Indiana University-Purdue University Indianapolis (IUPUI) / Streptococcus mutans, an inhabitant of oral biofilm or dental plaque, adheres to the tooth surface via protein antigen I/II (PA I/II). Pathologic lesions of atherosclerosis (AT) and infective endocarditis (IE) harbor S. mutans. Serotypes f and k strains with collagen binding protein genes cbm and cnm are uncommon in the mouth, but these are the most prevalent S. mutans strains in AT and IE tissues and can invade endothelial cells (EC) in vitro. Tobacco use increases the risk for cardiovascular and oral diseases. Oral S. mutans encounter many substances including nicotine. Arginine is present in saliva and the EC glycocalyx that coats and protects ECs from shear forces of blood flow. Prior studies demonstrated arginine alters S. mutans biofilm. This work characterizes S. mutans serotype k strains and serotype c strains, the most prevalent in the mouth. The effects of nicotine and arginine on biofilm mass, metabolic activity and EC invasion were investigated. Biofilm production by serotypes c and k strains did not differ; there were no differences in responses to nicotine and arginine between these serotypes. Increased production of biofilm was associated with the cbm and cnm genes. Nicotine increased biofilm for all strains whereas arginine plus nicotine reduced bacteria and the extracellular polymeric substances. Previous EC invasion studies were performed with planktonic cultures of S. mutans; therefore, EC invasion by biofilm was evaluated. Significant factors for EC invasion by S. mutans are presence of the cbm gene and lack of PA I/II expression on the bacterial cell surface. Presence of the cnm gene increased EC invasion by biofilm but not planktonic cells. Planktonic cells of six strains invaded better than biofilm, whereas four strains showed increased invasion by biofilm cells. Neither nicotine nor arginine significantly altered the ability of S. mutans biofilm cells to invade ECs. Not all strains with cbm or cnm and no PA I/II expression invaded EC. A strain with PA I/II expression and without cbm and cnm genes invaded EC. While cbm, cnm and PA I/II expression are predictors of EC invasion, additional mechanisms for EC invasion by S. mutans remain to be revealed. / 2021-08-21

BACKGROUND: Protein disulfide isomerase A3 (PDIA3, also named GRP58, ER-60, ERp57) is conserved across species and mediates protein folding in the endoplasmic reticulum. PDIA3 is, reportedly, a chaperone for STAT3. However, the role of PDIA3 in regulating mitochondrial bioenergetics and STAT3 phosphorylation at serine 727 (S727) has not been described. METHODS: Mitochondrial respiration was compared in immortalized human cerebral microvascular cells (CMEC) wild type or null for PDIA3 and in whole organism C. Elegans WT or null for pdi-3 (worm homologue). Mitochondrial morphology and cell signaling pathways in PDIA3-/- and WT cells were assessed. PDIA3-/- cells were subjected to oxygen-glucose deprivation (OGD) to determine the effects of PDIA3 on cell survival after injury. RESULTS: We show that PDIA3 gene deletion using CRISPR-Cas9 in cultured CMECs leads to an increase in mitochondrial bioenergetic function. In C. elegans, gene deletion or RNAi knockdown of pdi-3 also increased respiratory rates, confirming a conserved role for this gene in regulating mitochondrial bioenergetics. The PDIA3-/- bioenergetic phenotype was reversed by overexpression of WT PDIA3 in cultured PDIA3-/- CMECs. PDIA3-/- and siRNA knockdown caused an increase in phosphorylation of the S727 residue of STAT3, which is known to promote mitochondrial bioenergetic function. Increased respiration in PDIA3-/- CMECs was reversed by a STAT3 inhibitor. In PDIA3-/- CMECs, mitochondrial membrane potential and reactive oxygen species production, but not mitochondrial mass, was increased, suggesting an increased mitochondrial bioenergetic capacity. Finally, PDIA3-/- CMECs were more resistant to oxygen-glucose deprivation, while STAT3 inhibition reduced the protective effect. CONCLUSIONS: We have discovered a novel role for PDIA3 in suppressing mitochondrial bioenergetic function by inhibiting STAT3 S727 phosphorylation.

