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Ischaemic retinal vein occlusion (RVO) is a potentially blinding condition that is not fully understood, and entails a higher risk of developing complications and poorer prognosis than non-ischaemic RVO. This PhD project was designed to improve the level of understanding on ischaemic RVO as well as to further the knowledge on this disease. For this purpose, this PhD thesis comprises of three main components: systematic reviews (chapters II and III), a retrospective observational clinical study (chapter IV), and a cross-sectional/ prospective pilot observational clinical study (chapter V). The systematic reviews aim at compiling current knowledge on ischaemic RVO and identifying gaps on the understanding of this retinal disease based on published literature (Chapter II) and on experimental animal models of RVO (Chapter III). The retrospective observational clinical study compares baseline characteristics as well as outcomes following treatment between patients with ischaemic and those with nonischaemic RVO. Finally, the pilot cross-sectional observational clinical study evaluates anatomical and functional correlates, point to point, at areas of retinal ischaemia along with other structural lesions in patients with newly diagnosed RVO. Ischaemia secondary to RVO is strongly associated with development of neovascular complications and poor visual prognosis. Prognostic parameters to differentiate patients who would develop extensive areas of ischaemia from those who would not is still to be explored. Available current treatments target the complications of RVO and are being used clinically for both ischaemic and nonischaemic RVO, although data from randomised clinical trials (RCTs) refers only to the latter group of patients. In fact, intravitreal anti-VEGF, in particular, proved to be as beneficial in ischaemic forms of RVO as in non-ischaemic forms. However, due to the low baseline vision classically found in the ischaemic form, final vision remains lower than in non-iRVO despite significant improvement of vision achieved following treatment in most patients, suggesting damage of the retina that cannot be restored with current treatment. This information is useful for the counselling of patients with ischaemic RVO prior to initiating anti-VEGF therapy. Reperfusion of ischaemic retina was observed in very few cases following anti-VEGF therapy. Factors determining reperfusion are still unknown and need to be investigated. Restoration of function, however, could not be seen in reperfused retina within six months of followup. A new treatment to reperfuse ischaemic retina before loss of retinal function occur is urgently needed as damage of retinal cells as a result of ischaemia in RVO may be irreversible, and this needs to be further investigated and better understood. Experimental animal models, although imperfect, are available and can be utilised to facilitate the understanding of this potentially blinding condition.

Cardiac vulnerability to injury following ischemia/reperfusion (I/R) during postnatal development displays a biphasic pattern in rats, with 14-day olds being least vulnerable. The underlying cause for this change is not currently known. We hypothesise that developmental differences in survival signalling pathways (e.g. Reperfusion Injury Salvage Kinase (RISK) & Survivor Activating Factor Enhancement (SAFE)) contribute to these biphasic changes. The work presented therefore looked to identify any changes in the expression of relevant proteins over the course of postnatal development, as well as to examine the effects of targeted inhibition of these proteins on cardiac injury, and to assess the morphology and abundance of cardiac ultrastructures related to survival signalling and the response to I/R. Hearts from 7-day old, 14-day old, 28-day old and adult male Wistar rats were extracted and processed for measurements of protein expression, cardiomyocyte viability, cardiac injury, and cardiac structural/ultrastructural differences. This was done using a range of techniques, including proteomics, Langendorff perfusion, cardiomyocyte isolation, western blotting, histology and electron microscopy. Several key survival signalling proteins, including AKT, PKCε, and AMPK, showed a biphasic profile of expression that parallels the biphasic profile of cardiac vulnerability to I/R. Inhibition of these proteins reduced cardiomyocyte viability and increased cardiac infarct size in 14-day olds, but not in adults. Mitochondria, which are important endpoints for survival signalling, displayed changes in morphology indicative of less injury and disruption in 14-day old than adult hearts following I/R. Ultrastructures associated with cardioprotection (e.g. caveolae and exosome containing MVBs) were also more abundant in 14-day old hearts in comparison with adults. In conclusion, these data show that the least vulnerable age group (14-day olds) has an abundance of pro-survival proteins and that their inhibition rendered them more vulnerable to I/R, which was supported by ultrastructural changes.

Lung transplantation remains limited by a lack of donor organs available for transplant and a reduced median survival secondary to allograft rejection and graft dysfunction. Upon donor death, a catecholamine and cytokine storm ensues, resulting in inflammatory cell infiltration within the lung. Reperfusion of such immunologically primed donor lungs can then drive a heightened recipient immune response. Despite this, the donor immune compartment is often ignored. The aim of this PhD was to characterise the effects of reperfusion upon the donor immune compartment and the recipient response, to identify potential biomarkers and avenues for manipulation. A porcine model was used to determine whether EVLP could alter the inflammatory signalling profile of the donor lung upon transplantation. Indeed, a global up-regulation of cell-survival proteins was observed within EVLP transplanted lungs, yet not in lungs that underwent standard transplant. Increased levels of cell-free mtDNA were also detected in the recipient circulation following standard transplant. The capability of EVLP to facilitate the mobilisation of inflammatory mediators from the lung prior to transplantation was explored. A secondary preservation flush was performed to characterise the immune content of the donor lung following cold ischaemic storage. The migratory pattern of donor leukocytes during EVLP was also elucidated, to determine whether a continual non-recirculating perfusion flush could reduce the donor leukocyte burden prior to transplantation. In addition, cell-free levels of mtDNA were also quantified during human and porcine EVLP, and in the recipient circulation post human lung transplantation. These levels were then correlated with functional and clinical outcomes to determine whether mtDNA is a biomarker of lung function. The study demonstrated that the donor immune compartment is comprised of a large repertoire of donor leukocytes that readily migrate from the lung upon immediate revascularisation. A continual flush with non-recirculating perfusate facilitated the removal of 9 billion donor leukocytes, yet upon perfusate recirculation, donor leukocyte migration continued. The overwhelming propensity of the donor leukocyte repertoire to mobilise upon revascularisation demonstrates the need to consider donor leukocyte depletion prior to transplantation. Furthermore, cell-free mtDNA was observed to correlate with injury on the EVLP circuit, and the incidence of chronic graft dysfunction post-transplantation, highlighting its potential to serve as a biomarker in lung transplantation.

