Alpha Omega Alpha Honor Medical Society

Using RNA Interference to Prevent Cerebral Vasospasm after Subarachnoid Hemorrhage: Improving Vessel Relaxation Through Heat Shock Protein 20 Upregulation

Investigator: Travis Ladner

Mentor: Colleen Brophy, MD

Introduction:
Cerebral vasospasm is the leading contributor to the morbidity and mortality associated with subarachnoid hemorrhage (SAH). Despite this significant public health challenge, SAH-associated cerebral vasospasm still lacks a definitive explanatory model. We hypothesized that SAH-induced vasospasm is due to impaired relaxation in addition to pathologic vasoconstriction. The purpose of this investigation was to develop a therapeutic that will circumvent impaired vasodilatory pathways (e.g. cGMP/PKG) by restoring the activity of the downstream phosphorylated HSP20.

Methods:
Rat aorta were harvested, sectioned, and incubated for 24 hours in HEPES buffered DMEM in 1 of 3 treatment groups: 1) self-assembling micelle containing antagomir 320 2) micelle with miR 320 and 3) untreated (control). Western blot was used to quantify heat shock protein 20 (HSP20) levels, normalized to tissue GAPDH levels, across the three groups. Force measurements were obtained and recorded: Rings were progressively stretched to optimal resting tension (approximately 1 g), then maintained at the resting tension and equilibrated for another hour. Rings were contracted multiple times with high extracellular potassium; force generated was measured. Rings were equilibrated for an additional 30 min and dose response curves for 1) phenylephrine-induced contraction and 2) sodium nitroprusside (SNP)-induced vasorelaxation after phenylephrine-induced contraction.

Results:
Antagomir 320 resulted in increased expression of HSP20 in rat aortic tissue whereas miR 320 decreased HSP20 expression. Antagomir 320 also decreased phenylephrine- induced contraction of rat aorta compared to control and miR 320 groups. Antagomir 320 also led to increased sodium nitroprusside-induced relaxation of rat aortic tissue following phenylephrine-induced contraction.

Discussion:
We found that administration of antagomir 320, a chemically engineered oligonucleotide that silences microRNA 320 (miR 320), reduced vasoconstriction and enhanced vasorelaxation in ex vivo rat aorta, likely due to upregulation of heat shock protein 20 (HSP20).

Last modified: 2/01/2017

Updated on February 3, 2017.


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