yes / Interleukin-36 cytokines are predominantly expressed by epithelial cells. Significant upregulation of epidermal IL-36 is now a recognised characteristic of psoriatic skin inflammation. IL-36 is known to induce inflammatory responses in dendritic cells, fibroblasts and epithelial cells. Although vascular alterations are a hallmark of psoriatic lesions and dermal endothelial cells are well known to play a critical role in skin inflammation, the effects of IL-36 on endothelial cells are unexplored. We here show that endothelial cells including dermal microvascular cells express a functionally active IL-36 receptor. Adhesion molecules VCAM-1 and ICAM-1 are upregulated by IL-36γ stimulation and this is reversed by the presence of the endogenous IL-36 receptor antagonist. IL-36γ stimulated endothelial cells secrete the proinflammatory chemokines IL-8, CCL2 and CCL20. Chemotaxis assays showed increased migration of T cells following IL-36γ stimulation of endothelial cells. These results suggest a role for IL-36γ in the dermal vascular compartment and it is likely to enhance psoriatic skin inflammation by activating endothelial cells and promoting leukocyte recruitment.

Chronic inflammation is a complex immune response linked to several diseases. The first step in the inflammatory response is the recruitment of immune cells to the endothelium of the vascular wall. This process is mediated by E/P-Selectin, for which no antagonist efficiently interacts to limit the inflammatory response. Previous work identified transcription factor GATA5 as a key regulator of endothelial homeostasis and revealed an altered expression of inflammatory genes in human endothelial cells with loss of GATA5. The objective of my project is to understand the role of GATA5 in selectin-dependent vascular inflammation and to develop selectin inhibitors. I used biochemical, cellular and in vivo approaches to evaluate a series of novel small molecules for their ability to interfere with selectin binding to their ligand, PSGL-1. The work identified a new lead candidate, LCB 2248, for the development of new E/P-Selectin antagonists and contributed to the understanding of the role of GATA5 in cell recruitment and adhesion. The mechanistic insight gathered and the identification of an E/P-Selectin antagonist will hopefully pave the way for the development of effective treatments for patients with chronic inflammation.

Cardiovascular disease and the associated endothelial dysfunction are characterized by leukocyte activation, decrease endothelial nitric oxide synthase (eNOS) activity, and increased endothelial cell adhesion molecules expression. This leads to the release of myeloperoxidase (MPO) by activated neutrophils and monocytes. MPO is a peroxidase enzyme that plays an important role in innate immune host defense, however it has been shown to play a pathogenic role in cardiovascular disease, mainly by causing endothelial dysfunction. The molecular mechanisms through which MPO induces endothelial damage are not fully understood. Calpains are a family of calcium-dependent proteases. Two calpain isoforms, µ- and m-calpain, are expressed in the vascular wall, including endothelial cells. Activation of calpains has been recently implicated in inflammatory disorders of the vasculature. The goal of this study was to test the hypothesis of a role for calpains in the molecular mechanism(s) through which MPO causes endothelial dysfunction and vascular inflammation. To explore if MPO activates calpain and to identify the calpain isoform(s) involved, we first studied the effects of MPO treatment on calpain activity in mouse lung microvascular endothelial cells (MMVEC). MMVECs were stimulated with 10 nmol/L MPO for 60, 120, 180, and 240 min. Using a fluorescent calpain activity assay, we found that MPO time dependently activates calpain in endothelial cells, with peak activity reached within 180 min. Using immunoblot analysis techniques we demonstrated that the calpain isoform targeted by MPO is µ-calpain. Interestingly, using a biotin switch assay,10 nmol/L MPO appears to activate the µ-calpain isoform via denitrosylation of its C-terminus domain. Using MMVECs, we studied the effects of the MPO/µ-calpain signaling on endothelial