Obesity has reached worldwide epidemic proportions and with this increased incidence of obesity, comes an increase in incidence of the comorbidities associated with obesity such as diabetes and cardiovascular disease (CVD). The underlying mechanisms which connect these diseases are still poorly understood. One system which has been shown to be up-regulated in the setting of obesity and diabetes is that of the G-protein coupled receptor-55/Lysophosphatidylinositol (GPR55/LPI). Despite being upregulated in the setting of obesity, the function of GPR55 in obesity and other disease states remains elusive. Therefore, the present study aimed to 1) investigate the role of GPR55 in obesity by characterising the phenotype of the GPR55 knockout (GPR55-/-) mouse when challenged with a high fat diet (HFD) intervention, 2) elucidate any effect of the GPR55 knockout and HFD intervention on the myocardial infarct size sustained following a period of ischaemia/reperfusion (I/R) and 3) make use of an in vitro model to elucidate the mechanisms by which changes occur in the adipose tissue of mice fed a HFD. GPR55-/- mice fed a HFD for 12-weeks gained significantly more weight in the form of fat mass, compared to wild-type (WT) controls and consequently become obese. Obese GPR55-/- mice displayed hypertrophic adipose tissue concurrent with the significant dysregulation of plasma lipids, increases in specific circulating LPI species, increased lipid deposition within the liver and a change in adipose tissue gene expression profile. These changes were not observed in GPR55-/- mice fed a standard diet or WT mice fed a HFD. Following a period of I/R, the myocardial infarct size in hearts from WT HFD fed mice was significantly smaller than in hearts from WT standard diet fed mice. This reduction in infarct size due to HFD intervention was not dependent on RISK-pathway activation and was not observed in hearts from GPR55-/- mice, therefore demonstrating that the cardio-protective effect of a HFD on infarct size is dependent on GPR55. In vitro studies using 3T3-L1 cells determined that the changes in adipose tissue gene expression of HFD fed mice was not due to enhanced stimulation with LPI or via hypoxic mechanisms. The results of these studies demonstrate that GPR55 has an anti-obesity function in vivo and also mediates the cardio-protective effect of a HFD on myocardial infarct size, through currently unknown mechanisms.

The initial hours following an ischaemic event in the brain represent a critically important window in which therapeutic interventions to reduce neuronal damage and improve patient outcome can be made. Nevertheless, the dynamics of cerebral blood flow and oxygenation, as well as the local physiological changes, in the first few hours after reperfusion following ischaemic stroke are not well understood. In the first study, a remote filament approach was used to obtain multispectral imaging data before, during and after middle cerebral artery occlusion to investigate early changes in haemodynamic concentration of oxy-/deoxy-haemoglobin and total blood volume, in anaesthetised mice. We use immunohistochemistry to establish the extent of cortical injury and correlate the severity of damage with the change of oxygen perfusion during and after the ischaemic event. Increased numbers of platelets and activated microglia, expression of interleukin-1α, evidence of BBB breakdown and neuronal stress are all seen within the stroked hemisphere of MCAo mice and correlate with the severity of oxy-haemoglobin concentration deficit at experimental but not with the change in oxy-haemoglobin concentration during the acute stroke. In the second study, we used the same remote filament and optical imaging approach to investigate the effects of acute systemic inflammation on haemodynamics pre, during, and after induced cerebral ischaemia. We found that an acute systemic inflammatory challenge exacerbates oxy-haemoglobin deficit after 3 h of reperfusion following an ischaemic event. We investigated known pathophysiological markers to elucidate potential mechanisms that may contribute to this exacerbated oxygenation deficit and found hyper-coagulated platelets within the large and microvessels of the ipsilateral cortex. Our findings demonstrate that despite initial restoration of HbO2 supply after 30 min MCAo there is a delayed compromise that coincides with inflammatory processes that could be a future target for improved stroke outcome after thrombolysis. We also show that acute systemic inflammation exacerbates this oxy-haemoglobin deficit after an ischaemic challenge and increases pathophysiology.

