Date of Award

Spring 2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Lloyd Wolfinbarger

Committee Member

Christopher Osgood

Committee Member

Emilia Oleszak

Committee Member

Ralph Powers

Abstract

Cryopreserved allograft heart valves represent the best solution for a patient with a failing heart valve. However, the constituent cells become apoptotic and within months of transplant the heart valve becomes acellular and the recipient's cells do not repopulate the allograft (3, 51). A strategy to prevent this situation would be to minimize or prevent apoptosis from occurring by strategically altering steps during heart valve processing. Recently it has been demonstrated that: 1) Heat shock protein 70 is a negative modulator of the apoptotic cascade; 2) Cells in culture exposed to hypothermic conditions produce heat shock protein 70 upon rewarming; and 3) Glutamine can induce heat shock protein 70 production. The purpose of the current research is to apply these concepts to allograft heart valve tissues and begin to understand the mode(s) of action by investigating the resultant intracellular heat shock protein 70 concentration, presence of caspase 3, presence of cytochrome c outside of the mitochondria and compromised DNA.

Allograft heart valve conduit tissue was processed traditionally at 4°C and 37°C with and without glutamine and assessed prior to and after cyropreservation. Intracellular heat shock protein 70 and cytochrome c concentration inside and outside of the mitochondria was assessed utilizing enzyme-linked immunoassay; the presence of caspase 3 was assessed utilizing immunohistochemistry and histomorphometry; and the presence of compromised DNA was assessed utilizing the TUNEL assay and histomorphometry. It was found that treatment of allograft heart valve conduit tissue at 37°C plus glutamine lead to a statistically significant increase in heat shock protein 70 concentration, maintenance of the mitochondrial cytochrome c concentration, less caspase 3 positive cells and less TUNEL positive cells relative to the other three treatment groups immediately prior to cyropreservation and after thawing and subsequent incubation at 37°C for seventy-two hours.

Therefore it is concluded that allograft heart valves can be processed at, 37°C with glutamine, which will facilitate increased amounts of heat shock protein 70 thereby reducing the amount of apoptotic cells. The presence of heat shock protein 70 appears to modulate apoptosis at or upstream of cytochrome c release from the mitochondria.

DOI

10.25777/qb5y-p126

ISBN

9781267425492

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