<p> Endothelial cells are a vital region in the pathophysiology of the vasculature because it is the interface between blood flow and the vessel. One way that the structure of the vessels wall can change is by the accumulation of reactive oxygen species (ROS), which has been correlated to aneurysm formation. Four main ROS sources in endothelial cells are: NADPH oxidase, mitochondria electron transport chain, eNOS uncoupling, and xanthine oxidase. Endothelial cells are an essential component of vasculature that has distinct functions and morphology. The aorta and brain arteries are highly populated by endothelial cells but the morphology and cellular signaling has been shown to be different. This study focuses on the difference between brain and aorta ROS production and how flow affects ROS. Joeseph Moran-Guiati and Jason Kushner provided the brain and aortic endothelial cultures for these studies. NADPH oxidase complex is the main contributor in both cell types but more in brain. Surprisingly, both cell types contain approximately the same number of NOX subunits, suggesting that the difference in ROS production is dependent on how activated these subunits are. Mitochondrial ROS was only significantly generated in brain cells and is verified because brain endothelium contains higher numbers of mitochondria. Both uncoupling of eNOS and xanthine oxidase did not contribute to ROS generation in static cultures. ROS production increased even further in both cell types when cells were exposed to flow and even higher in brain, suggesting that flow effects ROS generation. These results provide useful information in the difference between ROS generation and how it can be harmful in possibly causing intracranial aneurysm formation.</p>

<p> Regulation of barrier function is critical for patients who suffer from inflammatory diseases such as acute respiratory distress syndrome (ARDS) and sepsis. A major regulator of endothelial barrier function is vascular endothelial cadherin (VE-cad). Cellular levels of VE-cad are known to be regulated by p120 catenin. Loss of p120 leads to decreased barrier function as a result of the endocytosis of VE-cad. However, recent work from our lab shows that expression of an endocytic defective VE-cad mutant was not able to rescue barrier function, as measured using transendothelial electrical resistance (TEER). In contrast, expression of a non-phosphorylatable VE-cad mutant was able to restore barrier function independent of p120 binding. These results suggest that endocytosis is not the only mechanism regulating VE-cad localization to the cell-cell junctions, but rather the phosphorylation state of the protein may play a more critical role to stabilizing VE-cad at the junction. In order to investigate junctional stability of VE-cad, we created a recombinant form of VE-cad by cloning mEos2 into a plasmid containing the VE-cad gene. This fluorophore is photoconvertible, thus allowing for tracking protein movement at the cell-cell junction. The VE-cad proteins, labeled with mEos2 at the C-terminus, were introduced via adenoviral infection into human umbilical vein endothelial cells (HUVEC). Initially, mEos2 fluoresces green, in order to induce photoconversion, a 405nm laser is directed in a specific region of interest (ROI) at the junction. A conformational change in the mEos2 protein will cause irreversible red fluorescence. Tracking the change in fluorescence intensity in the ROI will provide insight into the localization of VE-cad at endothelial cell junctions. We now have a model that can be used to test junctional localization and stability of endocytic defective and non-phosphorylatable mutants of VE-cad.</p><p>

<p> Blood supply is crucial for tumor growth and metastasis. However, current anti-angiogenic therapy is not as effective as predicted, thus a better understanding of the tumor angiogenic process and new anti-angiogenic agent are urgently required. Anthrax lethal toxin (LeTx) has an anti-angiogenic effect on tumors. Tumors treated with LeTx are smaller, paler, and have lower mean vessel density compared to control treated tumors. Most interestingly, compared to current anti-angiogenic treatment, LeTx does not cause normalization of tumor vessels. Instead, tumors treated with LeTx have massive hemorrhages, pointing to a potential alternative mechanism to inhibit tumor angiogenesis. I hypothesize that instead of causing &ldquo;normalization&rdquo; of tumor vasculature, LeTx&rsquo;s anti-angiogenic effects works in a manner similar to a hemorrhagic toxins. To test this hypothesis, I compared the effect of LeTx to snake venom metalloproteinase, a known hemorrhagic toxin, in tumor vasculature. Quantified by Nuance multispectral imaging system, both LeTx and SVMP caused an increase in tumor hemorrhage. Futher analysis of vasculature integrity using continued vessel length showed disruption of vessels by LeTx and SVMP. With these results, I conclude that the anti-angiogenic effects of LeTx are due to its hemorrhagic nature, and not due to normalization of tumor vasculature. Further understanding of LeTx mechanism can help design novel anti-angiogenic agent that compliments current therapy.</p><p>