The immunosuppressant Cyclosporin A (CsA) has been shown to protect against cell injury caused by ischaemia and reperfusion. A mitochondrial lesion in the form of a Ca2+ sensitive non-selective inner membrane pore is reputed to contribute to the progression of this form of injury and the pore is blocked by CsA. This study was aimed at resolving the CsA receptor of the pore to establish whether this protein was novel or whether it was already recognized. Mitochondrial CsA binding proteins were identified using a tritiated derivative of CsA, 3H[PA-CS], which contains a photoactive diazirine group at position 8 of the molecule. Many components were photolabelled. In order to identify the relevant protein use was made of the fact that CsA binding to mitochondrial membranes was potentiated by ADP and depressed by Ca2+. Heart mitochondria were photolabelled in the presence of ADP (2mM) or Ca2+ (>100μM). Fractionation of mitochondrial membranes extracted in detergent revealed components between 11 and 25kDa exhibiting ADP/Ca2+-sensitive labelling. ADP increased 3H[PA-CS] labelling of this fraction whilst Ca2+ reversed it. The ADP/Ca2+-sensitive labelled component was purified to a single band on SDS-PAGE migrating at approximately 22kDa Identical proteins were purified from sheep heart and rat liver mitochondria The 22kDa protein displayed peptidylprolyl cis trans isomerase (PPIase) activity suggesting it belonged to the cyclophilin (CyP) family of proteins. The protein displayed a Ki of 8nM for CsA and a kcat/Km of 5.8μM1 s-1. This agrees well with other known cyclophilins. Partial amino acid sequencing of the protein revealed sequence similarity with human cyclophilin D (CyPD). CyPD was prone to proteolytic attack during purification However removal of the outer mitochondrial membrane with low concentrations of digitonin and the subsequent treatment of the mitoplasts with 0.5M [NaCl] resulted in increased stability with an almost 10 fold increase in protein yield The localisation of CyPD was investigated using fractionated mitochondria This showed that it is localised in the matrix space Though CyPD is photolabelled in an ADP/Ca2+-sensitive manner, the [3H]CsA binding capacity of CyPD was unaffected by ADP [2mM] or Ca2+ [>100μM]. Also the PPIase activity of the protein was not influenced by high concentrations of ADP or Ca2+. This, as well as photolabelling studies, suggested that CyPD binds to another component in intact mitochondria that is sensitive to ADP and Ca2+, and that this interaction confers the same sensitivity to CyPD-CsA interaction The occasional elution of the ADP/Ca2+ sensitive component at 29kDa, rather than 22kDa, suggests that the target might be a low molecular weight protein. However a CyPD affinity column failed to retain any specifically interacting target protein from inner membrane extracts. These findings provide firm evidence that CyPD interacts with the Ca2+ sensitive pore and that it might be involved in pore regulation.

Heart failure (HF) is a chronic condition that is the result of pathological cardiac events including cardiac hypertrophy and myocardial infarction, being the most prevalent. Pathological cardiac hypertrophy is a mal-adaptive response contributing to heart failure via loss of cardiomyocytes, decreased contractile properties, and increased interstitial fibrosis. Along with hypertension, myocardial ischemia/reperfusion (I/R) injury is a common mediator of pathological cardiac hypertrophy and remodeling through cardiomyocyte cell death and the activation of the innate immune response. Human Antigen R (HuR) is a widely expressed mRNA binding protein that our lab has previously shown to play a key role in cardiomyocyte hypertrophy in vitro and in vivo. However, the specific mechanism, activation, and post-transcriptional modulation of HuR in pathological cardiac hypertrophy and post-ischemic cardiac remodeling has not been delineated. Firstly, we sought to determine the role of HuR in cardiomyocyte hypertrophy. We demonstrate that HuR undergoes cytoplasmic translocation following treatment with phenylephrine or angiotensin II, agonists of two independent Gaq-coupled GPCRs known to induce hypertrophy. Gq-mediated HuR activation is dependent on p38 MAP kinase, but not canonical Gq-PKC signaling. Furthermore, HuR activation is necessary for Gq-mediated hypertrophic growth of cardiomyocytes as siRNA-mediated knockdown of HuR inhibits in vitro cardiomyocyte hypertrophy. Additionally, overexpressing HuR in cardiomyocytes is sufficient to induce hypertrophic cell growth. Next, to delineate the downstream mechanisms of HuR’s translocation promoting cardiomyocyte hypertrophy, our results suggest that HuR mediates the transcriptional activity of NFAT (nuclear factor of activated T cells), a hallmark of pathological cardiac hypertrophy. The results presented here identify HuR as a novel mediator of cardiac hypertrophy downstream of the Gq-p38 MAPK pathway.To further advance the knowledge of HuR as a mediator of hypertrophic myocyte growth in vitro and delineate the functional role for HuR in mediating hypertrophic remodeling in vivo, the second half of this dissertation work focused on the functional and mechanistic role of HuR in mediating post-ischemic fibrotic and immune remodeling of the heart. Our results show HuR activation in the myocardium as early as two hours with an increase in HuR total protein expression sustained at seven days post-I/R. Next, we demonstrate that acute pharmacological inhibition of HuR in vivo, via administration of the novel inhibitor KH-3 at the start of reperfusion, significantly decreased cardiac mRNA expression of TNF-alpha and IL-6 two hours post-reperfusion. Furthermore, acute HuR inhibition significantly reduced cardiac monocyte recruitment into the myocardium at seven days post-I/R. Next, our NRVM model showing that HuR inhibition in cardiomyocytes blunts pro-inflammatory gene expression post-LPS treatment. Additionally, our results show that conditioned media from LPS-treated NRVMs mediates a direct change in the gene expression of pro-inflammatory cytokines in macrophages that is dependent on HuR activity in myocytes.Lastly, RNA-seq analysis gene expression following LPS treatment demonstrates that the increase in inflammatory gene expression profiles is largely dependent on HuR. In summary, this work demonstrates (1) a functional role of HuR in hypertrophic growth of cardiomyocytes, that (2) mediates myocardial remodeling following ischemia/reperfusion injury through a dampened myocyte-driven cytokine expression and subsequent macrophage infiltration.

A myocardial infarction occurs when blood flow to the heart is restricted and is termed “ischemia.” Reperfusion, the restoration of blood flow, is necessary for survival but can cause additional damage. PHLPP, a Ser/Thr protein phosphatase, includes two isoforms (PHLPP1 and PHLPP2), and was originally identified as a tumor suppressor in cancer (Gao, 2005). In the heart, PHLPP1 removal was found to be cardio-protective through increased Akt phosphorylation/activation basally leading to cell survival following insult (Moc, 2015). PHLPP2 removal does not alter Akt activity basally and has been found to increase cardiomyocyte hypertrophy in vitro (Yeh, 2018). We hypothesize that PHLPP2 removal alters cardiomyocyte growth signaling and is detrimental to the heart following ischemic injury. Wild-type, PHLPP1 and PHLPP2 KO mice were subjected to 1hr ischemia via occlusion of the left descending artery followed by reperfusion for various times. Whole hearts were removed following reperfusion and the left ventricle sectioned into ischemic and non-ischemic regions and used for gene and protein expression analysis. The extent of I/R injury was determined using triphenyltetrazolium chloride and Evan’s blue dye, followed by digitized imaging of the area of infarction (IA) versus the area at risk (AAR). Our initial studies demonstrate attenuation in infarct in PHLPP1 KO compared to WT with the same AAR; however PHLPP2 removal increased infarct injury. MRNA and protein expression analysis indicates PHLPP2 removal enhances injury through increased cell death, an imbalance in inflammation/antioxidant signaling, and dysregulated autophagy while PHLPP1 removal confers protection through apoptosis inhibition and increased mitochondrial biogenesis.

