Piperine, a pungent alkaloid found in the fruits of long and black pepper plants, has diverse physiological effects, including anti-inflammatory and anti-cancer activities. The effect of piperine on the function of T cells and endothelial cells, two important elements of inflammation, have not been examined previously and were the focus of this study. Piperine inhibited the proliferation of human endothelial cells, murine T cells, and IL-2-dependent CTLL-2 T cells, without affecting cell viability. Progression into the S phase of the cell cycle was inhibited in all three cell types. In T cells, piperine inhibited expression of the early activation marker CD25, production of IFN-?, IL-2, IL-4, and IL-17A, and the generation of cytotoxic effector cells. In endothelial cells, piperine inhibited migration and tubule formation in vitro and ex vivo, as well as breast cancer cell-induced angiogenesis in chick embryos. Piperine inhibited Akt phosphorylation in signaling pathways associated with growth factor receptors on endothelial cells, T cell receptor and CD28 on T cells, and IL-2 receptor on CTLL-2 cells. Additionally, piperine inhibited ERK1/2 and I?B phosphorylation in activated T cells, as well as STAT3, STAT5, and ERK1/2 phosphorylation in IL-2-stimulated CTLL-2 cells. However, piperine is not a broad-spectrum inhibitor of phosphorylation as it did not inhibit ZAP-70 phosphorylation in activated T cells or phosphorylation of JAK1 and JAK3 in IL-2-stimulated CTLL-2 cells. Piperine-mediated inhibition of T cell activation and IL-2 receptor signaling suppresses T cell proliferation and effector cell differentiation, suggesting possible utility in treating T cell-mediated autoimmune and chronic inflammatory conditions. Additionally, the potent anti-angiogenic activity of piperine warrants further study for the prevention of inflammation- and cancer-promoting angiogenesis.

Lysophosphatidylcholine (LPC) is a by product of phospholipid metabolism, that under physiological conditions is maintained at a low level. However, through an enhanced degradation of phospholipids and/or a reduced catabolism, LPC accumulates in the plasma and fluids of patients with disorders underscored by inflammation - such as atherosclerosis, diabetes, ischaemia and epilepsy. Previous studies have demonstrated LPC to possess vasoactive properties, able to both induce and inhibit vasodilation. Furthermore, a variety of proteins are sensitive to LPC, including non-selective cation (NSC) channels and Ca2+-activated K+ (KCa) channels. These channels are intimately associated with the maintenance and regulation of vascular tone. The aim of this study was to elucidate the mechanisms underlying the vascular effect of LPC.Aortic segments were constricted with phenylephrine and exposed to cumulative concentrations of LPC, with an ensuing endothelium-dependent, concentration-dependent vasodilation. Inhibitors of nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) abolished LPC-induced responses, implicating nitric oxide (NO) as the mediator. Two cation fluxes were implicated in the dilator activity of LPC - Ca2+ and K+. NSC channel antagonists and reduced extracellular Ca2+ concentration attenuated dilation and reduced the Ca2+ signal activated in isolated rat aortic endothelial cells (RAEC) by LPC, implicating endothelial Ca2+ influx in the response. In addition, LPC also evoked a robust hyperpolarisation of isolated RAEC membrane potential. The K+ channel antagonists TEA+, TRAM-34 and apamin, inhibitors of KCa channels, attenuated both the LPC-induced dilation and RAEC membrane hyperpolarisation, highlighting their potential role in mediating both these processes. HEK293 cells, which lack many of the channels and signalling pathways possessed by other cells, mimicked RAEC in their sensitivity to LPC, generating robust elevations of intracellular Ca2+ when exposed to this lysolipid. Likewise, membrane hyperpolarisations were also observed in HEK293 cells, however, these only occurred when cells expressed recombinant KCa channels. This suggests that KCa channel activation is dependent upon Ca2+ influx, not vice versa. Phospholipase C (PLC) inhibitor U73122, attenuated LPC-induced hyperpolarisation, raising the question as to the possible involvement of G-protein coupled receptors in the bioactivity of LPC. Alternately, LPC might initiate PLC activity, and subsequent NSC channel opening and Ca2+ influx via a perturbation of membrane integrity, like certain local anaesthetics. It is proposed that endothelial NSC-channel activation by LPC initiates endothelial cell signalling, with concomitant activation of Ca2+-sensitive proteins such as NOS, to bring about vasodilation, and KCa channels, which modulate membrane potential and in turn the driving force for Ca2+ entry.