<p> Integrins are trans-membrane receptors that form focal contacts and mediate the attachment of the smooth muscle cell cytoskeleton to the extracellular matrix. Focal adhesion kinase (FAK) regulates the recruitment and assembly of focal adhesion proteins. It has been established that integrin proteins and FAK play important roles in cell adhesion, tension generation, and mechanotransduction. Integrins and FAK are abundant in smooth muscles; however, the function of FAK in gastric smooth muscle cell contractile regulation remains unclear. To better understand the role of FAK in gastric fundus smooth muscle contractile regulation, we performed two major projects: (1) addressing gastric smooth muscles contractile regulation in cholinergic neurotransmission by FAK phosphorylation (Chapter 2) and (2) characterizing the change in Ca²⁺ sensitization proteins distribution at focal adhesion sites under electric field stimulation induced cholinergic neurotransmission and FAK phosphorylation regulation (Chapter 4). In carrying out the first project, the contractile responses of murine gastric smooth muscles were determined using standard myobath &ndash; isometric force transducer techniques and phosphorylated or total proteins determined by automated capillary electrophoresis and immunoblotting by Wes Simple Western. For the second project, we developed smooth muscle tissue <i>in situ </i> proximity ligation assay (PLA) for the quantitative PLA analysis of protein-protein interaction and protein phosphorylation (Chapter 3). From these studies, we revealed a novel role of FAK in gastric fundus smooth muscle contractile regulation by cholinergic stimulation. We also demonstrate quantifiable tissue level PLA that can be extended and applied to studies of protein-protein interaction, and protein phosphorylation in various tissues and signaling pathways.</p><p>

The overall goal of this dissertation was to examine inflammatory and regenerative responses to acute skeletal muscle damage and to define molecular mediators of repair. Study I examined the effects of an oral anti-inflammatory supplement on exercise-induced muscle damage (EIMD) and systemic inflammation in a human model. Quercetin has been shown in animal and in vitro models to downregulate nuclear factor-kappa beta (NF-κB) nuclear transactivation and monocyte chemoattractant protein 1 (MCP-1) secretion, which regulate muscle regeneration and inflammatory signaling between muscle and immune cells after injury. Subjects ingested quercetin (N=15) or placebo (N=15) before and after performing 24 eccentric contractions of the elbow flexors. Subjects experienced moderate strength losses and delayed onset muscle soreness, indicating damage, but no supplementation effect was observed. The null effect of quercetin in the human model (with its complex inflammatory response) encouraged us to explore basic injury-induced inflammation in a controlled in vitro model, to better understand the post-injury roles of NF-κB and MCP-1. Study II used an in vitro injury model (scratch of C 2C12 myotubes) to identify the roles and interplay of NF-κB and MCP-1 in muscle regeneration and inflammation following acute injury. Protein expression changes of NF-κB and MCP-1, and morphological changes in regenerating muscle cultures were monitored for 24-72 hours (h) post-injury (3-6 replicates per experiment). NF-κB activation was significantly downregulated (-30±1.4% to -44±1.1%) at 6-12h post-injury. Pharmacological blockade of NF-κB downregulated satellite cell proliferation by 19±9% after 19h and 72h, evidence for a role of NF-κB signaling in post-injury regeneration. Furthermore, NF-κB activation strongly correlated (R=0.69) with MCP-1 secretion from injured muscle cultures, and blockade of NF-κB reduced MCP-1 secretion at 1-24h (-33±0.1%) and strongly correlated (R=0.74) with NF-κB activation. These data support recent in vivo findings to demonstrate that NF-κB and MCP-1 signaling are critical regulators of inflammatory and regenerative responses following muscle injury. Moreover, this work provides the first kinetic profile of early (<24 hours) molecular responses of NF-κB and MCP-1 to acute muscle injury, and introduces novel evidence that NF-κB regulates MCP-1 protein secretion, indicating an indispensible role of NF-κB signaling in muscle inflammation in vitro.

The Testis Specific Serine Kinase (Tssk) family of proteins is a large group of kinases that present high level of conservation within paralogs, as well as within species. In addition, in all reported cases as well as in the analysis of expressed sequence tags available in databases, this family of proteins presents a very strict pattern of either testicular or male-gonadal expression. This high level of conservation prompted the postulate that these kinases ought to be important for either testicular function or fertilization. In this work we attempt a biochemical characterization of one family member (Tssk6) in the mouse. We also analyze the male infertility phenotype presented by mice null for Tssk6 revealing its requirement for actin dynamics and the relocalization of proteins necessary for gamete fusion. In this analysis we described Tssk6 as the second protein known to date to be necessary in the sperm for gamete fusion to take place. We also examined a novel member of the Tssk family in the mouse as well as ortholog proteins in two invertebrates (C. elegans and D. melanogaster ). Although our understanding of the function, activity and regulation of these kinases remains small, this work constitutes a significant advance towards the understanding of the identity of the Tssk family. The results that follow have far reaching effects that surpass the realm of the Tssk family. They influence the study of sperm biological processes like the changes in sperm cytoskeletal structures and the acrosome reaction. They also influence the field of developmental biology and scientist working in the molecular characterization of the process of gamete fusion and zygote formation. Lastly, the work here presented influences as well evolutionary developmental biology through the study of a highly conserved family of proteins that is essential for reproduction and could play a role in the process of speciation.

