Aortic valve (AV) calcification is an inflammation driven process that occurs preferentially in the fibrosa. To explore the underlying mechanisms, we investigated if key microRNAs (miRNA) in the AV are differentially expressed due to disturbed blood flow (oscillatory shear (OS)) experienced by the fibrosa compared to the ventricularis. To identify the miRNAs involved, endothelial-enriched RNA was isolated from either side of healthy porcine AVs for microarray analysis. Validation using qPCR confirmed significantly higher expression of 7 miRNAs (miR-100, -130a, -181a/b, -199a-3p, -199a-5p, and -214) in the fibrosa versus the ventricularis. Upon bioinformatics analysis, miR-214 was selected for further investigation using porcine AV leaflets in an ex vivo shear system. Fibrosa and ventricularis sides were exposed to either oscillatory or unidirectional pulsatile shear for 2 days and 3 & 7 days in regular and osteogenic media, respectively. Higher expression of miR-214, increased thickness of the fibrosa, and calcification was observed when the fibrosa was exposed to OS compared to the ventricularis. Silencing of miR-214 by anti-miR-214 in whole AV leaflets with the fibrosa exposed to OS significantly increased the protein expression of TGFβ1 and moderately increased collagen content but did not affect AV calcification. Thus, miR-214 is identified as a side- and shear-dependent miRNA that regulates key mechanosensitive gene in AV such as TGFβ1.

BackgroundThe understanding of endothelial cell biology has been facilitated by the availability of primary endothelial cell cultures from a variety of sites and species; however, the isolation and maintenance of primary mouse aortic endothelial cells (MAECs) remain a formidable challenge. Culturing MAECs is difficult as they are prone to phenotypic drift during culture. Therefore, there is a need to have a dependable in vitro culture system, wherein the primary endothelial cells retain their properties and phenotypes.MethodsHere, we developed an effective method to prepare immortalized MAEC (iMAEC) lines. Primary MAECs, initially isolated from aortic explants, were immortalized using a retrovirus expressing polyoma middle T-antigen. Immortalized cells were then incubated with DiI-acetylated-low density lipoprotein and sorted via flow cytometry to isolate iMAECs.ResultsiMAECs expressed common markers of endothelial cells, including PECAM1, eNOS, VE-cadherin, and von Willebrand Factor. iMAECs aligned in the direction of imposed laminar shear and retained the ability to form tubes. Using this method, we have generated iMAEC lines from wild-type and various genetically modified mice such as p47phox-/-, eNOS-/-, and caveolin-1-/-.ConclusionIn summary, generation of iMAEC lines from various genetically modified mouse lines provides an invaluable tool to study vascular biology and pathophysiology.

Technology (CRESMET), and an evaluator for several NSF projects. His first research strand concentrates on the relationship between educational policy and STEM education. His second research strand focuses on studying STEM classroom interactions and subsequent effects on student understanding. He is a codeveloper of the Reformed Teaching Observation Protocol (RTOP) and his work has been cited more than 1800 times and his publications have been published in multiple peer-reviewed journals such as Science is professor in the Materials Science Program in the Fulton School of Engineering at Arizona State University. He teaches in the areas of introductory materials engineering, polymers and composites, and capstone design. His research interests include evaluating conceptual knowledge, misconceptions and technologies to promote conceptual change. He has co-developed a Materials Concept Inventory and a Chemistry Concept Inventory for assessing conceptual knowledge and change for introductory materials science and chemistry classes. He is currently conducting research on an NSF faculty development program based on evidence-based teaching practices. The overall goal is to develop disciplinary communities of practice across the college of engineering. The approach is being promoted through semester-long faculty workshops and then through a semester of supported implementation of faculty classroom innovations. Changes in faculty beliefs and classroom practice should positively impact student performance and retention.

Class instruction is a living and ever evolving process aimed at providing students with a quality education. Instructors are responsible for analyzing their courses to ensure that delivery of information is effective. Changes made are usually based on student assessments; however, our reactions to assessments are flawed without student insight. One method to obtain student feedback is through muddiest point reflections. This activity asks students to reflect on what was just taught allowing students the opportunity to share what was "muddy". This mixed-methods study provides vignettes from faculty members on their use of muddiest point reflections and an assessment of what value students associate with such an intervention. Faculty members who have used this approach say it drives change within their classes. The analysis of student value beliefs revealed muddiest point reflections as an intervention that positively impacts interest, attainment, and utility value without negative cost. The appeal of muddiest points was also evident with 77% of students hoping to see muddiest point reflections in another class and 93% agreeing to recommend their course experience to a friend. These findings suggest that students agree more than disagree that muddiest point reflections are a valuable addition to their educational experience.