▼ The vertebral column is a complex anatomical structure in terms of form and function, and the majority of the loads applied to it during locomotion pass through the bodies of the vertebrae. Spontaneous vertebral fractures are a common skeletal pathology in humans, and are linked to low bone mineral density. In contrast, spontaneous vertebral fractures have not been reported in apes, even in cases of extremely low bone mineral density. Given these observations, it is likely that there is a structural difference in the vertebral bodies among humans and apes that causes humans to be more susceptible to spontaneous vertebral fracture. The focus of this study was to determine how the structure of the vertebral body in terms of gross morphology, trabecular microarchitecture and shell thickness differed among humans and apes, and whether these differences were related to body mass, locomotion and strength of the vertebral body. Humans have larger vertebral bodies along the entire length of the vertebral column than would be expected for body mass, and the vertebral bodies increase in size along the length of the vertebral column similar to the much larger gorilla. However, when vertebral body size is normalized within the vertebral column, there are no differences among the species. Additionally, although humans have large vertebral bodies, they are still weaker than would be expected for their size. Humans have lower trabecular bone volume fraction than gibbons and African apes, and displayed a negative correlation between bone volume fraction and degree of anisotropy in the ventral vertebral body unlike the other hominoids. Humans have thinner vertebral shells than would be expected for their body mass and vertebral body height. The combination of tall vertebral bodies containing unsupported columnar, craniocaudally oriented trabeculae that do not support the thinner than expected vertebral shell may be the structural differences in vertebral bodies that account for the increased susceptibility to spontaneous vertebral fractures in humans. Evolution of larger, more trabecularized vertebrae with a thinner shell in humans is likely linked to the evolution of bipedalism; however, an exact mechanism is currently unclear and requires further investigation. Advisors/Committee Members: Simpson, Scott.

▼ The epicardial lymphatic system of the heart is responsible for eliminating excess fluid in the interstitium. Its failure to function properly results in edema or tissue fibrosis. Despite its physiological importance, the development of the cardiac lymphatics as well as the cardiac lymphangiogenic process have not been thoroughly investigated. In this study, we used the nuclear-localized transcription factor Prox-1 as a lymphatic marker, given that it is frequently used to label adult lymphatic endothelial cells and can be used to induce various cell types to transform into a more lymphatic phenotype. We tracked the progressive formation of the cardiac lymphatic network in both avian and mouse models, respectively from HH Stages 24-40 and from ED 9.5 to adult stages. Additional lymphatic markers such as LYVE-1, VEGFR-3, and podoplanin were used to verify lymphatic identity. We thus observed that there were three types of cells with lymphatic phenotype in the mammalian heart: (1) incoming Prox-1-positive cells, (2) LYVE-1-positive epicardial cells, and (3) LYVE-1-positive/VEGFR-3-positive myocardial cells/vessels. Adult and embryonic epicardial cell lines and primary cultures of epicardial cells also expressed Prox-1, LYVE-1, and VEGFR-3 in a subset of cells, but not in the subcellular localization seen for mature lymphatics. We explored the mechanisms that may push these pluripotent epicardial cells into the lymphatic phenotype. Adult epicardial cells were treated with the lymphangiogenic growth factor VEGF-C, resulting in a dramatic increase in Prox-1 and phosphorylated ERK expression in the nuclei of a subset of cells. The nuclear accumulation of Prox-1 appeared to be factor specific. The ERK inhibitor UO126 induced a decrease in Prox-1 expression in the cell nuclei in both untreated and VEGF-C treated cells, suggesting that phosphorylated ERK may regulate Prox-1 localization and transcriptional function. Similar findings were observed with explanted embryonic epicardial cells. Tissue oxygen levels, mediated by the hypoxia inducible factor HIF-1, may also regulate lymphangiogenic signaling in the epicardium. Targeting components of the VEGFR-3/ERK or HIF pathways could therefore be instrumental in developing therapeutic strategies aimed at stimulating controlled lymphangiogenesis in cases of cardiac edema, or even inhibiting the process during tumor metastasis. Advisors/Committee Members: Freeman, Barbara.

