Browsing by Subject "Endothelium"
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Item Amelioration of oxidative stress in human endothelial cells by caffeic acid phenethyl ester (CAPE) and fluorinated derivatives (FCAPES) and pharmacokinetic characterization of CAPE and FCAPE in rats(2007-12) Wang, Xinyu, 1974 Aug. 12-; Stavchansky, SalomonTissue ischemia is a major cause of morbidity contributing to disease processes such as cardiovascular diseases, stroke, cancer, and traumatic injury and may lead to death. Failure to quickly reestablish flow to ischemic tissue results in tissue death. However, even timely return to normal flow has a downside in that the reintroduction of oxygen to ischemic tissue results in ischemia/reperfusion (I/R) injury that produces an oxidant stress. This pathological process requires new therapeutic strategies and agents to reduce the personal, social and economic loss. One of the most generally accepted mechanisms for the pathology of I/R injury is the production of the reactive oxygen species (ROS), suggesting antioxidants may ameliorate I/R injury. Caffeic acid phenethyl ester (CAPE), a plant derived polyphenolic compound, has been shown to protect organs from I/R induced damage in vivo, and this effect has been attributed to its antioxidant activity. To better understand the mechanism of CAPE protection, a model using menadione-induced oxidative stress in human endothelial cells to simulate I/R injury in vitro was developed. Gene expression analysis was performed with microarrays undergoing cytoprotection with CAPE. The dose-dependent cytoprotection of CAPE has been related to its induction of heme oxygenase-1 (HO-1). With the aim of improving the beneficial effect of CAPE and understanding structure activity relationship, six new catechol ring-fluorinated CAPE derivatives were synthesized and evaluated in the menadione-endothelial cell model. The data suggest good cytoprotective effects of CAPE and some analogues and indicate important structural features for cytoprotection. Further investigation of the mechanism of cytoprotection showed that cytoprotection profiles of CAPE and derivatives correlate better to their ability to induce HO-1 in human endothelial cells than free radical scavenging activity. One CAPE derivative (FCAPE) with cytoprotective effects similar to CAPE in vitro exhibited better stability in rat plasma. A validated ultra-performance liquid chromatography/tandem mass spectrometric method was developed that allowed for quantification of CAPE and FCAPE in plasma samples. Pharmacokinetic studies in male Sprague Dawley rats following intravenous bolus administration of 5, 10, and 20 mg/kg CAPE and 20 mg/kg FCAPE were performed. The results indicate that dose proportionality for CAPE does not exist in the dose range studied. Although the elimination half life was found not to be significant different between CAPE and FCAPE, significant difference was observed between the total body clearance of FCAPE and CAPE which may due to the difference in volume of distribution.Item Dissecting the Molecular Basis of the Antiphospholipid Syndrome(2008-05-13) Ramesh, Sangeetha; Shaul, P. W.The antiphospholipid syndrome (APS) is an autoimmune disorder characterized by circulating antiphospholipid antibodies (aPL), thrombotic events, recurrent pregnancy loss, and increased risk of coronary artery disease. The endothelium is a critical direct target of aPL, which cause increases in adhesion molecule expression and procoagulant activity. However, the molecular mechanisms underlying aPL actions on endothelium are unknown. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) prevents leukocyte adhesion and thrombosis. In the present study we determined if aPL-induced alterations in endothelial cell phenotype are mediated by aPL actions on eNOS. Normal human IgG (NHIgG) and human IgG containing polyclonal aPL were obtained from healthy individuals and APS patients, respectively, and purified by protein G-sepharose chromatography. We found that aPL prevents VEGF-mediated attenuation of monocyte adhesion to cultured endothelial cells and this is reversed by an NO donor, indicating a role for eNOS antagonism. In contrast, NHIgG has no effect on adhesion. Whereas NHIgG does not alter eNOS activation, stimulation of eNOS by VEGF and other agonists is fully antagonized by aPL. In intact mice, NO-dependent, acetylcholine-induced increases in carotid vascular conductance are unchanged by NHIgG treatment but impaired following aPL, indicating that these processes are operative in vivo. Additional studies in cultured endothelial cells demonstrated that aPL attenuates eNOS activation by inhibiting Ser1179 phosphorylation. We further found that deprivation of the cell surface protein beta 2 