant therapeutic implications, such that TRAIL may improve the angiogenic response to ischemia and increase perfusion recovery in patients with cardiovascular disease and diabetes.

Targeted subendothelial matrix oxidation by myeloperoxidase triggers myosin II- dependent de-adhesion and alters signaling in endothelial cells

During inflammation, myeloperoxidase (MPO) released by circulating leukocytes accumulates within the subendothelial matrix by binding to and transcytosing the vascular endothelium. Oxidative reactions catalyzed by subendothelial-localized MPO are implicated as a cause of endothelial dysfunction in vascular disease. While the subendothelial matrix is a key target for MPO-derived oxidants during disease, the implications of this damage for endothelial morphology and signaling are largely unknown. We found that endothelial-transcytosed MPO produced hypochlorous acid (HOCl) that reacted locally with the subendothelial matrix and induced covalent cross-linking of the adhesive matrix protein fibronectin. Real-time biosensor and live cell imaging studies revealed that HOCl-mediated matrix oxidation triggered rapid membrane retraction from the substratum and adjacent cells (de-adhesion). De-adhesion was linked with the alteration of Tyr-118 phosphorylation of paxillin, a key adhesion-dependent signaling process, as well as Rho kinase-dependent myosin light chain-2 phosphorylation. De-adhesion dynamics were dependent on the contractile state of cells, with myosin II inhibition with blebbistatin attenuating the rate of membrane retraction. Rho kinase inhibition with Y-27632 also conferred protection, but not during the initial phase of membrane retraction, which was driven by pre-existing actomyosin tensile stress. Notably, diversion of MPO from HOCl production by thiocyanate or nitrite attenuated de-adhesion and associated signaling responses, despite the latter substrate supporting MPO-catalyzed fibronectin nitration. These data show that subendothelial-localized MPO employs a novel “outside-in” mode of redox signaling, involving HOCl-mediated matrix oxidation. These MPO-catalyzed oxidative events are likely to play a previously unrecognized role in altering endothelial integrity and signaling during inflammatory vascular disorders.

Indoleamine-2,3-dioxygenase elevated in tumor-initiating cells is suppressed by mitocans.

Tumor-initiating cells (TICs) often survive therapy and give rise to second-line tumors. We tested the plausibility of sphere cultures as models of TICs. Microarray data and microRNA data analysis confirmed the validity of spheres as models of TICs for breast and prostate cancer as well as mesothelioma cell lines. Microarray data analysis revealed the Trp pathway as the only pathway upregulated significantly in all types of studied TICs, with increased levels of indoleamine-2,3-dioxygenase-1 (IDO1), the rate-limiting enzyme of Trp metabolism along the kynurenine pathway. All types of TICs also expressed higher levels of the Trp uptake system consisting of CD98 and LAT1 with functional consequences. IDO1 expression was regulated via both transcriptional and posttranscriptional mechanisms, depending on the cancer type. Serial transplantation of TICs in mice resulted in gradually increased IDO1. Mitocans, represented by α-tocopheryl succinate and mitochondrially targeted vitamin E succinate (MitoVES), suppressed IDO1 in TICs. MitoVES suppressed IDO1 in TICs with functional mitochondrial complex II, involving transcriptional and posttranscriptional mechanisms. IDO1 increase and its suppression by VE analogues were replicated in TICs from primary human glioblastomas. Our work indicates that IDO1 is increased in TICs and that mitocans suppress the protein.

Arrested Hematopoiesis and Vascular Relaxation Defects in Mice with a Mutation in Dhfr

ABSTRACT Dihydrofolate reductase (DHFR) is a critical enzyme in the folate metabolism pathway and also plays a role in regulating nitric oxide (NO) signaling in endothelial cells. Although both coding and noncoding mutations with phenotypic effects have been identified in the human DHFR gene, no mouse model is currently available to study the consequences of perturbing DHFR in vivo. In order to identify genes involved in definitive hematopoiesis, we performed a forward genetic screen and produced a mouse line, here referred to as Orana, with a point mutation in the Dhfr locus leading to a Thr136Ala substitution in the DHFR protein. Homozygote Orana mice initiate definitive hematopoiesis, but expansion of progenitors in the fetal liver is compromised, and the animals die between embryonic day 13.5 (E13.5) and E14.5. Heterozygote Orana mice survive to adulthood but have tissue-specific alterations in folate abundance and distribution, perturbed stress erythropoiesis, and impaired endothelium-dependent relaxation of the aorta consistent with the role of DHFR in regulating NO signaling. Orana mice provide insight into the dual roles of DHFR and are a useful model for investigating the role of environmental and dietary factors in the context of vascular defects caused by altered NO signaling.

