Cardiovascular disease (CVD) is the leading cause of death in the United States, and the risk and severity of CVD are increased with type 2 diabetes. However, the exact mechanisms that result in the enhanced association between CVD and type 2 diabetes have not been clearly elucidated. Inflammation and oxidative stress are strongly implicated in both diseases. In type 2 diabetes, there is a dysregulation of inflammatory mediators where an anti-inflammatory molecule adiponectin and a pro-inflammatory cytokine TNF-alpha, are reduced and increased, respectively. Even lower plasma adiponectin concentrations are associated in type 2 diabetic patients with cardiovascular disease. Thus, adiponectin may be a significant link between the two diseases. The studies in this dissertation examined the in vivo cardioprotective actions of adiponectin and apocynin, a NADPH oxidase inhibitor, in the Zucker diabetic fatty (ZDF) type 2 diabetic model. A model of coronary artery occlusion was utilized to induce myocardial ischemia-reperfusion (I/R) injury. The mechanisms of protective actions were assessed by measures of inflammation, oxidative stress and DNA damage. Further, in vitro actions of adiponectin in human whole blood were investigated. We found that in vivo treatments of adiponectin and apocynin significantly reduced myocardial infarction in the type 2 diabetic heart by 40% and 68%, respectively. The cardioprotective action of adiponectin was associated with 2 to 4 fold significant attenuations in several inflammatory characteristics, such as neutrophil adhesion molecule CD11b expression, myocardial adhesion molecule ICAM-1 expression, myocardial neutrophil accumulation and plasma TNF-alpha concentration. The cardioprotective action of apocynin was associated with a significant reduction in myocardial oxidative stress by 25%. In vitro adiponectin actions demonstrated the ability of adiponectin to reduce neutrophil ROS production in human whole blood. These studies were the first to report the cardioprotective action of adiponectin in the type 2 diabetic heart. In addition, adiponectin was found to modulate neutrophil-mediated myocardial I/R injury. Collectively, these findings indicate the significant role of adiponectin in inflammation and oxidative stress in type 2 diabetes. Further, it can be concluded that inflammation and oxidative stress significantly contribute to the enhanced severity of injury observed in the type 2 diabetic heart.

Osteoarthritis, which affects over 21 million people and costs the US $61 billion/yr, is devastating the US population and taxing the health care system. These numbers will increase exponentially as the population ages. It is reported that previous trauma to cartilage resulting in focal chondral defects progresses to osteoarthritis if treatment is delayed or unsuccessful. Current treatment modalities for focal chondral defects have had variable success rates. As such scaffold based therapies in combination with tissue engineering are being developed as an alternative therapy for focal chondral defects. One important area of research to be addressed for these therapies to be successful is rapid integration of native tissue with the implant. An advantage of using scaffold based therapies is that scaffolds provide a stable surface for tissue to grow on and integrate with the existing tissue. In addition, there is the opportunity to use scaffolds for measuring joint loading. These measurements are crucial for a better understanding of the loading environment leading to osteoarthritis as well as for development of rehabilitation regimens when tissue engineering is used to treat defects. It is the goal of this research to determine if mimicking the native trabecular bone structure can be utilized to promote rapid bone ingrowth into implants and to determine whether these implants can be used to directly measure in vivo joint loads. To address the goals of this study, polybutylene terephthalate scaffolds were designed and then built using a fused deposition modeling system. Two different scaffold designs were utilized to determine if mimicking bone structure results in improved bone ingrowth. One scaffold was a biomimetic scaffold that replicated the trabecular bone structure and the other utilized a simple porous structure. These scaffolds were also equipped with strain gauges so that they could be used to monitor joint loading within the knee joint. The strain gauges were used in combination with implantable miniature radio transmitters to allow a fully internal measurement system to be used to determine joint loads during gait as well as other weight bearing activities. Using histology and μCT it was observed that the biomimetic scaffolds increased bone ingrowth into the scaffold over 500% compared to the simple porous scaffolds. These biomimetic scaffolds also increased bone growth in the areas adjacent to the scaffold. Additionally, it was demonstrated that these scaffolds when outfitted with strain gauges could measure axial joint loads occurring within the knee joint during various activities. It was noted that the temporal measurements were highly correlated with video analysis and that peak loads increased as a function of time post implantation. The ability of biomimetic scaffolds to increase bone ingrowth is important for anchoring the scaffold in place and allowing successful integration of tissue engineered cartilage with the native tissue. This will improve success rates of scaffold based tissue engineering therapies. The ability of implants to measure joint loads is crucial to developing a better understanding of osteoarthritis as well as improving rehabilitation protocols. Additionally, by monitoring the change in peak loads with time it will be possible to monitor the healing response at the implant site. Overall, this research demonstrates that polybutylene terephthalate scaffolds have the ability to be used in combination with tissue engineering constructs to treat focal chondral defects and are capable or providing direct in vivo loading measurements.

