Defense Date

3-14-2024

Graduation Date

Summer 8-10-2024

Availability

One-year Embargo

Submission Type

dissertation

Degree Name

PhD

Department

Pharmaceutics

School

School of Pharmacy

Committee Chair

Devika S Manickam

Committee Member

Rehana K. Leak

Committee Member

Wilson Meng

Committee Member

R. Anne Stetler

Committee Member

Peter L. D. Wildfong

Keywords

Extracellular vesicles, exosomes, microvesicles, mitochondria, ischemic stroke, engineered extracellular vesicles, heat shock proteins, brain endothelial cells

Abstract

Ischemic stroke is the second leading cause of death worldwide, and the lack of effective treatment options is an unmet clinical need for stroke survivors. Ischemic stroke-induced oxygen-glucose deprivation (OGD) in the affected brain tissue leads to mitochondrial dysfunction and adenosine triphosphate (ATP) depletion in the brain endothelial cells (BECs) lining the blood-brain barrier (BBB). ATP deprivation in the BECs dysregulates actin cytoskeleton dynamics, resulting in BBB breakdown. Restoration of blood flow (i.e., reperfusion) upon the first-line stroke treatment further leads to BBB breakdown and widens the paracellular spaces between BECs. Subsequent reperfusion infiltrates harmful blood toxins and immune cells into the brain, resulting in severe neurovascular injuries and cognitive impairments. Therefore, enhancing BEC mitochondrial functions and protecting its structural integrity is a promising approach to alleviating ischemia/reperfusion-mediated neurovascular injury.

Mitochondrial component-containing extracellular vesicles (EVs), including exosomes (EXOs) and microvesicles (MVs), are intriguing and promising strategies for delivering mitochondria to the target site. EV-mediated transfer of mitochondrial components to the injured cells has shown increased recipient cell ATP levels, oxidative phosphorylation, mitochondrial biogenesis, and cell survival in various cell culture studies and preclinical disease models. In addition to the innate mitochondrial cargo, EVs can be engineered to enrich innate mitochondrial components as well as to load exogenous therapeutic nucleic acids or proteins that can protect BBB integrity post-ischemic stroke. Engineered EVs containing an enriched mitochondrial load may be a more potent treatment strategy to increase BEC metabolic functions compared to naïve EVs. Published studies have shown that brain-derived neurotrophic factor (BDNF) released from cerebral endothelial cells enhance post-stroke neuronal cell viability due to its neuroprotective effects. Preclinical studies have demonstrated that overexpression of 27 kDa heat shock protein (HSP27) in brain BEC inhibits actin polymerization and restores BBB tight junctions, eliciting long-lasting protection against stroke-induced BBB disruption. Hence, engineering mitochondria-containing EVs with exogenous plasmid BDNF or HSP27 could enhance EV-mediated BBB and neurovascular protection post-ischemic stroke.

The central hypothesis of this work is that delivery of mitochondria-containing naïve EVs or BDNF/HSP27-loaded engineered EVs to the injured BECs could increase cellular ATP levels, mitochondrial respiration, and blood-brain barrier integrity, which will ultimately decrease mouse brain infarct volume post-ischemic stroke. This dissertation tested this hypothesis via three primary aims: (1) investigating mitochondria-contacting naïve EV-mediated decrease in mouse brain infarct volume post-stroke, (2) evaluating whether enriched mitochondrial load (mito-EVs) or plasmid BDNF-loaded EVs (pBDNE-EVs) could outperform naïve EV’s mitochondrial functionality, and (3) investigating if HSP27-loaded EVs (HSP27-EVs) could protect BBB integrity during ischemia/reperfusion injury. The results showed that intravenously injected BEC-derived MVs showed a significant reduction in mouse brain infarct volume and significantly improved neurological functions in a mouse model of ischemic stroke. Mito-MVs significantly increased recipient ischemic BEC ATP levels compared to naïve MVs. The data showed the capability of EVs to load an exogenous plasmid DNA and pBDNF-EV-mediated increases in intracellular ATP levels in recipient BECs. Lastly, HSP27-EVs significantly reduced ischemia-induced paracellular permeability of small and large tracer molecules across primary BEC monolayers. In conclusion, mitochondria-containing naïve and engineered MVs demonstrate potential to restore ischemic BBB metabolic functions and structural integrity to improve post-stroke outcomes.

Language

English

Additional Citations

  1. Dave KM, Stolz DB, Manickam DS. Delivery of mitochondria-containing extracellular vesicles to the BBB for ischemic stroke therapy. Expert Opinion on Drug Delivery.1-20.
  2. Dave KM, Stolz DB, Venna VR, Quaicoe VA, Maniskas ME, Reynolds MJ, Babidhan R, Dobbins DX, Farinelli MN, Sullivan A, Bhatia TN, Yankello H, Reddy R, Bae Y, Leak RK, Shiva SS, McCullough LD, Manickam DS. Mitochondria-containing extracellular vesicles (EV) reduce mouse brain infarct sizes and EV/HSP27 protect ischemic brain endothelial cultures. J Control Release. 2023;354:368-93.
  3. Dave KM, Dobbins DX, Farinelli MN, Sullivan A, Milosevic J, Stolz DB, Kim J, Zheng S, Manickam DS. Engineering Extracellular Vesicles to Modulate Their Innate Mitochondrial Load. Cell Mol Bioeng. 2022;15(5):367-89.
  4. Dave KM, Zhao W, Hoover C, D’Souza A, S Manickam D. Extracellular Vesicles Derived from a Human Brain Endothelial Cell Line Increase Cellular ATP Levels. AAPS PharmSciTech. 2021;22(1):18.
  5. D'Souza A, Dave KM, Stetler RA, S. Manickam D. Targeting the blood-brain barrier for the delivery of stroke therapies. Advanced Drug Delivery Reviews. 2021;171:332-51.
  6. D'Souza A, Burch A, Dave KM, Sreeram A, Reynolds MJ, Dobbins DX, Kamte YS, Zhao W, Sabatelle C, Joy GM, Soman V, Chandran UR, Shiva SS, Quillinan N, Herson PS, Manickam DS. Microvesicles transfer mitochondria and increase mitochondrial function in brain endothelial cells. Journal of Controlled Release. 2021.

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