Graduation Year

2018

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Jerome Breslin, Ph.D.

Co-Major Professor

Sarah Y. Yuan, M.D., Ph.D.

Committee Member

Thomas Taylor-Clark, Ph.D.

Committee Member

Srinivas Tipparaju, Ph.D.

Committee Member

Hana Totary-Jain, Ph.D.

Keywords

glycocalyx, hemorrhagic shock, mitochondria, sphingosine-1-phosphate

Abstract

Excessive microvascular permeability is a serious complication involved in traumatic injury and inflammatory diseases. Alcohol intoxication can exacerbate the physiological derangements produced by microvascular endothelial barrier dysfunction in such disease conditions. Sphingosine-1-phosphate (S1P) has known endothelial barrier-protective properties, and has been shown to ameliorate microvascular leakage in a model of combined alcohol intoxication and hemorrhagic shock and resuscitation (HSR). However, whether the barrier-protective properties of S1P extend to endothelial cells of the blood-brain barrier (BBB) is unclear. The mechanisms of S1P-induced barrier protection during alcohol intoxication or HSR are also unknown. In the current study, we tested the hypothesis that S1P could enhance endothelial barrier during alcohol intoxication or hemorrhagic shock by preserving the integrity of junction proteins and the endothelial glycocalyx, and protecting mitochondrial function. Cultured primary human brain microvascular endothelial cell (HBMEC) monolayers were used to characterize endothelial-specific mechanisms of S1P protection of the BBB during alcohol treatment.

Transendothelial electrical resistance (TER) and apparent permeability coefficients for albumin, dextran-4 kDa, and sodium fluorescein were used as indices of barrier function. Junctional localization was determined by immunofluorescence confocal microscopy. We also used an established in vivo rat model of conscious HSR and assessed microvascular leakage, endothelial glycocalyx integrity, and mitochondrial function by intravital microscopy. Cultured rat intestinal microvascular endothelial cell (RIMEC) monolayers were used to test the ability of S1P to protect against glycocalyx shedding and endothelial barrier dysfunction caused by direct disruption of mitochondrial integrity due to inhibition of mitochondrial complex III. The results show that alcohol significantly impaired HBMEC TER and increased solute permeability, which was reversed with application of S1P after alcohol treatment. Alcohol caused the formation of gaps between cells. Treatment with S1P (after alcohol) increased junctional localization. Our in vivo results show that S1P protects against HSR-induced hyperpermeability, preserves the expression of adherens junctional proteins, and protects against glycocalyx degradation. S1P treatment during HSR also protects against mitochondrial membrane depolarization. Besides that, S1P protects RIMECs against mitochondrial dysfunction-induced endothelial barrier dysfunction and glycocalyx degradation by acting through mitochondrial complex III.

Our results indicate that S1P may be useful for restoring BBB function during alcohol intoxication. Moreover, S1P protects against HSR-induced mitochondrial dysfunction in endothelial cells, which in turn improves the structure of the endothelial glycocalyx after HSR and allows for better junctional integrity to prevention of excess microvascular permeability.

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