dysfunction. MMVECs were stimulated with 10 nmol/L MPO for 180 min. Expression levels of Protein Phosphatase 2 (PP2A), total 5' AMP-activated protein kinase (AMPK), Thr172 phospho-AMPK, total endothelial nitric oxide synthase (eNOS),Ser1177 phospho-eNOS, total protein kinase B (AKT), Ser473 phospho AKT, Adiponectin receptor 1 (AdipoR1), and Adiponectin receptor 2 (AdipoR2), were measured by immunoblot analysis. Interestingly, MPO impaired Thr172AMPK, Ser1177eNOS, but not Ser 473 AKT phosphorylation in a calpain dependent manner. On the other hand, MPO significantly increased the expression levels of PP2A. Inhibition of PP2A with okadiak acid decreased the phosphorylation levels of AMPK, and eNOS, indicating that PP2A is a downstream target of the MPO/calpain system. MPO treatment significantly increased the expression of vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells. Pharmacological inhibition of calpain activity attenuated expression of VCAM-1. MPO also decreased protein levels of AdipoR1, and AdipoR2 in a calpain dependent manner. The treatment of MMVEC with adiponectin in the presence of MPO was not able to restore AdipoR2 expression levels. Using genetically modified mice, we found evidence of reduced leukocyte adhesion to the aortic endothelium in response to MPO in µ-calpain deficient mice, compared to wild-type mice . These effects appears to be attributed to the endothelial calpain, since incubating wild type aortas with calpain deficient leukocytes did not reduce leukocyte adhesion to the endothelium. The data in this study first establish a role for calpain in the endothelial dysfunction and vascular inflammation of MPO, with MPO activating the µ-calpain isoform via denitrosylation. Our data also report that increased calpain activity downregulats the expression of a number of signaling molecules important for endothelial cell function. This study may provide the MPO/calpain/PP2A signaling pathway as a novel pharmacological targets for the treatment of inflammation-driven vascular disorders. / Biomedical Sciences

活性氧在內皮細胞生理發展比如細胞生長增殖和病理中起到非常重要的作用。在病理條件下，活性氧在血管功能失調和重構起到關鍵作用。氧化應激現在被認為存在於多種形式的心血管疾病中。諸多證據表明著活性氧誘導的心血管系統中很多功能異常之前會伴隨有細胞內鈣離子濃度的上升。 / 在本論文的第一個部分，我比較了活性氧在大血管（主動脈）和小血管（腸系膜動脈）的內皮細胞裡引起的鈣應激的相似和差異之處。在這兩種細胞中，活性氧均可引起細胞內鈣離子濃度的上升。這種鈣離子濃度增加可被磷酸酯酶C (PLC) 的抑製劑U73122或者磷酸肌醇受體 (IP₃R) 抑製劑 （Xestospongin C， XeC）大幅度的減弱。此外，用過氧化氫預處理後的細胞會降低細胞對ATP的鈣應激反應。這種鈣應激反應的抑制可能是由於過氧化氫引發的鈣庫流失。令人關注的是，腸系膜動脈的內皮細胞對過氧化氫的作用更為敏感。次黃嘌呤 (hypoxanthine; HX) 加上黃嘌呤(xanthine; XO) 也能引起這兩種內皮細胞鈣離子濃度的上升，而這種鈣離子的增加源於超氧陰離子而不是氫氧離子。在腸系膜動脈的內皮細胞中，過氧化氫在此事件中起到的作用明顯比在主動脈細胞大。總之，過氧化氫可以引起大血管和小血管的內皮細胞裡磷酸酯酶C-磷酸肌醇受體依賴的鈣應激反應。而這種鈣應激後的鈣庫耗竭會對ATP引起的鈣應激起作用。綜上所述，小血管的內皮細胞的鈣應激比大血管的內皮細胞對過氧化氫更為敏感。 / 基於以上的結果，在第二部分的內容中，我們以培植的微血管內皮細胞系（H5V）為小血管內皮細胞的模型，研究了TRPM2通道在過氧化氫誘導的的鈣應激和凋亡中的作用。TRPM2是表達在動物是血管內皮組織中的氧化敏感的和陽離子無選擇性通道。我們開發了TRPM2通道的抑制性抗體 (TM2E3)，這種抗體可以結合到TRPM2通道的離子孔道的E3區域。對H5V細胞進行TM2E3的預處理後，可以降低細胞對過氧化氫刺激下的鈣離子的增加。用TRPM2特異的短發卡核糖核酸 （shRNA）也有同樣的抑制反應。我們用了3種方法來檢測過氧化氫誘導的細胞凋亡：四甲基偶氮唑盐（MTT）檢測，脫氧核糖核酸凋亡片段的檢測和4,6-联脒-2-苯基吲哚(DAPI) 核染色。基於以上的試驗結果，TM2E3 和TRPM2特異的shRNA都表現出了對過氧化氫引起的細胞凋亡的保護作用。相反，在細胞中過表達TRPM2會導致過氧化氫引起的鈣離子濃度上升的增加和細胞凋亡程度的加重。 這些發現強有力的證明了TRPM2 介導了過氧化氫引起的鈣離子濃度的上升和細胞凋亡。此外，我們還研究了TRPM2激活後的下游事件：半胱氨酸蛋白酶-3，-8和9是否參與到這個過程。我的數據表明過氧化氫誘導細胞凋亡是通過內源和外源通路導致半胱氨酸酶-3激活，而TRPM2在這個過程中起到了重要的決定作用。總括而言，TRPM2 介導了過氧化氫誘導的內皮細胞凋亡，下調內源性的TRPM2的表達會保護血管內皮細胞。 / Reactive Oxygen Species (ROS) play a key role in normal physiological processes such as cell proliferation and growth, as well as in pathological processes. Under pathological conditions ROS contribute to vascular dysfunction and remodeling through oxidative damage. Oxidative stress is now thought to underlie many cardiovascular diseases. Accumulating evidence also demonstrate that many ROS-induced functional abnormalities in the cardiovascular system are preceded by an elevation of intracellular Ca²⁺. / In the first part, I compared the Ca²⁺ responses to ROS between mouse endothelial cells derived from large-sized artey aortas (aortic ECs), and small-sized mesenteric arteries (MAECs). Application of hydrogen peroxide (H₂O₂) caused an increase in cytosolic Ca²⁺ levels ([Ca²⁺]i) in both cell types. The [Ca²⁺]i rises diminished in the presence of U73122, a phospholipase C inhibitor, or Xestospongin C (XeC), an inhibitor for inositol-1,4,5-trisphosphate (IP₃) receptors. In addition, treatment of endothelial cells with H₂O₂ reduced the Ca²⁺ responses to subsequent challenge of ATP. The decreased Ca²⁺ responses to ATP were resulted from a pre-depletion of intracellular Ca²⁺ stores by H₂O₂. Interestingly, we also found that Ca²⁺ store depletion was more sensitive to H₂O₂ treatment in endothelial cells derived from mesenteric arteries than those of derived from aortas. Hypoxanthine-xanthine oxidase (HX-XO) was also found to induce [Ca²⁺]i rises in both types of endothelial cells, the effect of which was mediated by superoxide anions and H₂O₂ but not by hydroxyl radicals. H₂O₂ made a greater contribution to HX-XO-induced [Ca²⁺]i rises in endothelial cells from mesenteric arteries than those from aortas. In summary, H₂O₂ could induce store Ca²⁺ release via phospholipase C-IP₃ pathway in endothelial cells. Emptying of intracellular Ca²⁺ stores contributed to the reduced Ca²⁺ responses to subsequent ATP challenge. Furthermore, the Ca²⁺ responses in endothelial cells of small-sized arteries were more sensitive to H₂O₂ than those of large-sized arteries. / In the second part, I used murine heart microvessel endothelial cell line H5V as a model of endothelial cells from small-sized arteries to investigate the role of Melastatin-like transient receptor potential channel 2 (TRPM2) channels in H₂O₂-induced Ca²⁺ responses and apoptosis. TRPM2 is an oxidant-sensitive cationic non-selective channel that is expressed in mammalian vascular endothelium. A TRPM2 blocking antibody channel (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the Ca²⁺ responses to H₂O₂. Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect in H₂O₂-induced Ca²⁺ responses. H₂O₂-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect on H₂O₂-induced apoptotic cell death. In contrast, overexpression of TRPM2 in H5V cells increased the Ca²⁺ responses to H₂O₂ and aggravated the apoptotic cell death in response to H₂O₂. These findings strongly suggest that the TRPM2 channel mediates Ca²⁺ overload in response to H₂O₂ and contributes to oxidant-induced apoptotic cell death in vascular endothelial cells. I also examined the downstream cascades of TRPM2 activation and explored whether caspase-3, -8 and -9 were involved in this process. My data indicates that H₂O₂-induced cell apoptosis through both intrinsic and extrinsic apoptotic pathways, leading to activation of caspases-3. Furthermore, TRPM2 played an essential role in the process. Together, my data suggest that TRPM2 mediates H₂O₂-induces endothelial cell death and that down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Sun, Lei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 101-114). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Declaration of Originality --- p.I / Abstract --- p.II / 論文摘要 --- p.IV / Acknowledgments --- p.VI / Abbreviations and Units --- p.VII / Table of Contents --- p.IX / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Reactive oxygen species and Reactive nitrogen species --- p.1 / Chapter 1.1.1 --- What is oxidative stress? --- p.1 / Chapter 1.1.2 --- Types of ROS --- p.2 / Chapter 1.1.2.1 --- Hydroxyl radical (*OH) --- p.2 / Chapter 1.1.2.2 --- Hydrogen peroxide (H₂O₂) --- p.3 / Chapter 1.1.2.3 --- Superoxide (O₂*⁻) --- p.4 / Chapter 1.1.2.4 --- Nitric oxide (NO) --- p.5 / Chapter 1.1.3 --- ROS-producing systems --- p.6 / Chapter 1.1.3.1 --- NAD(P)H oxidase --- p.6 / Chapter 1.1.3.2 --- Xanthine oxidase (XO) --- p.7 / Chapter 1.1.3.3 --- The mitochondrial respiratory chain --- p.8 / Chapter 1.1.3.4 --- Uncoupled endothelial NO synthase --- p.8 / Chapter 1.1.4 --- Antioxidant defense mechanisms in the cardiovascular systems --- p.9 / Chapter 1.1.4.1 --- SOD --- p.9 / Chapter 1.1.4.2 --- Catalase --- p.10 / Chapter 1.1.4.3 --- Glutathione peroxidase (GPx) --- p.10 / Chapter 1.1.4.4 --- Small molecules --- p.11 / Chapter 1.1.5 --- Role of oxidative stress in human diseases --- p.12 / Chapter 1.1.6 --- Endothelium dysfunction in oxidative stress-relating human diseases --- p.12 / Chapter 1.1.7 --- Role of Ca²⁺ in oxidative stress-relating human diseases --- p.14 / Chapter 1.1.8 --- Differential effects of ROS on endothelial calcium signaling --- p.15 / Chapter 1.1.8.1 --- Multiple