▼ The outflow tract myocardium and other regions corresponding to the location of the major coronary vessels of the developing chicken heart, display a high level of hypoxia as assessed by the hypoxia indicator EF5. The EF5 positive tissues are also specifically positive for nuclear-localized hypoxia inducible factor-1 alpha (HIF-1a), the oxygen-sensitive component of the hypoxia inducible factor-1 (HIF-1) heterodimer. In this study we altered ambient oxygen levels (hypoxia 15%; hyperoxia 75-40%) during developmental stages (ED 4.5-9, HH 25-35) critical to avian coronary vessel development in order to alter tissue hypoxia, HIF-1a protein expression and some of its downstream targets. We also altered gene expression in the embryonic outflow tract myocardium by injecting an adenovirus containing a constitutively active form of HIF-1a (AdCA5). Under all experimental conditions, HIF-1a reached significant levels. Downstream targets of HIF-1a, such as VEGF, PDGF-B and VEGF receptor 2 (VEGFR2), were significant after exposure to the experimental conditions as well. We assayed for coronary anomalies and used 3D reconstructions to specifically identify altered morphology with the coronary vasculature. When incubated for 4.5 days under abnormal oxygen levels or injected with the AdCA5, coronary arteries displayed deviations from their normal proximal connections to the aorta. These deviations were similar to known clinical anomalies of coronary arteries. These findings indicate that developing coronary vessels may be subject to a level of regulation that is dependent on differential oxygen levels within cardiac tissues and subsequent HIF-1 regulation of gene expression. Advisors/Committee Members: Watanabe, Michiko.

▼ Cardiac arrest and resuscitation results in ischemia/reperfusion injury associated with oxidative stress, leading to post-resuscitation mortality and poor neurological outcome, especially in the elderly. A 12-minute cardiac arrest results in about 50% survival rate in the resuscitated young adult rats over a 4-day recovery period. I investigated hypoxic ventilatory response (HVR) by measuring the minute volume during nomoxic condition and a brief exposure to mild hypoxia (10% oxygen) before and following cardiac arrest and resuscitation. The results indicated that following resuscitation HVR in the non-survivor group was essentially absent while the brainstem responsiveness to hypoxia is fully maintained in the survivor group. Thus, the HVR was shown to be a reliable indicator of survival vs. non-survival during early days of recovery following cardiac arrest and resuscitation. I also investigated recovery following cardiac arrest and resuscitation in the aged rat. The results showed in the 24-month old rats, the overall survival rate, hippocampal CA1 neuronal counts, HVR and brain mitochondrial respiratory control ratio were significantly decreased compared to the younger rats following cardiac arrest and resuscitation. The post-resuscitation with an antioxidant, alpha-phenyl-tert-butyl-nitrone (PBN) improved outcome. The data suggested that (i) there was increased susceptibility to ischemia/reperfusion in aged rat brain; (ii) HVR was decreased in the aged rats; (iii) brain mitochondrial respiratory function related to coupled oxidation was decreased following cardiac arrest and resuscitation in rats, more so in the aged; and (iv) treatment with an antioxidant, such as PBN, reduced the oxidative damage following cardiac arrest and resuscitation. I also studied the effect of adenosine (ADO) and an adenosine A1 receptor agonist, 2-chloroadenosine (2CADO) on the recovery of the brain in a rat model of cardiac arrest and resuscitation. The results suggest that the improved post-ischemic brain blood flow and ADO-induced hypothermia may be the two major contributors to the neuroprotective effects of adenosine following cardiac arrest and resuscitation. Adenosine treatment delayed the hippocampal neuronal loss and promoted long-term survival and may be used as adjuncts to broaden the window of opportunity in the treatment of the ischemia/reperfusion injury. Advisors/Committee Members: LaManna, Joseph C.