Endoglin potentiates nitric oxide synthesis to enhance definitive hematopoiesis

ABSTRACT During embryonic development, hematopoietic cells develop by a process of endothelial-to hematopoietic transition of a specialized population of endothelial cells. These hemogenic endothelium (HE) cells in turn develop from a primitive population of FLK1+ mesodermal cells. Endoglin (ENG) is an accessory TGF-β receptor that is enriched on the surface of endothelial and hematopoietic stem cells and is also required for the normal development of hemogenic precursors. However, the functional role of ENG during the transition of FLK1+ mesoderm to hematopoietic cells is ill defined. To address this we used a murine embryonic stem cell model that has been shown to mirror the temporal emergence of these cells in the embryo. We noted that FLK1+ mesodermal cells expressing ENG generated fewer blast colony-forming cells but had increased hemogenic potential when compared with ENG non-expressing cells. TIE2+/CD117+ HE cells expressing ENG also showed increased hemogenic potential compared with non-expressing cells. To evaluate whether high ENG expression accelerates hematopoiesis, we generated an inducible ENG expressing ES cell line and forced expression in FLK1+ mesodermal or TIE2+/CD117+ HE cells. High ENG expression at both stages accelerated the emergence of CD45+ definitive hematopoietic cells. High ENG expression was associated with increased pSMAD2/eNOS expression and NO synthesis in hemogenic precursors. Inhibition of eNOS blunted the ENG induced increase in definitive hematopoiesis. Taken together, these data show that ENG potentiates the emergence of definitive hematopoietic cells by modulating TGF-β/pSMAD2 signalling and increasing eNOS/NO synthesis.

ePortfolios, Assessment and Professional Skills in the Medical Sciences

Medical Science students are generally unaware of their developing professional skills related to graduate capabilities during their initial training and at a program level it is a challenge for administrators to evidence development of such capabilities. In the Bachelor of Medical Science program at the University of New South Wales, staff have instigated alignment of assessment with graduate capabilities, combined with program-wide tracking of student achievement in teamwork tasks. Teamwork was chosen as a focus as anecdotal evidence suggested that this graduate capability is hard to master. Tracking was achieved by mapping and aligning assessment tasks that built authentic teamwork skills, and by implementing standards-based criteria addressing development of teamwork skills. This curriculum strategy is program-wide and cross-disciplinary, integrating content knowledge and technical skills that articulate with professional skills across all medical sciences. Implementation was via Workshop UNSW (a guided learning space) in Moodle with Wordpress used as a digital site to create ePortfolios that facilitated and captured student reflective practice related to developing deeper understanding of key teamwork elements such as contribution, collaborative behaviour and role play. ePortfolios allow students to curate evidence that facilitates recognition of teamwork skills and use of Workshop UNSW allowed monitoring of student narratives in skills development, and enabled student self and peer evaluation. Student peer evaluation is an important aspect of the intended process for learners with complimentary implementation of ePortfolio pedagogy to engage students in professional skills development in teamwork. This is a first stage approach to building professional skills for Medical Science students that will have life-long learning effects and can be easily adapted to other skills and other programs.

TEAMWORK SKILLS BUILDING AND REFLECTIVE PRACTICE IN THE MEDICAL SCIENCES USING EPORTFOLIO LEARNING AND AUTHENTIC ASSESSMENT

Development of co-curricular professional skills and capabilities by undergraduates that are linked to formal academic learning is difficult to capture at the program level within higher education institutions. Feedback from employers, educators and students suggests that the teamwork graduate capability is highly sought after, but it is hard for students to prove mastery. In the Bachelor of Medical Science (BMedSci) program at UNSW Australia, we initiated a program-wide Comprehensive Teamwork Learning and Assessment (CTLA) model by aligning assessment tasks focussed on building teamwork capabilities combined with self and peer evaluation of teamwork skills awareness and development, ePortfolio implementation and a teamwork satisfaction survey. These CTLA model elements support student reflective practice and awareness of teamwork skills attainment. Our CTLA model involved tracking, mapping and aligning assessment tasks that built relevant, authentic skills for teamwork and incorporated standards-based criteria that directly addressed teamwork skills development using an adapted Association of American Colleges & Universities (AAC&U) VALUE rubric for teamwork; the UNSW Teamwork Skills Development Framework. This process integrated content knowledge and technical skills that articulated with professional skills development across all the medical sciences disciplines. We implemented this system using Workshop UNSW in Moodle for teachers to monitor and students to evaluate teamwork skills progression for themselves and their peers. ePortfolio/reflective blogging using WordPress to document and reflect on their personal-professional development of teamwork skills was implemented as a key part of this approach. Student satisfaction was surveyed at the completion of the teamwork assessment tasks across the program. Study outcomes demonstrated the innovative CTLA model and assessment approaches improved teamwork skills awareness and attainment in the medical science program at UNSW. A cross-disciplinary skills awareness and development capture strategy developed as part of a UNSW Innovation Research Project will be presented. ePortfolio/reflective blogging coupled to the Teamwork Skills Development Framework, an adapted version of the AAC&U teamwork value rubric for use in the medical science program as well as application of the Reflective Rubric UNSW. There scope to implement this mechanism for extracting data across any ePortfolio platform for application as metadata that sits behind badges of skills recognition. This mechanism is useful for capturing, quantifying and recognising skills development when coupled to authentic assessment tasks in the sciences and beyond.