Oxidative Stress-induced Ca²⁺ signaling pathway --- p.16 / Chapter 1.1.9 --- Effects of ROS on Agonist-induced endothelial calcium signaling --- p.19 / Chapter 1.1.10 --- Role of H₂O₂ as EDHF --- p.20 / Chapter 1.1.11 --- Differential effect of ROS on cells derived from large-sized and small-sized artries --- p.21 / Chapter 1.2 --- The transient receptor potential (TRP) Channels --- p.21 / Chapter 1.2.1 --- TRP Channel structure --- p.22 / Chapter 1.2.2 --- TRP Channel function --- p.23 / Chapter 1.2.3 --- TRPM subfamily --- p.23 / Chapter 1.2.3.1 --- TRPM2 Property and Structure --- p.24 / Chapter 1.2.3.2 --- TRPM2 Expression --- p.25 / Chapter 1.2.3.3 --- TRPM2 Activator --- p.25 / Chapter 1.2.3.4 --- TRPM2 Physiological and pathophysiological function --- p.28 / Chapter Chapter 2 --- Objectives of the Present Study --- p.35 / Chapter Chapter 3 --- Materials and methods --- p.37 / Chapter 3.1 --- Ethics statement --- p.37 / Chapter 3.2 --- Materials --- p.37 / Chapter 3.3 --- Methods --- p.38 / Chapter 3.3.1 --- Cell culture --- p.38 / Chapter 3.3.1.1 --- Primary Cell Culture --- p.38 / Chapter 3.3.1.2 --- H5V endothelial cell line --- p.39 / Chapter 3.3.1.3 --- Human embryonic kidney 293 (HEK293) cells --- p.39 / Chapter 3.3.4. --- TRPM2-specific shRNA, TRPM2 and transfection --- p.39 / Chapter 3.3.5 --- Western blotting --- p.40 / Chapter 3.3.6 --- [Ca²⁺]i Studies --- p.43 / Chapter 3.3.6.1 --- Fluo-4/AM- Measuring intracellular [Ca²⁺]i --- p.43 / Chapter 3.3.6.2 --- Fura-2/AM-Measuring intracellular [Ca²⁺]i --- p.44 / Chapter 3.3.6.3 --- Mag-fluo-4-Measuring Ca²⁺ Content in Intracellular Ca²⁺ Stores --- p.45 / Chapter 3.3.7 --- IP₃ measurement --- p.45 / Chapter 3.3.8 --- Electrophysiology --- p.46 / Chapter 3.3.9 --- TRPM2 blocking antibody (TM2E3) and Pre-immune IgG Generation --- p.46 / Chapter 3.3.10 --- DNA fragmentation assay --- p.47 / Chapter 3.3.11 --- DAPI Staining --- p.48 / Chapter 3.3.12 --- MTT assay --- p.48 / Chapter 3.3.13 --- Statistical analysis --- p.49 / Chapter Chapter 4 --- Effect of Hydrogen Peroxide and Superoxide Anions on Cytosolic Ca²⁺: Comparison of Endothelial Cells from Large-sized and Small-sized Arteries --- p.50 / Chapter 4.1 --- Introduction --- p.50 / Chapter 4.2 --- Materials and methods --- p.52 / Chapter 4.2.1 --- Primary Cell Culture --- p.52 / Chapter 4.2.2 --- [Ca²⁺]i Measurement --- p.52 / Chapter 4.2.3 --- Measuring Ca²⁺ Content in Intracellular Ca²⁺ Stores --- p.52 / Chapter 4.2.4 --- IP₃ measurement --- p.53 / Chapter 4.2.5 --- Data Analysis --- p.53 / Chapter 4.3 --- Results --- p.53 / Chapter 4.3.1 --- Both Ca²⁺ entry and store Ca²⁺ release contributed to H₂O₂-induced [Ca²⁺]i rises.. --- p.53 / Chapter 4.3.2 --- H₂O₂ enhanced IP₃ production and store Ca²⁺ release --- p.54 / Chapter 4.3.3 --- H₂O₂ reduced the Ca²⁺ responses to ATP in a H₂O₂ concentration and incubation time dependent manner --- p.54 / Chapter 4.3.4 --- H₂O₂ induced Ca²⁺ store depletion --- p.55 / Chapter 4.3.5 --- Ca²⁺ responses to ATP in the absence of H₂O₂ --- p.56 / Chapter 4.3.6 --- Non-involvement of hydroxyl radical --- p.56 / Chapter 4.3.7 --- HX-XO-induced [Ca²⁺]i rises were caused by superoxide anion and hydrogen peroxide --- p.56 / Chapter 4.4 --- Discussion --- p.68 / Chapter Chapter 5 --- Role of TRPM2 in H₂O₂-induced cell apoptosis in endothelial cells --- p.72 / Chapter 5.1 --- Introduction --- p.72 / Chapter 5.2 --- Materials and Methods --- p.73 / Chapter 5.2.1 --- Cell Culture --- p.74 / Chapter 5.2.2 --- [Ca²⁺]i measurement --- p.74 / Chapter 5.2.3 --- DNA fragmentation assay --- p.74 / Chapter 5.2.4 --- MTT assay --- p.74 / Chapter 5.2.5 --- TRPM2-specific shRNA, TRPM2 and transfection --- p.75 / Chapter 5.2.6 --- Electrophysiology --- p.75 / Chapter 5.2.7 --- Western blotting --- p.75 / Chapter 5.2.8 --- DAPI Staining --- p.76 / Chapter 5.2.9 --- Data analysis --- p.76 / Chapter 5.3 --- Results --- p.76 / Chapter 5.3.1 --- Involvement of TRPM2 channels in H₂O₂-induced Ca²⁺ influx in H5V cells --- p.76 / Chapter 5.3.2 --- Involvement of TRPM2 channels in H₂O₂-elicited whole-cell current change in H5V cells --- p.77 / Chapter 5.3.3 --- Role of TRPM2 channels in H₂O₂-induced apoptotic cell death in H5V cells --- p.78 / Chapter 5.3.4 --- Involvement of