glycoprotein I (beta 2GPI) from endothelial cells prevents aPL inhibition of eNOS, and that FC1, a monoclonal antibody against beta 2GPI, mimics the effects of aPL. Receptor-associated protein or RAP, an antagonist of the LDL receptor (LDLR) family, fully prevents aPL antagonism of eNOS in endothelial cells. Moreover, eNOS antagonism by aPL as indicated in vivo in carotid vascular conductance studies is absent in apoER2 -/- mice. Thus, aPL-induced changes in endothelial cell phenotype are mediated by eNOS antagonism, which is due to impaired Ser1179 phosphorylation and requires beta 2GPI and apoER2. eNOS antagonism through these mechanisms represent a novel, potentially critical proximal process in the pathogenesis of thrombosis and cardiovascular disease in APS.Item Intracellular pH regulation in microvascular endothelial cells(Texas Tech University, 2000-05) Rojas, Jose DMany advances in vascular biology are a result of the use of cultured endothelial cells as a model. This model system has shown that endothelial cells synthesize and secrete various substances involved in regulating blood flow. There can be considerable heterogeneity in the properties and function of endothelial cells. These heterogeneities are a result of the tissue of origin and whether they are from macro- or microcirculation. Nitric oxide, a vasoactive substance synthesized by endothelial cells, has been shown to activate other second messenger systems in both a calcium-dependent and –independent fashion. There is a substantial body of work on intracellular calcium [Ca^2+] regulation in endothelial cells. Similarly, it is known that intracellular pH (pH'") is important in signal transduction following hormonal stimulation, and in regulating many physiological processes including cell growth, secretion, contraction, and invasion/migration. Significant work has been done on pH'" regulation in macrovascular endothelial cells; however, there remains a large gap in the knowledge of pH'" regulation in microvascular endothelial cells. Since there have been reported differences in [Ca^2+ ] regulation and nitric oxide production in microvascular and macrovascular endothelial cells, one specific aim of this dissertation was to perform an in-depth study of pH^in regulation in microvascular endothelial cells from different vascular beds. Microvascular endothelial cells are intimately involved in the invasive process of angiogenesis and there have been reports of plasma membrane vacuolar-type ATPase (pmV-ATPase) in several invasive cell types. These observations led to the formulation of the hypothesis that pmV-ATPase is required for invasion and was tested in this study. To our knowledge, this study is the first reported observation of pmV-ATPase as a pH^10 regulatory mechanism in microvascular endothelial cells along with evidence that the mechanism is involved in the process of invasion. Diabetes affects the cardiovascular system with complications that include acidosis, abnormalities in blood flow regulation, and altered microvascular proliferation and angiogenesis. Part of this study examined pH'" regulation in microvascular endothelial cells from a model of spontaneous diabetes. As a result of this work we determined that diabetic cells have decreased pmV-ATPase activity. Along with decreased pmV-ATPase activity diabetic cells also exhibit decreased invasive Potential and angiogenesis. Pharmacologic inhibition of pmV-ATPase activity in normal cells renders them as non-invasive as diabetic cells and unable to form capillary-like structures in an angiogenesis model. Finally, the study examines endothelial cells from lung micro vasculature. This vascular bed is known to undergo extensive vascular remodeling with prolonged hypoxia. Since prolonged hypoxia results in acidosis, it was predicted that these cells would have a dynamic pH'" regulatory that allows them to cope with this situation. Moreover, because vascular remodeling is an invasive process, it was predicted that these endothelial cells would also express pmV-ATPase. In support of the hypothesis; immunocytochemical, fluorescence spectroscopy, and pharmacologic studies reveal pmV-ATPase as a major pH'" regulatory mechanism in these cells. In summary this work demonstrates the presence of pmV-ATPase in several microvascular beds and provides functional evidence for its role in the invasive process of vascular remodeling.Item Modeling the growth and dissolution of clots in flowing blood(Texas A&M University, 2006-10-30) Mohan, AnandMultiple interacting mechanisms control the formation and dissolution of clots to maintain blood in a state of delicate balance. In addition to a myriad of biochemical reactions, rheological factors also play a crucial role in modulating the response of blood to external