BME Seminar: Scott Younger
Monday, February 27th, 2023 - 12:00 p.m.
Scott Younger
Ph.D. Candidate of Biomedical Engineering
"Biophysical Mechanisms of Vascular and Membrane Permeability Studied by Microscopy and Electrophysiology Techniques"
Keating 103
Live Zoom link | Passcode: BearDown
Hosts: Dr. Beth Hutchinson and Dr. Shang Song
(Instructor permission required for enrolled students to attend via Zoom)
Persons with a disability may request a reasonable accommodation by contacting the Disability Resource Center at 621-3268 (V/TTY).
Abstract: Increased permeability of vascular tissues and vascular cell plasma membranes to circulating solutes is involved in numerous health disorders. Loss of normally impermeable membrane barriers causes ionic homeostasis and cell death in individual cells, as well as edema and other potentially fatal pathophysiologies in tissues, such as acute respiratory distress syndrome. This work investigated vascular tissue permeability at two levels: artificial membrane ionic permeability of individual lipid bilayers, and paracellular gap formation in vascular endothelial cell monolayers (ECs). These were tested mostly with electrophysiology, optical microscopy, and atomic force microscopy (AFM) to understand what allowed solutes through both forms of barriers and image how this occurred.
Increased oligomeric levels of the poorly understood peptide, medin aortic amyloid, are found in patients with altered aortic wall integrity, and medin has been implicated in both aortic aneurysms and vascular dementia. Amyloidogenic proteins can form large fibrillar structures, but previous work suggested smaller oligomeric forms are key to several diseases. We found medin oligomers to be capable of forming membrane-spanning ionconductive pores via lipid bilayer electrophysiology, suggesting an oligomer-mediated toxicity mechanism could cause these pathologies. AFM imaging and transmission electron microscopy showed compact oligomers and non-fibrillar medin aggregates, along with a low thioflavin T fluorescence emitted by aggregates compared to other amyloids, suggested that medin aggregation into pores follows a nonamyloidogenic pathway. In silico modeling by molecular dynamics simulations provided atomic-level structural detail of medin pores with a CNpNC barrel topology and diameters like the values estimated by experimental pore conductances. The loss of ionic homeostasis from this pore activity could thus lead to vascular dysfunction and barrier breakdown, so reducing this pore behavior is a possible drug target.
The general appearance of EC barrier recovery was previously studied, which this work furthered by analyzing the biophysical properties of lamellipodia in modulating EC spatial-specific contractile properties and gap closure. EC lamellipodia are thin, sheet-like membrane projections that play a major role in the initial phases of gap closure and barrier restoration. By fabricating micropillars with soft lithography, we formed reproducible paracellular gaps in human lung EC monolayers to observe gap closure behavior. Comparison of unaltered control gap closure and gaps treated with the barrier-enhancing sphingolipid, sphingosine-1-phosphate (S1P), allowed us to track EC gap closure and motility rates in differing conditions via time lapse optical microscopy. From there, AFM fast force mapping let us breakdown the viscoelastic behavior of lamellipodia and the perpendicular-facing ruffle structures they sometimes create. These measurements showed lower solid-like stiffness and increased liquid-like viscous behavior in the peripheral structures pushing gaps closed, compared to internal cellular regions. This separation of elasticity and viscosity indicates a radically different composure of structural protein and fluid components in cellular regions. We hypothesize this dissipative EC behavior implies decreased actomyosin cross-linking and rapid rearrangement. These insights could be used in drug design or modeling barrier breakdown and restoration, and thus restore vascular barrier integrity in patients.