Purpose: There is an ongoing debate concerning whether ischemic preconditioning elicits consistent and meaningful exercise performance benefits. We have previously demonstrated no performance benefits of ischemic preconditioning at submaximal aerobic exercise intensities. It is likely that the beneficial effects of ischemic preconditioning on performance only involve supramaximal anaerobic exercise bouts, which elicit greater metabolic and neuromuscular stress. The aim of the study was to test the hypothesis that ischemic preconditioning improves maximal accumulated oxygen deficit (aO2D), an indicator of anaerobic capacity, in NCAA Division I middle-distance runners. Methods: A randomized sham-controlled crossover study was employed in which 10 NCAA Division I middle-distance (800 to 1600 meter) track athletes (age: 21±1 yr; VO2 max: 65±7 mlO2·kg·-1·min-1) completed three supramaximal treadmill running trials (110% VO2max; ~12.6 mph @ 5% grade) to volitional exhaustion coupled with indirect calorimetry to assess maximal aO2D at baseline, after a sham control trial (mock preconditioning), and with limb-based ischemic preconditioning (4×5 min cycles of brachial artery ischemia/reperfusion). Maximal aO2D (mlO2·kg-1) for each trial was determined by first calculating the theoretical oxygen demand required for the supramaximal running bout (linear regression extrapolated from 9×5 min submaximal running stages). The actual oxygen demand measured during the supramaximal bout was then subtracted from the theoretical value to obtain the aO2D. Statistical Analysis: A three-way repeated-measures ANOVA with adjustment for multiple comparisons was used for within-group differences (i.e., baseline vs sham vs ischemic preconditioning) in aO2D. Results: Ischemic preconditioning (122±38 sec) increased (P=0.0001) supramaximal time to exhaustion by 22% compared with both baseline (99±23 sec, 95% CI: 4.8-40.6, P=0.014) and sham (101±30 sec 95% CI: 6.7-34.2, P=0.001). Effect size for these trial differences as estimated by the Partial Eta2 (0.58) were large. Furthermore, the aO2D was considerably greater (P=0.009) with ischemic preconditioning. During their supramaximal run in the presence of ischemic preconditioning, aO2D was 46±35 mlO2·kg-1, a substantial (Partial Eta2 = 0.43) increase compared with baseline (35±27 mlO2·kg-1, P=0.025, 95% CI:1.3-19.2) and sham (38±32 mlO2·kg-1, P=0.046, 95% CI: 0.13-14.0). There were no statistical differences in time to exhaustion and maximal aO2D between baseline and sham. Conclusions: Limb-based ischemic preconditioning considerably improves time to exhaustion and anaerobic capacity as measured by the maximal aO2D in NCAA Division I middle-distance track athletes. Additional work is needed to confirm whether these laboratory-based performance benefits can be translated to better outcomes in real track meets in elite athletes seeking a competitive advantage.

Ca2+ dysregulation underlies many forms of cardiac stress and contributes significantly to arrhythmia and dysfunction during heart disease. Ca2+/CaM-dependent protein kinase II (CaMKII) is a multifunctional kinase that is activated by intracellular Ca2+, and signals through a variety of substrates to regulate cardiomyocyte biology. Elevated CaMKII activity has been associated with a range of pathologies including Ca2+ leak from the sarcoplasmic reticulum, mitochondrial dysfunction, and cell death. The predominant form of CaMKII in the heart is CaMKIIδ, which is alternatively spliced to yield the CaMKIIδB and CaMKIIδC subtypes. This dissertation describes the role of CaMKIIδ in cardiac stress generally, and specifically the role of the CaMKIIδB and CaMKIIδC subtypes in ischemia/reperfusion injury. Chapter 1 provides background information on the role of CaMKIIδ in cardiovascular disease and the structure and function of CaMKIIδB and CaMKIIδC. Chapter 2 describes the majority of the dissertation, focusing on studies on the subcellular distribution of CaMKIIδ subtypes and their role in ischemia/reperfusion injury and inflammatory signaling downstream of NF-κB. Chapter 3 contains studies on the involvement of CaMKIIδ in pathological signaling during cardiac stress elicited by chronic β-adrenergic stimulation and Gq overexpression. Chapter 4 describes ongoing and future studies and the relevance of these findings to human disease.