Orientador: Sara Teresinha Olalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T20:55:53Z (GMT). No. of bitstreams: 1 Lazarini_Mariana_D.pdf: 4089908 bytes, checksum: bf5ee8f74ac7dfe4c18c3e84bdeeecff (MD5) Previous issue date: 2010 / Resumo: Importante passo para a compreensão dos processos fisiopatológicos das neoplasias é a identificação de genes diferencialmente expressos e das funções biológicas de cada proteína codificada por estes genes. Rho GTPase activating protein 21 (ARHGAP21) é uma proteína pertencente à família RhoGAP, que interage com ARF-GTPases e com alfa-catenina, modulando a dinâmica da actina associada às membranas do Golgi e à integridade das junções aderentes. O objetivo geral do presente estudo foi investigar as funções da proteína ARHGAP21 em células endoteliais e de adenocarcinoma de próstata com relação à viabilidade celular, ciclo celular, migração, adesão e expressão gênica. Neste trabalho foi demonstrado que ARHGAP21 possui atividade RhoGAP para RhoA e RhoC. Em células HUVECs (Human Umbilical Vein Endothelial Cells), foi observada uma localização nuclear e citoplasmática de ARHGAP21 e sua depleção induziu alterações no ciclo celular. Além disso, ensaios de microarranjos de DNA em HUVECs demonstraram modulação de genes como PAI-1, BNIP3, staniocalcina 1 e podocalyxin. Em linhagens celulares de adenocarcinoma de próstata (LNCaP e PC-3), também foi observada uma localização nuclear e citoplasmática de ARHGAP21. A diminuição da expressão desta proteína em células PC-3 resultou em redução da viabilidade e da migração celular em fibronectina. Com relação à viabilidade celular, a inibição de ARHGAP21 tem efeito adicional ao agente quimioterápico cisplatina. Em células LNCaP, por sua vez, foi observada uma menor adesão em matrigel e fibronectina das células submetidas à inibição da expressão de ARHGAP21, em comparação às células controle. Experimentos de microarranjos de DNA e RT-PCR quantitativo em tempo real em células de adenocarcinoma de próstata submetidas à inibição de ARHGAP21 demonstraram expressão alterada dos genes TGF-beta induced e dos genes BNIP3, staniocalcina 1 e podocalyxin, que também foram modulados nas células HUVECs com inibição da expressão de ARHGAP21. Em conclusão, o presente estudo identificou ARHGAP21 como uma proteína com importantes funções em células endoteliais e de adenocarcinoma de próstata, através da regulação da viabilidade, adesão e migração celular. Os achados aqui descritos sugerem que ARHGAP21 pode ser uma molécula alvo para a terapia de neoplasias, provavelmente através da sua função como reguladora da atividade de RhoA e RhoC / Abstract: One step in the path towards building a comprehensive molecular portrait of human cancer is the definition of differentially expressed genes and the function of their coding proteins. Rho GTPase activating protein 21 (ARHGAP21) is a RhoGAP protein, which interacts with ARF-GTPases and alpha-catenin, controlling actin dynamics on Golgi membranes and the integrity of adherens junctions, respectively. The aim of the present study was to investigate ARHGAP21 functions on endothelial and prostate cancer cells regarding to cell viability, cell cycle, migration, adhesion and gene expression. This study demonstrated that ARHGAP21 has RhoGAP activity for RhoA and RhoC. ARHGAP21 localized in the nucleus and cytoplasm of HUVECs (Human Umbilical Vein Endothelial Cells) and its depletion alteres the cell cycle phases. Furthermore, microarrays assays in HUVECs ARHGAP21 knockdown demonstrated modulation of genes such as PAI-1, BNIP3, stanniocalcin 1 and podocalyxin. In the prostate adenocarcinoma cell lines (LNCaP and PC-3), ARHGAP21 is also located in the nucleus and cytoplasm. Depletion of this protein in PC-3 cells resulted in decrease of cell viability and migration in fibronectin. Regarding to cell viability, inhibition of ARHGAP21 has an additional effect on cisplatin chemotherapeutic agent. In LNCaP cells, a lower adhesion was observed in matrigel and fibronectin of cells subjected to inhibition of ARHGAP21 expression compared to control cells. DNA microarray and quantitative RT-PCR experiments on prostate adenocarcinoma cells ARHGAP21 knockdown showed modulation of TGF-beta induced gene expression. The expression of BNIP3, stanniocalcin 1 and podocalyxin was also altered in HUVECs cells ARHGAP21 knockdown. In conclusion, this study identified ARHGAP21 as a protein with important functions in endothelial cells and prostate adenocarcinoma, by regulating cell viability, adhesion and migration. The findings described here suggest that ARHGAP21 may be a molecule target for cancer therapy, probably due to its GAP activity for RhoA and RhoC / Doutorado / Doutor em Fisiopatologia Medica