stimuli. The broad stimuli for clot formation were laid out, more than a century ago, in, what is now referred to as, Virchow??????s triad. To date, a comprehensive model for clot formation and dissolution, that takes into account the biochemical, medical and rheological factors, has not been put into place, the existing models emphasizing either one or the other of the factors. In this dissertation, a model is developed for clot formation and dissolution that incorporates many of the relevant crucial factors that have a bearing on the problem. The model, though just a first step towards understanding a complex phenomenon goes further than previous models in integrating the biochemical, medical and rheological factors that come into play. The model is tested in some simple flow situations as part of an attempt to elucidate Virchow??????s triad. Extensions to the model, along with detailed numerical studies, will hopefully aid in a clearer understanding of the phenomenon, and in making relevant clinical correlations.Item Regulation of calcium stores in normal and diabetic endothelial cells(Texas Tech University, 2000-12) Sanka, Shankar ChittaranjanCytosolic Ca^^ ([Ca^^]*'^^) mediates many cellular ftinctions, e.g.. cell growth, motility, secretion, etc. In many cell types, ion transport processes appear to be dependent on metabolism of glucose for maximal activity. In certain cell types, a strict coupling between glycolysis and the acfivity of Endoplasmic Reticulum Ca^"-ATPases (SERCA). involved in regulating Ca^^ homeostasis, has been suggested. In diabetes, glucose homeostasis is altered. We hypothesize that Ca^^ homeostasis in microvascular endothelial cells from diabetic animals is altered due to a dysfunction of glycolysis coupling the activity of SERCA. We further hypothesize that endosomal/lysosomal (E/L) compartments exhibiting SERCA are involved in this dysfunction. Our data indicated that agonist stimulation (ATP, vasopressin, angiotensin-II)elicited [Ca^"]^^* increases (independent of extracellular Ca^^) that were larger in endothelial cells from diabetic than from normal animals. Simultaneous measurements of [Ca^^]'^^' and Ca^^ in E/L compartments ([Ca^^]^) using fluorescence spectroscopy, indicated that E/L compartments released Ca^^ following agonist-stimulation. The magnitude of the Ca'* release was significantly larger in microvascular endothelial cells from diabetic rats. SERCA inhibitors elicited Ca^^ releases from E/L compartments in both normal and diabetic models. The magnitude of the [Ca^^]^ release was however similar among normal and diabetic cells. Immunocytochemical experiments demonstrated that 60% of E/L compartments exhibited SERCA. These data indicate that (a) E/L compartments are important for Ca^^ homeostasis in microvascular endothelial cells from both normal and diabetic models; (b) Ca^^ regulation in E/L compartments is different in cells from a diabefic model, (c) the compartment involved in altered Ca'* homeostasis in diabetes is unknown.Item Regulation of inflammation: the role of the endothelial cells(Texas Tech University, 1999-08) Roberts, Erin DeniseThe endothelium plays an important role during the inflammatory response. Through the interaction with leukocytes, endothelial cells (EC) can be exposed to numerous pro-inflammatory mediators including peroxidases resulting in enhancement of the inflammatory process. In this study, EC were exposed to various peroxidases to determine their effects on cytokine secretion and respiratory burst (RB). Chemiluminescence was employed to determine the effects of various peroxidases on EC RB. Recombinant enzymatically inactive human myeloperoxidase (iMPO) and enzymatically inactive horseradish peroxidase(dHRP) increased RB. Enzymatically active porcine eosinophil peroxidase (pEPO) demonstrated a rapid increase in RB followed by no effect over the course of time. Other peroxidases tested had no effect on RB by EC. Recombinant enzymatically active human myeloperoxidase (MPO) and iMPO are derived from neutrophil degranulation and have been reported to augment cytokine secretion by macrophages. Endothelial cells exposed to iMPO in vitro exhibited dose-and time-dependent increases in interleukin-6 (IL-6), interleukin-8 (IL-8), and granulocyte-macrophage colony-stimulating factor (GM-CSF); however, MPO did not have a significant effect. A ribonuclease protection assay (RPA) indicated this enhancement by iMPO was due to an increase in mRNA levels. Also, EC were exposed to pEPO and an enzymatically inactive fragmented form of human eosinophil peroxidase; both pEPO and fEPO observed a slight decrease in IL-8 secretion by EC. Taken as a whole, these data provide further insight into the effects of peroxidases on the development of inflammation.