Ischemia is characterized by reduced blood flow to an area of the body which can then cause cellular injury through the generation of reactive oxygen species (ROS), activation of inflammation, and induction of apoptosis. Although rapid reestablishment of flow is required to prevent organ death, the reperfusion phase of this injury can cause its own deleterious effects often exacerbating the initial insult. The combined action of the two injuries is termed ischemia/reperfusion (I/R) injury. Oxidative stress that results from ischemia/reperfusion injury is a common pathological condition that accompanies many human diseases including stroke, heart attack and traumatic injury. In addition, neurodegenerative diseases including Parkinson’s, Alzheimer’s, and Huntington’s disease appear to involve oxidative stress. Although actively investigated by the medical and pharmaceutical industry; limited progress has been made to ameliorate I/R injury and to date there is no drug approved for treatment for I/R injury. Therapeutic approaches to treat I/R injury have included the administration of compounds to scavenge ROS or induce protective pathways or genetic responses. It was previously reported that caffeic acid phenethyl ester (CAPE), a plant-derived polyphenol, displayed cytoprotective effects against menadione (MD)-induced oxidative stress in human umbilical vein endothelial cells (HUVEC), and the induction of heme oxygenase-1 (HMOX1), a phase II enzyme, played an important role for CAPE cytoprotection. In an effort to improve this cytoprotection, other phase II enzyme inducers were investigated and, 2-cyano-3,12 dioxooleana-1,9 dien-28-imidazolide (CDDO-Im) and 2-cyano-3,12-dioxooleana-1,9-dien-28-oyl methyl ester (CDDO-Me), were found to be potent inducers with a rapid onset of action. CDDO-Im and CDDO-Me, synthetic olenane triterpenoids, developed as anticancer agents were compared to CAPE revealing that CDDO-Im was a more potent inducer of Phase II enzymes including HMOX1 and provided better cytoprotection than CAPE. Gene expression profiling showed that CDDO-Im was more potent inducer of protective genes like HMOX1 than CAPE and additionally induced heat shock proteins. To better understand the mechanism of action of CDDO-IM, a gene expression time-course was undertaken to identify early initiators of the transcriptional response preceding cytoprotection. Application of systems pharmacology identified molecular networks of cell mediating processes.

Ischemic postconditioning has been shown to reduce injury in response to ischemia/reperfusion. Because of limitations on the clinical use of ischemic postconditioning protocols, pharmacological agents that elicit a postconditioning (PostC) effect are highly desired. Previous studies have shown that stimulation of alpha1-adrenergic receptors (α1-ARs) is cardioprotective, thus, the first aim of this dissertation was to examine the effects of post-ischemic stimulation of α1-ARs on cardiac myocyte cell death. Adult rat ventricular myocytes and HL-1 cardiac myocytes were subjected to simulated ischemia-reperfusion injury. Cell membrane permeability, evaluated by measuring released lactate dehydrogenase (LDH) or propidium iodide uptake (PI), was used as an estimate of cell death. Lower amounts of LDH and PI uptake were detected when α1-ARs were stimulated at the onset of reperfusion. Further, lower levels of apoptosis were measured using TUNEL and DNA laddering to evaluate DNA cleavage and Annexin V staining to evaluate outer membrane phosphatidylserine. Prior studies suggest that increased autophagy following ischemia is protective. The second aim of this dissertation was to determine whether post-ischemic α1-AR stimulation inhibits cardiac myocyte death through modulation of autophagy. Alpha1-AR-mediated reductions in cell death were reversed in the presence of ATG inhibitor, 3-Methyladenine. Western blot for autophagosomal marker, LC3-II indicated modulation of autophagy, and two methods were used to measure autophagic flux. LC3-II turnover examined with and without autophagosome-lysosome fusion inhibitor chloroquine revealed an increase in autophagic flux or induction. HL-1 cells transfected with plasmid to express a tandem fluorescent-tagged LC3 molecule also indicated an increase in autophagic flux or induction. Finally, the third aim of this dissertation was to examine the molecular pathways stimulated by α1-ARs that lead to decreased cell death. PI fluorescence assays indicated possible involvement of ERK1/2, PKC, and PLC. Western blotting to detect phosphorylated ERK1/2 or Akt at various time points was not conclusive. Overall, these results suggest that α1-AR stimulation at the onset of reperfusion reduces cardiac myocyte death, an effect that appears to be dependent upon increased induction of autophagy. ERK1/2, PKC, and PLC activation are potential pathways involved in α1-AR-mediated cell survival.

Skeletal muscle regeneration involves a tightly regulated coordination of cellular and signaling events to remodel and repair the site of injury. When this coordination is perturbed, the regenerative process is impaired. The expression of insulin-like growth factor-I (IGF-I) is robust in the typical muscle regenerative program, promoting cell survival and increasing myoblast activity. In this project, we found that severely depressed IGF-I expression and intracellular signaling in aged skeletal muscle coincided with impaired regeneration from ischemia/reperfusion (I/R). To hasten muscle regeneration, we developed the PEGylated fibrin gel (PEG-Fib) system as a means to intramuscularly deliver IGF-I in a controlled manner to injured muscle. This strategy resulted in greatly improved muscle function and histological assessment following 14 days of reperfusion, which are likely mediated by improved myofiber survival. Recent evidence suggests macrophages (MPs) are responsible for the upregulation of IGF-I following injury, therefore we developed a rapid, reproducible, and cost-effective model of investigating MP profiles in injured muscle via flow cytometry. Using information gathered from this model, we found that increasing the number of a non-inflammatory MP population improves the recovery of muscle from I/R. These data demonstrate that immunomodulatory therapies have the potential to greatly improve the recovery of skeletal muscle from injury.