Disruption of the blood-brain barrier (BBB) is hypothesized to play an important role in the disease biology of schizophrenia (SZ). Brain microvascular endothelial cells (BMECs) have paracellular and transcellular proteins, transporters, as well as important extracellular matrix proteins, which collectively contribute to maintaining proper BBB function. While previous studies have provided some insights into the role of the BBB in SZ pathophysiology, there is a significant gap in our understanding of the cellular-molecular underpinnings of its major component, BMECs. Human induced pluripotent stem cells (hiPSCs) provide an exciting new avenue for exploring the role of BMECs in SZ. We hypothesize that BMECs have intrinsic deficits that lead to BBB dysfunction in SZ. In this study, we first aimed to test whether the existing hiPSC-derived BMEC protocols work with our patient-specific hiPSC samples. Secondly, we sought to investigate any potential deficits between BMECs derived from healthy control (HC) and SZ subjects. We successfully adapted the established protocol and confirmed the identity of these hiPSC-derived BMECs with relevant cell markers such as CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. We also evaluate barrier function by measuring trans-endothelial electrical resistance (TEER) and efflux transporters activity of ABCB1 and ABCC1. We observed evidence of poor cellular adhesion and disrupted tight junctions in a subset of SZ hiPSC-derived BMECs, where approximately 70% of them demonstrated extensive BBB disruption (reduced TEER). These findings suggest that there may be cell-autonomous disease-specific deficits in BMECs in SZ that result in BBB dysfunction. / 2022-06-07T00:00:00Z

Objective: We proposed that women who develop preeclampsia have a low ratio of 'protective' toxicity preventing activity (TxPA) to 'toxic' very low density lipoproteins (VLDL) late in pregnancy. Having confirmed this hypothesis, we then tested whether this low ratio would manifest itself early in the pregnancy of women who develop preeclampsia. Methods: Serially collected plasma from women who developed preeclampsia and from matched controls was assayed blind for TxPA, triglycerides, cholesterol, high-density lipoproteins, albumin, and nonesterified fatty acids (NEFA). Main Outcome Measures: Plasma concentrations of lipids, NEFA, and proteins which bind NEFA (TxPA and albumin) were measured in normal and preeclamptic women. These parameters were formulated prior to data collection because of the low albumin/triglyceride' ratios and the elevated NEFA levels reported to occur in preeclampsia. Results: In late pregnancy, TxPA was lower (1.82 ± 0.63 vs. 2.30 ± 0.40 g/dL, P = 0.008) and VLDL higher (292 ± 130 vs. 206 ± 60 mg/dL, P = 0.013) in preeclamptics than in controls. Discrimination analysis (TxPA and triglyceride), correctly classified 95% of the preeclamptics and 79% of the controls in late pregnancy. The ratio of TxPA to non-TxPA and triglyceride correctly classified 92% of the preeclamptics and 85% of the controls in early pregnancy. Conclusions: The ratio of TxPA to VLDL accurately distinguishes preeclamptic from normal pregnant women, suggesting that both these factors are involved in the development of preeclampsia.