Gap junctions are aggregates of intercellular channels each formed of protein subunits termed connexins (Cx). Recently published data show that junctional dye permeability relative to conductance (permselectivity) varies across several orders of magnitude for Cx43 junctions, suggesting variable selectivity of the comprising Cx43 channels. Logical candidates for this variable selectivity are variability in charge or size selectivity. Consequently, junctional charge and size selectivities were determined in the current study by simultaneous measurement of junctional permeance to dyes of differing size or charge.The results show that for a number of dyes differing in size, charge, chemical composition, and structure the primary determinant for selectivity through Cx43 gap junctions was the size of the dye permeant with this selectivity showing essentially no variability beyond that seen between incompletely divided cells, presumably representing the variability inherent to the measurement. As such, selectivity of dye-permeable Cx43 channels is well described by the physical dimensions of the channel pore acting essentially as a simple molecular sieve. The seemingly disparate dye selectivity and permselectivity results can be reconciled by the variable presence of a dye-impermeable but electrically conductive channel conformation for Cx43 channels, affording a possible mechanism for independent regulation of diffusion of larger molecules versus electrical conductance to smaller ions.Cx40 junctions, known to be cation selective, also showed minimal variability in charge selectivity indicating that Cx40 charge selectivity is also an essentially fixed parameter. Co-expression of Cx40 and Cx43 lead to charge selectivities ranging from Cx43 to Cx40 with an average intermediate between the two. Activation of PKC leads to an increase in cationic selectivity of Cx40/Cx43 composed junctions by specifically reducing permeability through non-selective Cx43 channels favoring permeation through cation-selective Cx40 channels, allowing for junctional charge selectivity regulation.The combined data suggest that selectivity properties for dye permeable channels composed of Cx43 or Cx40 are essentially fixed parameters of the channel pore. Only upon co-expression of these connexins is significant variability in selectivity seen. The differential effects of PKC-mediated phosphorylation on permeability of Cx43 and Cx40 channels then allows for regulation of junctional charge selectivity but only in cells expressing both connexins.

Hypertension is involved in the exacerbation of stroke. Increased blood-brain barrier (BBB) permeability and cerebral edema formation are potentially lethal complications of cerebral infarction. It is unclear how BBB tight junction (TJ), ion transporter, and protein kinase C (PKC) signaling pathway proteins critical for maintaining brain homeostasis contribute to cerebral infarction during hypertension development. The hypothesis of this study is that hypertension leads to molecular changes in the BBB which predispose the brain to increased cerebral infarct damage following ischemic stroke. Studies were undertaken to investigate the effect of hypertension development on (1) physiological parameters of the spontaneously hypertensive rat (SHR) and on the expression levels of BBB TJ, ion transporter, and PKC proteins potentially involved in ischemia-induced infarct damage; (2) ischemia-induced infarct volume following permanent middle cerebral artery occlusion (MCAO); and (3) the effect of inhibition of Na+/H+ exchanger isoform 1 (NHE-1) on ischemia-induced infarct volume following permanent MCAO in hypertensive SHR (15 weeks). Early hypertension development was determined in SHR and compared to normotensive, age-matched Wistar-Kyoto (WKY) rats at 5 (pre-hypertension), 10 (early stage hypertension), and 15 (later stage hypertension) weeks of age. Characterization of BBB TJ and ion transporter proteins known to contribute to edema and fluid volume changes in the brain show differential protein expression patterns during hypertension development. Western blot analysis of TJ zonula occludens-2 (ZO-2) showed decreased expression while ion transporter, NHE-1 was markedly increased in hypertensive SHR (15 weeks) compared to age-matched controls. Hypertensive SHR (10 and 15 weeks) showed greatly increased necrotic volume with impaired neurological deficits and edema formation. Increased NHE-1 expression in hypertensive SHR (15 week) suggests a potential role for this ion transporter in the promotion of ischemic brain injury. Selective inhibition of NHE-1 using 5-(N,N-Dimethyl)amiloride (DMA) showed significant attenuation in ischemia-induced infarct volume in hypertensive SHR following MCAO. These data suggest a novel role for NHE-1 at the BBB/neurovascular unit in the regulation of ischemia-induced infarct volume in hypertensive SHR suggesting that modulation of NHE-1 may be a factor important in the potentiation of MCAO infarct size and a novel therapeutic target in the prevention of ischemic stroke.