Reperfusion of the ischemic myocardium occurs in nearly 2 million people annually in the United States as a complication of cardiovascular disease, in patients experiencing cardiac arrest, myocardial infarction or undergoing cardioplegic arrest during cardiac surgery. Various levels of low flow are induced by such ischemic events, most notably in the moments following ischemia. However, the post-ischemic low flow period has hardly been examined in the literature. The work presented in this dissertation explores the role of reactive oxygen species (ROS) generation in global ischemia, post-ischemic low flow, and subsequent full reperfusion of the myocardium. This dissertation consists of three parts. In Chapter 2, an isolated buffer-perfused rat heart model was employed to explore both low pressure and low coronary flow as interventions in global cardiac ischemia. The ROS burst was observed upon full reperfusion in rat hearts, with differences in ROS generation between the cellular and vascular compartments. Differences in recovery of left ventricular (LV) function were also observed, with LV functional preservation at 10% low flow, but not at 0.5% low flow intervention. In Chapter 3, a novel model of the isolated heart is described, in which whole blood is recirculated throughout experimentation. Blood-perfused and buffer-perfused hearts were noted to respond similarly to global ischemia and low flow intervention. Also, a method describing detection of the ROS burst at reperfusion in blood-perfused hearts is described. A characteristic acute, short-lived formation of hydrogen peroxide was discovered in blood-perfused hearts subjected to ischemia and reperfusion. In Chapter 4, the neutrophil elastase inhibitor Sivelestat is examined for putative cardioprotective properties. Sivelestat was noted to possess dramatic infarct-sparing properties in the isolated heart, along with the ability to preserve LV function. ROS production was examined, and Sivelestat was shown to reduce ROS in ischemic-reperfused hearts and also in hypoxic-reoxygenated aortic endothelial cells. The aim of Chapter 4 is to introduce Sivelestat to the heavily investigated field of cardiac pharmacology.

Bilirubin was considered to be a toxin that accumulates after catabolism of heme by the enzyme heme oxygenase. However, a mounting body of evidence suggests that bilirubin, at physiological (non-toxic) doses, is a powerful antioxidant and anti-atherosclerotic agent. Recent clinical studies have shown that human beings with mild hyperbilirubinemia (Gilbert Syndrome) are protected against coronary heart disease. The purpose of this study was to investigate whether administration of exogenous bilirubin to normal rats would convey similar protective effects in an experimental model of coronary ischemia. Our hypothesis was that bilirubin administration (20uM/kg, IP, 1 hour before injury) would decrease infarct area and preserve left ventricular function when compared to non-treated rats. Coronary ischemia was induced by temporary (30 min) ligation of the left anterior descending coronary artery in control rats (n=5) and in bilirubin treated rats (n=5), followed by a 1hour period of reperfusion. Left ventricular function was estimated non-invasively using echocardiographic measurements of fractional shortening and percent area shortening. Effects of anesthesia on cardiac function were controlled by using a sham group (n=5). There was a significant reduction of infarct size in the bilirubin treated group compared to the non-treated group (p

Ouabain and other digitalis drugs are specific inhibitors of the purifiedNa+,K+-ATPase, the ubiquitous enzyme that transports Na+ and K+ across theplasma membrane by hydrolysis of ATP. Independent of changes in ionpumping activity, ouabain binding to the cardiac Na+, K+-ATPase also triggers theassembly and activation of a cardioprotective signaling cascade initiated by theformation of the Na+,K+-ATPase/c-Src binary receptor. Activation of this complexby low doses of ouabain before ischemia/reperfusion protects the heart againstinfarction, a phenomenon called ouabain preconditioning. The aim of this studywas to investigate whether ouabain preconditioning affects the fate of the cardiacmembrane Na+,K+-ATPase enzyme complex following ischemia/reperfusion (IR)injury. In our model of Langendorff-perfused rat heart preparation exposed to 30min of zero flow ischemia, Na+,K+-ATPase activity was significantly decreased inthe IR group after 30 min of reperfusion, but not after 5 min of reperfusion. Thisdecrease was prevented by ouabain preconditioning. This was correlated withtotal Na+,K+-ATPase α1 and α2 isoforms protein contents, which were notaffected after 0 or 5 min, but decreased after 30 min of reperfusion and protectedby ouabain. Finally, immunodetection following differential cell fractionationshowed that, after IR, Na+, K+-ATPase α1 was enriched in fractions enriched withthe Early Endosome Antigen 1 (EEA1) marker in the IR group, and this wasprevented by ouabain pre-treatment. Taken together, these data suggest that,during the first 5 min of reperfusion that follow 30 min of ischemia, the Na+,K+-ATPase complex is redistributed into intracellular compartments. After 30 min,Na+,K+-ATPase is degraded. Such redistribution may explain previous reports ofdecreased sarcolemmal activity without changes in total protein contents andcontribute to the increase in intracellular Na+ observed duringischemia/reperfusion injury. Ouabain preconditioning prevented the redistributionof α1 at 5 min reperfusion and protected against α1 and α2 protein degradationand loss of enzyme activity.

This study was aimed at characterizing the apoptotic response equine small intestine subjected to experimental ischemia-reperfusion injury and determining if use of an angiotensin converting enzyme inhibitor (enalaprilat) would ameliorate the apoptotic response. It was designed to determine if mucosal epithelial cells undergo apoptosis during the ischemia phase and also examined if apoptosis is significantly exacerbated by reperfusion. It also investigated whether administration of enalaprilat decreased reperfusion injury secondary to reduced enterocyte apoptosis. Injury was induced using a low flow model of I-R. During celiotomy a single loop of jejunum was isolated and arterial flow decreased to 20% of baseline for one hour and complete occlusion for a second hour. Reperfusion was monitored for 3 hours. A control group (n=6) were not treated while the treatment group (n=6) received 0.5 mg/kg enalaprilat in 0.9% NaCl immediately following ischemia. Jejunal samples were taken prior to the induction of ischemia, immediately post-ischemia and at 1, 2 and 3 hours of reperfusion. Samples were evaluated for gross tissue pathology with standard hematoxylin and eosin staining, the presence of apoptotic cells via TUNEL staining, and gene expression of three apoptosis related genes (bax, bcl-2, p53) using qPCR. Serum enalaprilat and ACE concentrations were determined from blood samples drawn concurrent with jejunal sampling using HPLC/MS and standard HPLC. Plasma enalaprilat concentrations were comparable to previous reports in awake horses. Enalaprilat appeared to have no effect on serum ACE concentrations, however a significant spike in ACE concentration occurred in the treatment group at 1 hour of reperfusion (P=0.0001). Grade of mucosal damage was not significantly different between control and treatment groups at any time point. Subjectively apoptotic index appeared to be higher in the treatment group at end ischemia and during reperfusion. There were no changes in expression of p53 or bcl-2 in either group. Bax expression was significantly decreased (P= 0.02) in the control group at 2 hours of reperfusion. Based on our data administration of an ACE inhibitor during anesthesia in horses with an ischemic segment of intestine confers no protective benefit and may be associated with increased intestinal injury and apoptosis. Lack of expression of p53, bax and bcl-2 suggests another apoptotic mechanism in equine ischemic intestine.