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20562号 / 医博第4247号 / 新制||医||1022(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 江藤 浩之, 教授 開 祐司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21003号 / 医博第4349号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 木村 剛, 教授 小西 靖彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Chapter 1: Molecular Mechanism of Mitochondrial Fragmentation and Glucose Metabolism under Disturbed Flow in Endothelial Cells: Focus on the Role of Dynamin-Related Protein 1. The luminal surface of the endothelium is continually exposed to dynamic blood flow patterns that is known to alter immunometabolic phenotypes of the endothelial cells (ECs). Recent literature reported that inhibition of the metabolic reprogramming to glycolysis or enhancement of oxidative phosphorylation (OXPHOS) is considered as an effective strategy to prevent EC proinflammatory activation and eventually the progression of vascular diseases. Endothelial mitochondria are highly dynamic organelles playing versatile roles in maintaining endothelial cell homeostasis working as bioenergetic, biosynthetic, and signaling organelles. The balance between fusion and fission processes modulates mitochondrial network, which is essential for maintaining mitochondrial homeostasis. Disruption of the orchestrated balance, especially toward excessive fission resulting in fragmented and dysfunctional mitochondria, has been shown to be associated with atheroprone phenotypes of ECs. However, there is a key knowledge gap with respect to morphology of EC mitochondria under different flow conditions and its role on EC immunometabolic phenotypes.In chapter 1, the purpose of this study was to investigate the effect of different flow patterns on mitochondrial morphology in ECs and its implication in immunometabolic endothelial phenotype. The overarching hypothesis of the Chapter 1 was that disturbed flow (DF) will increase mitochondrial fragmentation, which will facilitate glycolysis and inflammatory activation in ECs. In the study, mitochondrial morphology was analyzed in ECs at multiple segments of the aorta and arteries in EC-specific photo-activatable mitochondria (EC-PhAM) mice. Increased mitochondrial fragmentation was observed at atheroprone regions (e.g., lesser curvature of the aortic arch, LC) with increased dynamin-related protein 1 (Drp1) activity, compared with the atheroprotective regions (e.g., thoracic aorta, TA). The atheroprone regions also showed a higher level of endothelial activation and glycolysis. Carotid artery partial ligation surgery, as a surgical model of DF, significantly induced mitochondrial fragmentation with elevated Drp1 activity and increased EC activation. in vitro experiments recapitulated in vivo observations. Inhibition of Drp1 activity by mdivi-1 attenuated the DF-induced atheroprone EC phenotypes, showing the close relationship between mitochondrial morphology and atheroprone phenotypes of ECs. As for the molecular mechanism, hypoxia-inducible factor 1 α (HIF-1α) stabilization and its nuclear translocation was significantly increased under DF, which was attenuated by mdivi-1 treatment. Mitochondrial reactive oxygen species (mtROS) and succinate, which are known to reduce prolyl hydroxylase domain 2 (PHD2) activity thereby increasing HIF-1α stabilization, were significantly elevated under DF, but those were attenuated by mdivi-1 treatment. Finally, a 7-week voluntary wheel-running exercise training significantly decreased mitochondrial fragmentation with a down-regulation of VCAM-1 expression at the LC. In conclusion, our data suggest that DF induces mitochondrial fragmentation with increased Drp1 activity, which is associated with an atheroprone EC phenotype. In addition, regular practice of aerobic exercise reduces mitochondrial fragmentation and prevents ECs from an atheroprone endothelial phenotype at the atheroprone regions. Chapter 2: Molecular Mechanisms for Unidirectional Flow (UF)/Exercise-Induced improvement of Mitochondrial Integrity: Focus on phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) /PARKIN-Dependent Mitochondrial Autophagy (Mitophagy) Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) is an essential molecule in the mitophagy process and known to act as a cytoprotective protein involved in several cellular mechanisms in mammalian cells. It has been documented that the loss of PINK1 expression in mice and various cell types enhance