The rate and severity of hypertension are much lower in women than men of a similar age. However, the incidence of hypertension and its complications increase dramatically after menopause, matching and then surpassing that of age-matched males. While current anti-hypertensive therapeutics can improve blood pressure in males, they have proven to be less effective in postmenopausal women. Clinical trials in menopausal women utilizing hormone replacement therapy have also produced controversial results, thus other approaches are necessary to control blood pressure in women after menopause. Targeting the endothelin system can attenuate hypertension in male mice, and components of this system are known to be upregulated in females after menopause. Recent evidence in male mice also demonstrates that T lymphocytes promote the development of hypertension. However, research into the role of the endothelin and immune systems during hypertension in females is lacking, and is necessary to better understand how blood pressure regulation changes after menopause and identify novel targets for anti-hypertensive drug development. Therefore, we sought to determine how the progression to menopause in the novel VCD mouse model of menopause impacts the degree of angiotensin II (Ang II) hypertension and whether antagonizing the ET-1 system could attenuate hypertension in menopausal animals. We also hypothesized that prevention of T cell-mediated responses contributes to sex differences in hypertension and that the increased degree of hypertension after menopause requires T cells. To determine how the gradual progression to menopause in VCD-treated mice impacts hypertension, we infused Ang II into premenopausal and VCD-treated peri- and postmenopausal animals. Compared to premenopausal mice, Ang II-induced hypertensive responses were significantly increased after menopause, but were unchanged during the perimenopause transition. 17𝛽-estradiol replacement during perimenopause prevented the increased hypertensive response in menopausal animals, demonstrating that upregulation of hypertension in this model is driven by the loss of estrogen-induced protective actions. To test the hypothesis that ETA receptor-mediated signaling promotes postmenopausal hypertension, VCD-treated menopausal mice were administered either the ETA receptor antagonist ABT-627, 17𝛽-estradiol replacement, or vehicle. The increased hypertensive response in menopausal mice was equally prevented by either ETA receptor antagonism or 17𝛽-estradiol replacement, supporting the notion that ET-1-targetted drugs may improve blood pressure control in postmenopausal women. To address the hypothesis that prevention of T cell-mediated responses contributes to sex differences in hypertension, Ang II was infused into T cell-deficient male and premenopausal or VCD-treated menopausal female Rag-1^(-/-) mice with or without CD3⁺ T cell adoptive transfer. The results support this hypothesis, demonstrating that T cells promote the increased hypertensive response in males, and that the T cell-dependent response is prevented in premenopausal females, establishing sex differences in hypertension. After menopause, T cells are required for the increase in hypertension. To test the hypothesis that anti-inflammatory regulatory T cells are required for resistance against hypertension in premenopausal females, PC-61 was administered to deplete regulatory T cells during 14 days of Ang II infusion. We found that regulatory T cell depletion significantly increased the degree of Ang II hypertension, supporting a critical anti-hypertensive role for regulatory T cells in premenopausal female mice.

Heart failure is characterized by the inability of the heart to meet the demands of the body, often through inadequate ventricular filling or pumping. Progression into this compromised state is marked by several structural and biochemical changes to the myocardium. A modification germane to the study at hand is the altered energetic and metabolic status of the cardiomyocyte. Therefore, the purpose of this study is to understand how regulators of energetic status, liver kinase B 1 (LKB1) and AMP-activated protein kinase (AMPK), interact with and alter function of the greatest energy consumer in the heart, the myofilament. Using measures of muscle myofilament function and post-translational modification status, relative activation of the LKB1/AMPK pathway was found to differentially alter contractility, cross-bridge kinetics, Myosin Binding Protein C phosphorylation, and Troponin I phosphospecies distribution. LKB1 complex treatment of cardiac trabeculae was shown to blunt contractility and maximum tension generation. Furthermore, regulation of the LKB1 complex was examined. The LKB1 complex was shown to alter its association with myofibrillar proteins, depending on the energetic state of the cardiomyocyte. The LKB1 complex was found localize in the region of the Z-Disk of the cardiac sarcomere, and potentially associate with both mitochondrial and mechanotransduction regulatory proteins. These results indicate potential roles for the LKB1/AMPK signaling axis to modify myofilament function, potentially though alterations in key post-translational modifications and protein-protein associations, in response to energetic stress. As such, targeting the LKB1/AMPK pathway could be beneficial in treating energetically impaired hearts.