Cardiovascular disease, which remains the leading cause of mortality in the Western world, is manifested in contractile dysfunction, myocardial infarction and arrhythmias. These detrimental effects are partially attributed to impaired Ca cycling and cell death. Since calsequestrin (CSQ) and inhibitor-1 (I-1) are key regulators of Ca cycling and the anti-apoptotic small heat shock protein 20 (Hsp20) is an important mediator of cardioprotection, this dissertation sought to gain further insights into the role of these proteins in cardiac pathophysiology.CSQ is a crucial regulator of Ca load in the sarcoplasmic reticulum and the Ca-release channel, the ryanodine receptor. Interestingly, human CSQ null mutations have been associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). To further address the significance of CSQ and CSQ null mutations, this dissertation attempted to generate a mouse model with ablation of CSQ. I-1 and its phosphorylation at Thr35 are important mediators of the heart's beta-adrenergic responses. To address any potential beneficial effects of enhanced I-1 activity in the adult heart, a mouse model with inducible expression of active I-1 (T35D) was generated. Active I-1 expression significantly enhanced contractility, associated with preferential phospholamban (PLN) and myosin light chain 2a phosphorylation. Upon ischemia/reperfusion (I/R), active I-1 augmented contractile function, associated with increased pThr17-PLN phosphorylation. Further examination revealed that the infarct region and apoptotic as well as necrotic injury were significantly attenuated by enhanced I-1 activity, associated with suppression of the endoplasmic reticulum stress response. These findings indicate that active I-1 may represent a potential therapeutic strategy in myocardial infarction. Hsp20 and its phosphorylation at Ser16 protect cardiomyocytes against apoptosis. To determine whether genetic variants exist in human Hsp20, which may modify these beneficial effects, the Hsp20 gene was sequenced in dilated cardiomyopathic patients and individuals with no heart disease. A C59T substitution was identified in Hsp20 in one patient and three individuals without heart disease. This mutation changes a conserved proline residue into leucine at position 20 (P20L), resulting in secondary structural alterations. The significance of this variant was assessed by subjecting cardiomyocytes, infected with Ad.GFP, Ad.WT-Hsp20 and Ad.P20L-Hsp20, to simulated ischemia/reperfusion. Expression of WT-Hsp20 significantly attenuated apoptotic injury, while the P20L-Hsp20 mutant showed no protection. This was associated with the mutant's diminished phosphorylation at Ser16. Furthermore, stimulation of cardiomyocytes with isoproterenol and protein kinase A-dependent phosphorylation in vitro confirmed the impaired ability of the mutant Hsp20 to become phosphorylated at Ser16. These findings indicate that the P20L substitution in Hsp20, may adversely affect the ability of human carriers to cope with cellular stress. Furthermore, a C29T transversion was identified exclusively in black cardiomyopathic males. This mutation results in substitution of serine at position 10 to phenylalanine (S10F), with similar structural alterations to the P20L mutant, suggesting that the S10F substitution may also have adverse effects on Hsp20's cardioprotective properties. Collectively, this dissertation provides novel insights into the role of these key regulatory proteins in the diseased myocardium, further illuminating the molecular and genetic defects, associated with cardiovascular disease, which may provide potential therapeutic strategies to alleviate its detriment.

The purpose of this dissertation was to demonstrate that a new method of direct intracellular energy delivery was effective in maintaining viability of in vitro Human Umbilical Vein Endothelial Cells (HUVECs) when exposed to chemical ischemia for prolonged periods of time and in preventing in vivo ischemia reperfusion injury in a composite tissue transplantation model. Ischemia and reperfusion injury is a well recognized phenomenon that occurs in situations like organ transplantation, shock, cardiac surgery, etc, where tissues are temporarily deprived of nutrients and oxygen for energy production. When exposed to short periods of ischemia, cells utilize their stored energy to survive. If ischemia is extended beyond certain limits, cellular energy stores become depleted leading to metabolic and structural changes. One of the effects of this lack of energy is the malfunctioning of the membrane adenosine triphosphate (ATP)-dependent ionic pumps which are ultimately responsible for maintaining cellular volume, intracellular pH and ionic homeostasis. Thus it is hypotesized that the negative effects of energy depletion on cellular homeostasis could be overcome by delivering energy directly into the cell. This method of direct energy delivery into cells is based on fusogenic lipid vesicles (FLVs) composed of lipids very similar to those that form the cellular membrane. These vesicles are very small and when put in contact with cell membranes, rapidly fuse to the cell membrane delivering their content into the cell interior. Making use of this phenomenon, the vesicles are loaded with ATP magnesium chloride (Mg-ATP) that is delivered directly into the cell, providing it with a readily usable form of energy. We hypothesized that, under chemical hypoxia, endothelial cell viability could be preserved and the Na + K +- ATPase pump activity could be maintained by delivering Mg-ATP directly into the cells. Further, we hypothesized that ischemia reperfusion injury due to tissue transplantation could be reduced by perfusing tissues ex vivo before reimplantation with a solution containing Mg-ATP-loaded FLVs. The first two hypotheses were tested in the following manner. First, we determined the optimal concentration of Mg-ATP that the FLVs needed to be loaded with to maintain the viability of HUVECs exposed to 4 hours of chemical hypoxia. Second, we tested if the Na + K + ATPase pump activity could be maintained by delivering Mg-ATP to HUVECs exposed to chemical hypoxia. The results of these experiments demonstrated that direct delivery of Mg-ATP using FLVs was effective in maintaining cell viability and Na + K + ATPase pump activity in cells exposed to chemical hypoxia. The third hypothesis was tested using a rat hind limb transplantation model. Hind limbs were harvested and perfused ex vivo with a preservation solution containing Mg-ATP-loaded FLVs. After 13 or 21 hours of ischemia limbs, were transplanted to the recipient. Limbs perfused with the Mg-ATP FLV solution had a greater survival rate than limbs perfused with control solutions. The application of this method of direct cellular energy delivery could have great implications in clinical situations where tissues are exposed to long periods of ischemia such as transplants or limb amputations. Further investigation is needed to optimize this preservation solution to allow its use as routine therapy in these situations.