caspases in H₂O₂-induced apoptotic cell death --- p.79 / Chapter 5.3.5 --- Involvement of TRPM2 in TNF-α-induced cell death in H5V cells --- p.79 / Chapter 5.3 --- Discussion --- p.90 / Chapter Chapter 6 --- General Conclusions, Disscussion and Future work --- p.94 / Chapter 6.1 --- General Conclusions --- p.94 / Chapter 6.2 --- Discussion --- p.95 / Chapter 6.2.1. --- Comparative study --- p.95 / Chapter 6.2.2. --- IP₃ receptor (IP₃R) --- p.95 / Chapter 6.2.3. --- TM2E3-Specific blocking antibody of TRPM2 --- p.95 / Chapter 6.2.4. --- Pathological effect of H₂O₂ at high concentration --- p.96 / Chapter 6.2.5 --- Non-change on Basal [Ca²⁺]i --- p.97 / Chapter 6.3. --- Future work --- p.98 / References --- p.101

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed on June 24, 2009). Includes bibliographical references (p. 99-116).

REGENERATIVE MEDICINE: VASCULARIZED SKELETAL MUSCLE Tissue engineering is a compelling strategy to create replacement tissues and in this study, skeletal muscle. One major hurdle in the field is how to vascularize large tissue-engineered constructs exceeding the nutrient delivery capability of diffusion. Endothelial colony forming cells and mesenchymal progenitor cells form blood vessels de novo and were co-injected with satellite cells in Matrigel, an extracellular matrix, or PuraMatrix, a synthetic hydrogel. Our approach focused on the ability of bioengineered vascular networks to induce murine and human satellite cells to differentiate and form organized skeletal muscle when injected. We found that perfused human blood vessels were formed in both Matrigel and PuraMatrix and that murine satellite cells differentiated and formed organized myotubes with striations, indicative of adult skeletal muscle. Mesenchymal progenitor cells also induced differentiation of satellite cells in vitro. Human Satellite cells, however, did not show signs of differentiation in either Matrigel or Puramatrix. These data have provided a proof of concept of engineering vascularized skeletal muscle using murine satellite cells. INDUCTION OF CARDIOMYOGENESIS The heart's regenerative capabilities are not robust enough to repair the amount of damaged tissue from myocardial infarction. A novel approach to relieve the ischemia is to deliver cells with vasculogenic ability, endothelial colony forming cells and mesenchymal progenitor cells, to assemble de novo blood vessels and support recovery of cardiomyocytes. In our study, we used an in vitro transwell system that prevent cell contact, but allow diffusion of soluble factors to investigate if endothelial colony forming cells or mesenchymal progenitor cells secrete factors that induce cardiomyogenesis. We found that neonatal rat cardiomyocyte proliferation is enhanced in the presence of endothelial colony forming cells and mesenchymal progenitor cells; however, presence of these cells without fetal bovine serum is not sufficient to initiate cardiomyogenesis. PERSONALIZED THERAPY FOR RENAL CELL CARCINOMA TESTING IN AN ENDOTHEIAL CELL MODEL Sunitinib and Pazopanib are both tyrosine kinase inhibitors with high specificity for vascular endothelial growth factor receptor 2 and are used in the treatment of Renal Cell Carcinoma to inhibit angiogenesis. Recent clinical findings suggest that a subset of the population with a single nucleotide polymorphism in vascular endothelial growth factor receptor 2 respond better to Pazopanib treatment. We used a standard in vitro angiogenesis assay, endothelial cell proliferation, to test the effects of the single nucleotide polymorphism on responsiveness to Sunitinib and Pazopanib. We found that cells containing the polymorphism are more sensitive to Pazopanib than Sunitinib, confirming the clinical finding. We also analyzed the inhibition of phosphorylated vascular endothelial growth factor receptor 2 and confirmed drug activity on the phosphorylated protein. These findings could have personalized clinical implications for the 3% of the population with the polymorphism.

