Casey Jane Ankeny

Identification of side- and shear-dependent microRNAs regulating porcine aortic valve pathogenesis

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.

“Unmuddying” course content using muddiest point reflections

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.

Mechanotransduction in small intestinal submucosa scaffolds: fabrication parameters potentially modulate the shear-induced expression of PECAM-1 and eNOS.

In small intestinal submucosa (SIS) scaffolds for functional tissue engineering, the impact of scaffold fabrication parameters on cellular response and tissue regeneration may relate to the mechanotransductory properties of the final arrangement of collagen fibres. We previously proved that two fabrication parameters, (a) preservation (P) or removal (R) of a dense collagen layer present in SIS, and (b) SIS in a final dehydrated (D) or hydrated (H) state, have an effect on the micromechanical environment of SIS. In a continuation of our studies, we herein hypothesized that these fabrication parameters also modulate early mechanotransduction in cells populating the scaffold. Mechanotransduction was investigated by seeding human umbilical vein endothelial cells (HUVECs) on scaffolds, exposing them to pulsatile shear stress (12 ± 4 dyne/cm2) for 1 h (n = 5) in a cone‐and‐plate shear system, and evaluating the expression of the mechanosensitive genes Pecam1 and Enos by immunofluorescence and qPCR. Expression of mechanosensitive genes was highest in PD grafts, followed by PH and RH grafts. The RD group had similar expression to that of unsheared control cells, suggesting that the RD combination potentially reduced mechanotransduction of shear to cells. We concluded that the two fabrication parameters studied, which modify SIS micromechanics, also potentially modulated the early shear‐induced expression of mechanosensitive genes in seeded HUVECs. Our findings suggest that fabrication parameters influence the outcome of SIS as a therapeutic scaffold. Copyright