Neurological disorders caused by acute disruption of cerebral blood flow are a major clinical concern in both the adult and newborn. Loss of oxygen utilization despite adequate blood flow marks brain tissue fated for death in stroke, but the reason for this is not clear. In this study, we used a cerebral ischemia-hypoxia model in mice to determine the effects of hypoxia on partially ischemic tissue. We found this engaged simultaneous cell survival and death signaling, as well as autophagy, a process by which the cell digests its contents to stay alive during an energy crisis. This was associated with oxidative stress, extensive clotting, and impaired reperfusion after ischemia-hypoxia. This perfusion impairment was prevented in fibrinogen deficient mice, who also exhibited less injury. These results indicate the loss of tissue oxygen utilization in stroke may lead to clotting in the vasculature, and that this must be resolved for effective recovery. In contrast, in neonatal rat hypoxia-ischemia, a model of hypoxic-ischemic encephalopathy, perfusion impairment was restored by 4 hours recovery, and the mechanism for this is also unclear. We found this restoration was associated with an induction of the clot busters tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. tPA deficient mice had greater fibrin deposition and mortality during hypoxia than wildtype mice, confirming a protective role. However, the induction of tPA, which can also be potentially damaging, was sustained throughout the future lesion in rats, and associated with blood-brain-barrier damage, white matter degradation, neuronal axon loss, and apoptosis. Inhibition of the downstream plasmin with alpha-2 antiplasmin offered significant protection, and the injection of additional tPA made the injury worse. These results indicate that inhibition of deleterious plasminogen activator / plasmin effects may aid in treatment of hypoxic-ischemic encephalopathy. All together, this study shows a differential response between the neonate and adult brain to hypoxia in a partially ischemic environment, the consequences of which may provide prime targets for clinical intervention.

BACKGROUND: Ischemia-reperfusion (I/R) injury associated with hepatic resections and liver transplantation remains a serious complication in clinical practice, in spite of several attempts to solve the problem. AIMS: To evaluate the response of the hepatocyte to ischemia METHODS: Published data are thus revised. RESULTS: The response of the hepatocyte to ischemia is based on the sensitivity of hepatocytes to different types of ischemia, the kind of cell death of the hepatocyte when it is subjected to ischemia, and on the response of the hepatocyte to the different times and extents of ischemia. Clinical factors including starvation, graft, age, and hepatic steatosis, all of which contribute to enhancing liver susceptibility to ischemia/reperfusion injury. CONCLUSION: Ischemic preconditioning, based on the induction of a brief ischemia to the liver prior to a prolonged ischemia, has been applied in tumor hepatic resections for reducing hepatic I/R injury and recent clinical studies suggest that this surgical strategy could be appropriate for liver transplantation.

Reperfusion of an ischemic myocardium is associated with a number of deleterious events that are thought to contribute to myocardial dysfunction. Cellular events that predominate during reperfusion include calcium overload, mitochondrial dysfunction and enhanced production of free radicals. Cardiac mitochondria are believed to play an important role in the pathophysiology of reperfusion by contributing to calcium overload as well as free radical production. In an initial in vivo study, the effect of in vivo ischemia/reperfusion on mitochondrial function was investigated. The LAD coronary artery of male SD rats was ligated to induce ischemia, and reperfusion was initiated by removal of the ligature. Cardiac mitochondria were isolated from the affected area of the left ventricular free wall and were utilized for assays of ADP linked respiration as well as individual mitochondrial enzyme activities and western blot analysis of mitochondrial proteins. We were able to demonstrate that reperfusion was associated with a significant decline in ADP linked respiration. The mechanism of decline in overall mitochondrial respiration was due to selective inactivation of the redox sensitive Krebs cycle enzymes KGDH and aconitase. Reperfusion was also associated with a significant decline in activity of complex I of the electron transport chain. The mechanism of inactivation of complex I was further investigated in an in vitro setting by simulating conditions that predominate during ischemia and reperfusion such as pH changes and calcium overload. Results indicate that mitochondrial calcium regulates complex I activity by inducing an endogenous superoxide burst. This leads to a sulfhydryl modification of complex I resulting in its inactivation. In the presence of NADH, and after chelation of calcium, complex I is capable of self-reactivation. The findings of our in vivo and in vitro studies suggest a scenario in which calcium overload during reperfusion results in an endogenous superoxide burst at the complex I site. This radical burst potentially contributes to inactivation of redox sensitive enzymes during reperfusion. The inhibitory effect of calcium on complex I activity also suggest a regulatory effect of calcium on mitochondrial proton gradient, which in turn regulates mitochondrial calcium uptake