susceptibility to stress-induced cell damage, while the overexpression of PINK1 significantly attenuates stress-induced mitochondrial and cellular dysfunction.In chapter 2, the purpose of this study was to investigate PINK1 expression and its subcellular localization under an exercise-mimic laminar shear stress (LSS) condition in human primary endothelial cells and in exercizing mice, and its implications on endothelial homeostasis and cardiovascular disease (CVD) prevention. The overarching hypothesis of the Chapter 2 was that unidirectional flow (UF) will increase cytosolic PINK1 expression through which UF-preconditioned ECs will be more protective against an accumulation of dysfunctional mitochondria via a greater mitophagy induction. In this study, we measured the full-length PINK1 (FL-PINK1) mRNA and protein expression levels in ECs under unidirectional laminar shear stress (LSS). LSS significantly elevated both FL-PINK1 mRNA and protein expressions in ECs. Mitochondrial fractionation assays showed a decrease in FL-PINK1 accumulation in the mitochondria with an increase in the cytosolic FL-PINK1 level under LSS. Confocal microscopic analysis confirmed these subcellular localization patterns suggesting downregulation of mitophagy induction. Indeed, mitophagy flux was decreased under LSS, determined by a mtKeima probe. Mitochondrial morphometric analysis and mitochondrial membrane potential determined by tetraethylbenzimidazolylcarbocyanine iodide (JC-1) showed mitochondrial elongation and increased mitochondrial membrane potential under LSS respectively, suggesting that an elevation of cytosolic PINK1 is not related to an immediate induction of mitophagy. However, increased cytosolic PINK1 elevated mitophagic sensitivity toward dysfunctional mitochondria in pathological conditions. Preconditioned ECs with LSS showed lower mtDNA lesions under angiotensin II stimulation. Moreover, LSS-preconditioned ECs showed rapid Parkin recruitment and mitophagy induction in response to mitochondrial toxin (i.e., carbonyl cyanide chlorophenylhydrazone, CCCP) treatment compared to the control. We measured PINK1 expression at ECs of the thoracic aorta in exercised mice, a physiological LSS-enhanced model, which was significantly elevated compared to sedentary animals. In addition, exercise-preconditioned mice were more protective to angiotensin II-induced mtDNA lesion formation in the mouse abdominal aorta than sedentary mice, suggesting a potential protective mechanism of exercise in a PINK1-dependent manner. In conclusion, LSS increases a cytosolic pool of FL-PINK1, which may elevate the mitophagic sensitivity toward dysfunctional mitochondria or activate other cytoprotective mechanisms in ECs. Our data suggest that exercise may support mitochondrial homeostasis in vascular ECs by enhancing PINK1-dependent cell protection mechanisms. / Kinesiology

No / Our objectives were to obtain monoclonal anti-endothelial cell antibodies (AECA) from systemic lupus erythematosus (SLE) patients, to characterize their antigen specificity, and their capability to induce a pro-inflammatory and pro-adhesive endothelial phenotype, and to investigate the mechanism of endothelial cell (EC) activation in vitro. Monoclonal IgG AECA were generated by hybridoma formation with human SLE B cells. Antigen specificity was characterized by immunoblotting with enriched cell membrane fractions, by cytofluorimetry and by cell solid-phase ELISA. Endothelial activation was evaluated by measuring increases in U937 cell adhesiveness, adhesion molecule (E-selectin and ICAM-1) expression and IL-6 production. In addition, mechanisms of endothelial activation were investigated by assessment of NF-B by measuring the loss of its inhibitor I-B. mAb E-3 bound live EC and recognized a 42 kDa EC membrane protein, it enhanced U937 adhesiveness, E-selectin and ICAM-1 expression and IL-6 production, and caused the loss of I-B. We conclude this is the first in vitro demonstration that a human monoclonal AECA from a SLE patient reacts with a constitutive endothelial membrane antigen and induces a pro-inflammatory endothelial phenotype through NF-B activation.