A panel of monoclonal antibodies (MAbs) was generated against bovine oviduct cell surface antigens. Male mice were immunized with intact oviduct cells which had been collected by collagenase dissociation. Mice were boosted twice at three week intervals and then fused with Sp2/0-Ag14 hybridoma cells according to conventional procedures. The resulting antibody-secreting hybrid clones were identified by an indirect enzyme-linked immunoadsorbant assay (ELISA) of cell supernatants on intact oviduct cells. Sixty-seven clones were found to be positive. Of these, thirty-three stable clones were established. Indirect immunofluorescence on live cells was used to visualize the staining patterns of each antibody. This panel of MAbs was tested by ELISA for cross-reactivity to cells from other bovine tissues, namely ovary, uterus, trachea, spleen, liver and lymph node. The majority of these MAbs (20) were specific for antigens on the oviduct cell surface. One MAb (#27) cross-reacted only with uterine cells. Eight MAbs cross-reacted with tracheal cells as well as uterine cells. The remaining four MAbs cross-reacted with a number of different cell types. In addition, this panel cross-reacted with sheep oviduct cells (14/33) but not those of rabbit. Also, eleven MAbs cross-reacted with cow oviduct fluid (ODF) while fourteen cross-reacted with sheep ODF. To determine whether any of the MAbs recognized epitopes which were cycle stage-specific, ODF from both the luteal and estrus phases of the reproductive cycle was examined. ELISA data of ODF from cows did not indicate any obvious cycle stage variations. In contrast, ELISA data of sheep ODF did indicate some variations in antigen appearance between the estrus and luteal phases. Antigens that reacted with the MAbs were characterized by immunoblotting analysis of detergent-solubilized membrane antigens after SDS-PAGE fractionation under reducing conditions. Soluble antigens in cow and sheep ODF were also characterized by similar methods. Several MAbs detected broad diffuse bands which suggests that these antigens may be carbohydrate in nature. In an in vitro cell adhesion assay, MAb #27 prevented apical sperm head attachment to oviduct cells at concentrations as low as 5 ug/ml of purified antibody. None of the other MAbs appeared to have an effect on sperm binding. This effect is not due to nonspecific toxicity since inhibition of attachment is reversible. The oviduct can now be examined in greater detail using these MAbs as probes for the molecular components of the system. Such studies should bring to light details of cell surface composition, which will provide further insights into possible oviduct cell surface functions such as sperm storage.

<p> Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo <i>de novo</i> vessel formation.&nbsp;Quantitative assessment of vasculogenesis is a central readout of endothelial progenitor cell functionality. However, current assays lack kinetic measurements. To address this issue, new approaches were developed to quantitatively assess <i> in vitro</i> endothelial colony forming cell (ECFC) network formation in real time. Eight parameters of network structure were quantified using novel Kinetic Analysis of Vasculogenesis (KAV) software. KAV assessment of structure complexity identified two phases of network formation. This observation guided the development of additional vasculogenic readouts, including a tissue cytometry approach to quantify the frequency and localization of dividing ECFCs within cell networks. Additionally, FIJI TrackMate was used to quantify ECFC displacement and speed at the single cell level during network formation. These novel approaches were then applied to determine how intrauterine exposure to maternal type 2 diabetes mellitus (T2DM) impairs fetal ECFC vasculogenesis, and whether increased Transgelin 1 (TAGLN) expression in ECFCs from pregnancies complicated by gestational diabetes (GDM) was sufficient to impair vasculogenesis. Fetal ECFCs exposed to maternal T2DM formed fewer initial network structures, which were not stable over time. Correlation analyses identified that ECFC samples with greater division in branches formed fewer closed network structures and that reductions in ECFC movement decreased structural connectivity. To identify specific cellular mechanisms and signaling pathways altered in ECFCs following intrauterine GDM exposure, these new techniques were also applied in TAGLN expression studies. Similarly, ECFCs from GDM pregnancies and ECFCs overexpressing TAGLN exhibited impaired vasculogenesis and decreased migration. Both ECFCs from GDM pregnancies as well as ECFCs over-expressing TAGLN exhibited increased phosphorylation of myosin light chain. Reduction of myosin light chain phosphorylation via Rho kinase inhibition increased ECFC migration; therefore, increased TAGLN was sufficient to impair ECFC vasculogenic function. Overall, identification of these novel phenotypes provides evidence for the molecular mechanisms contributing to aberrant ECFC vasculogenesis. Determining how intrauterine exposure to maternal T2DM and GDM alters fetal ECFC function will enable greater understanding of the chronic vascular pathologies observed in children from pregnancies complicated by diabetes mellitus.</p><p>