O hormônio melatonina produzido pela glândula pineal no período de escuro, participa na regulação circadiana de processos, fisiológicos e fisiopatológicos envolvendo vasos sanguíneos. Alguns destes estudos sugerem que as células endoteliais, que revestem os vasos sanguíneos são alvo para a melatonina circulante e medeiam a regulação do tônus vascular, em condições fisiológicas, e da interação neutrófilo-endotélio, em resposta a um estímulo injuriante. O óxido nítrico produzido pelas células endoteliais é um dos responsáveis por grande parte dos eventos vasculares, e a melatonina inibe a produção de óxido nítrico em diversos modelos. O objetivo deste estudo foi verificar o efeito da melatonina na produção de óxido nítrico induzido por bradicinina em células endoteliais em cultura. Para tanto, utilizamos uma técnica de cultura primária de células endoteliais de rato e através de um marcador fluorescente de óxido nítrico intracelular, mensuramos a fluorescência em microscópio confocal. Foi verificado que a melatonina e seu precursor N-acetilserotonina inibem a produção de óxido nítrico induzido por bradicinina e este efeito não ocorre pela inibição do aumento de cálcio que induz a produção de óxido nítrico. O análogo de receptores MT2 (4P-PDOT) e MT3 (5-MCA-NAT) não provocaram qualquer alteração sobre o aumento de óxido nítrico induzido por bradicinina, e a utilização do antagonista de receptores MT1 e MT2 (luzindol) não reverteu o efeito inibitório da melatonina. Portanto, nossos dados indicam que o efeito da melatonina sobre a atividade da NOS constitutiva não é mediado por receptores de membrana. Considerando que a melatonina é capaz de ligar-se à calmodulina, inibindo desta maneira a atividade da NOS endotelial constitutiva, poderíamos sugerir que este seria o mecanismo de ação. No entanto, é preciso ressaltar que tal atividade não é comprovada para a N-acetilserotonina, assim, apesar de ser este um possível mecanismo de ação, há a necessidade de demonstrar que a N-acetilserotonina está se ligando a calmodulina extraída de células endoteliais. Em resumo, neste trabalho mostramos que a melatonina em concentrações compatíveis com o pico noturno encontrado na circulação, pode modular eventos vasculares no organismo, através da inibição da produção de óxido nítrico em células endoteliais induzida por bradicinina. / Melatonin, the hormone synthesized by the pineal gland at night, signalizes darkness and modulates, in a circadian basis, blood vessels activity. Previous studies suggest that endothelial cells are the target for circulating melatonin and mediate changes in vascular tone and leukocyte-endothelial adherence properties. Melatonin effects can be mediated by several pathways, such as G protein-coupled receptors (MT1 and MT2 receptors), a putative membrane receptor, most probably an enzyme-binding site (MT3 receptor), and several intracellular mechanisms, including calmodulin binding and inhibition of constitutive and induced nitric oxide synthase. The aim of the present study was to characterize melatonin effect on the production of nitric oxide by bradykinin-stimulated endothelial cells in culture. Nitric oxide production was measured in real time at cellular level by detecting fluorescent stimulation of the probe DAF by confocal microscopy. After determining the ideal conditions for recording cumulative dose-response curves for bradykinin (1 – 100 nM) the effect of pre-incubated (1 min) melatonin and analogs was evaluated. Melatonin and its precursor, N-acetylserotonin, but not the selective ligands for receptors MT2 (4P-PDOT) and MT3 (5-MCA-NAT) receptors inhibited bradykinin-stimulated nitric oxide production. This effect was not blocked by the classical antagonist of MT1 and MT2 receptors, luzindol; excluding therefore the participation of membrane receptors. Taking into account that melatonin inhibits calmodulin activation of several enzymes, including constitutive nitric oxide synthase in brain and cerebellum, it could be suggested a similar mechanism for endothelial cells. However, this hypothesis is discussed taking into account that N-acetylserotonin was shown to do not bind neural cells calmodulin. In addition, here we discuss the relevance of the present finding according to physiological and physiopathological roles of endothelial nitridergic system. This analysis point melatonin modulation of constitutive nitric oxide synthase activity as a putative mechanism for explaining melatonin control of vascular tone.