Understanding renal transport mechanisms is essential to predicting molecular interactions and targeting drugs to specific transporters. This study focuses on characterizing rbOAT1 and rbOAT3 with respect to interactions with compounds that varied in charge, length and hydrophobicity. Straight chain dicarboxylates of five carbons or more inhibited both transporters well. Maximum inhibition occurred with glutarate (5C). Monocarboxylates interacted poorly with both transporters, yet exhibited greater inhibition as the chain length increased. Aromatic dicarboxylates inhibited both transporters optimally at a charge separation similar to that of glutarate. Both OAT1 and OAT3 were inhibited by reduced and oxidized 2,3-dimercapto-1-propane sulfonate (DMPS). To test the chemical properties of DMPS that facilitated its interaction with rbOAT1 and rbOAT3, I used 3-mercapto-1-propane sulfonate and found that the SH groups are essential in stabilizing DMPS to the binding sites. These results indicate that the size, hydrophobicity, charge, and H-bonding capabilities of a molecule work together to stabilize it to the transporter binding site.

The purpose of this study was to investigate the function of the protein bestrophin-1 (best-1). Previous studies have suggested that best-1 is either a Ca++-activated Cl- channel (ClCa) and/or that it is a regulator of Ca++ induced responses in the retinal pigment epithelium (RPE).Experiments were performed on whole monolayers of cultured primary fetal human retinal pigment epithelium (fhRPE). The amount and polarity of the transduced proteins exposed at the cell surface, as assessed by domain selective biotinylation, was not altered by overexpression of best-1 or the best-1 mutants W93C or R218C. Best-1 (Wt) transduction of fhRPE increased baseline short circuit current (Isc) over Controls and best-1 mutant experimental groups during electrophysiology on whole monolayers. Cl-ion substitution in the Wt group caused a reduction in Isc that was reversible upon re-introduction of Cl- to the bathing solution. Cl-ion substitution did not significantly alter Isc in any other experimental group. This data supports a regulatory function for best-1 for Cl- transport across the epithelium.Application of the Ca++-ionophore ionomycin resulted in a biphasic response in Control monolayers, which was reduced in monolayers overexpressing best-1. Following exposure to ionomycin, NFA was used to block ClCa currents. NFA resulted in a decrease in the TEP of Controls and Wt monolayers to approximately half the value observed at the maximal response to ionomycin (Peak 2 or P2). The Isc response of Control and Wt monolayers mimicked the TEP response, resulting in an Isc at the end of exposure to NFA that was the same as the Isc at Peak 1 (P1) of the biphasic response to ionomycin. This suggests that ClCa currents are at most responsible for P2 of the ionomycin response, but this is not exclusive of other channels sensitive to NFA being involved. R218C expressing cells showed little response to Ca++ and NFA, and W93C cells were not responsive.In conclusion, with the cellular expression and polarity of best-1 and best-1 mutants the same, the data from the present study support a Ca++ regulatory role for best-1 that functions to prime the RPE to respond to changes in intracellular Ca++.

Stroke is the third leading cause of death and disability in US. Cerebral edema is a major consequence of brain ischemia. Despite the importance of cerebral edema, no effective pharmacological treatments have been developed.Previous research indicates that the aquaporin-4 channel facilitates water movement during cerebral edema formation. Mice lacking the normal expression of aquaporin-4 have decreased cerebral edema, reduced infarct formation and improved neurological outcome induced for classic models of cerebral edema (1, 2, 3). To our knowledge, no compounds that effectively block water permeability through aquaporin-4 have been discovered.My hypothesis is that an aquaporin-4 blocker would significantly decrease the cerebral edema formation after ischemic stroke in mice. My objective was to identify and characterize a novel aquaporin-4 blocker using Xenopus laevis. I also proposed that dystrophin deficient mice, a mouse strain that has a decreased expression of aquaporin-4 channels would have a decrease formation of cerebral edema after transient ischemic stroke when compared with a strain matched controls.I found that bumetanide, a well-described Na+, K+, Cl- cotransporter inhibitor, reversibly and dose dependently inhibited water permeability through aquaporin-4 channels. These results indicated that the protective effect of bumetanide seen in rats after ischemic stroke (4) might be through a combined effect on aquaporin-4 channels and the Na+, K+, Cl- cotransporter.In order to identify the relative amount of protection conferred from the aquaporin-4 channels compared to the Na+, K+, Cl- cotransporter, I characterized the dystrophin deficient mouse after ischemic stroke. I found that dystrophin-deficient mice had a decrease in the formation of cerebral edema after transient brain ischemia when compared with strain-matched controls. Dystrophin-deficient mice had an increased mortality and seizure-like activity after transient brain ischemia. One hypothetical mechanism might be that increased plasma potassium is associated with a presumably decreased ability to buffer potassium after neuronal stimulation, due to its lack of aquaporin-4 and potassium channels (Kir4.1) at the end feet of astrocytes. Because of these additional effects, I concluded that the mdx mouse is not an ideal model for the study of a protective effect of an aquaporin-4 blocker after ischemic stroke.

Thesis (Ph. D.)--Oklahoma State University, 2007. / Vita. Includes bibliographical references.