A melatonina é produzida pela glândula pineal somente durante o escuro e atinge rapidamente a circulação, além disso, outros tecidos e células são capazes de produzir melatonina. As células endoteliais, devido a sua localização, são excelentes alvos para as ações da melatonina. O entendimento dos mecanismos de ação pelos quais a melatonina desenvolve seus efeitos sobre as células endoteliais, possibilitaria o uso desta indolamina e de seus análogos como uma importante ferramenta farmacológica. No presente trabalho, demonstramos que a melatonina em concentrações compatíveis com as encontradas na circulação durante o pico noturno de produção pela pineal, atua sobre as células endoteliais inibindo a produção de NO proveniente da enzima constitutiva (eNOS), enquanto altas concentrações de melatonina, que podem ser atingidas por exemplo pela produção por células imunocompetentes ativadas, inibem a produção induzida de NO mediada pela iNOS. A melatonina (1 nM) inibe a produção constitutiva de NO induzida por agonistas que atuam através da ativação de receptores acoplados à proteína G (histamina, carbacol e ATP/P2Y), e este efeito deve-se à inibição do aumento de [Ca2+]i por liberação de estoques intracelulares, sendo independente da ativação de receptores de melatonina. A melatonina inibe os efeitos decorrentes da produção de NO induzida por bradicinina como a produção de GMPc por células endoteliais e a vasodilatação de arteríolas \"in vivo\". A melatonina inibe a produção de NO induzida por LPS também de maneira independente da ativação de seus receptores, porém, em concentrações muito maiores (1-10 µM) do que a necessária para inibir a produção constitutiva. Estes efeitos devem-se à inibição da expressão da enzima iNOS por impedir a translocação do NF-kB ao núcleo. A vasodilatação de aortas induzida por LPS também é inibida por melatonina. Podemos concluir até o momento que as células endoteliais, devido a sua localização, são excelentes sensores para as ações da melatonina e podem auxiliar no melhor entendimento do conceito \"eixo imune-pineal\". Os estudos sobre os mecanismos pelos quais a melatonina atua em condições fisiológicas e fisiopatológicas são essenciais para se conhecer o potencial terapêutico da melatonina. / Melatonin, the darkness hormone, produced at night by the pineal gland, is also synthesized in a non-rhythmic manner by other cells. Pineal and extra-pineal melatonin reaches endothelial layer, and the understanding of its mechanism of action will improve the possibilities of using this indolamine and derivates as pharmacological tools. Here we showed that melatonin, in concentrations compatible to nocturnal melatonin surge impairs the activity of eNOS, while much higher concentrations, which can be attained by activated immune competent cells, impair the induction of iNOS synthesis. As a consequence of inhibiting eNOS we showed that melatonin inhibits vasodilation of the microcirculation induced by bradykinin. The inhibitory effect of melatonin is observed only when eNOS is activated by triggering G protein-coupled receptors (bradykinin B2, muscarinic and P2Y purine receptors). Activation of eNOS by calcium-channel operated receptors (P2X) is not blocked by melatonin. Inhibition of the transcription of iNOS results in inhibition of the LPS-induced vasodilation of rat aorta. As a matter of fact, here we show that LPS effect is dependent on the endothelial layer. The mechanism of action of melatonin in inhibiting iNOS transcription is due to block of the NF-kB pathway. Our work contributed to unravel the role of endothelium cells as targets for melatonin and as a key player in the \"immune-pineal axis\". The understanding of the concentrations ranges reached by endogenous production, i.e., the discrimination between the levels achieved during physiological and physiopathological responses, are essential for using these substances as analogous therapeutical tools.