Eicosanoid Signaling and Vascular Dysfunction: Methylmercury-Induced Phospholipase D Activation in Vascular Endothelial Cells

Mercury, especially methylmercury (MeHg), is implicated in the etiology of cardiovascular diseases. Earlier, we have reported that MeHg induces phospholipase D (PLD) activation through oxidative stress and thiol-redox alteration. Hence, we investigated the mechanism of the MeHg-induced PLD activation through the upstream regulation by phospholipase A₂ (PLA₂) and lipid oxygenases such as cyclooxygenase (COX) and lipoxygenase (LOX) in the bovine pulmonary artery endothelial cells (BPAECs). Our results showed that MeHg significantly activated both PLA₂ (release of [³H]arachidonic acid, AA) and PLD (formation of [³²P]phosphatidylbutanol) in BPAECs in dose- (0–10 μM) and time-dependent (0–60 min) fashion. The cPLA₂-specific inhibitor, arachidonyl trifluoromethyl ketone (AACOCF₃), significantly attenuated the MeHg-induced [³H]AA release in ECs. MeHg-induced PLD activation was also inhibited by AACOCF₃ and the COX- and LOX-specific inhibitors. MeHg also induced the formation of COX- and LOX-catalyzed eicosanoids in ECs. MeHg-induced cytotoxicity (based on lactate dehydrogenase release) was protected by PLA₂-, COX-, and LOX-specific inhibitors and 1-butanol, the PLD-generated PA quencher. For the first time, our studies showed that MeHg activated PLD in vascular ECs through the upstream action of cPLA₂ and the COX- and LOX-generated eicosanoids. These results offered insights into the mechanism(s) of the MeHg-mediated vascular endothelial cell lipid signaling as an underlying cause of mercury-induced cardiovascular diseases.     

Role of Nitric Oxide Isoforms in Vascular and Alveolar Development and Lung Injury in Vascular Endothelial Growth Factor Overexpressing Neonatal Mice Lungs

Background

The role of vascular endothelial growth factor (VEGF)-induced 3 different nitric oxide synthase (NOS) isoforms in lung development and injury in the newborn (NB) lung are not known. We hypothesized that VEGF-induced specific NOS pathways are critical regulators of lung development and injury.

Methodology

We studied NB wild type (WT), lung epithelial cell-targeted VEGF165 doxycycline-inducible overexpressing transgenic (VEGFTG), VEGFTG treated with a NOS1 inhibitor (L-NIO), VEGFTG x NOS2^-/- and VEGFTG x NOS3^+/- mice in room air (RA) for 7 postnatal (PN) days. Lung morphometry (chord length), vascular markers (Ang1, Ang2, Notch2, vWF, CD31 and VE-cadherin), cell proliferation (Ki67), vascular permeability, injury and oxidative stress markers (hemosiderin, nitrotyrosine and 8-OHdG) were evaluated.

Results

VEGF overexpression in RA led to increased chord length and vascular markers at PN7, which were significantly decreased to control values in VEGFTG x NOS2^−/− and VEGFTG x NOS3^+/- lungs. However, we found no noticeable effect on chord length and vascular markers in the VEGFTG / NOS1 inhibited group. In the NB VEGFTG mouse model, we found VEGF-induced vascular permeability in the NB murine lung was partially dependent on NOS2 and NOS3-signaling pathways. In addition, the inhibition of NOS2 and NOS3 resulted in a significant decrease in VEGF-induced hemosiderin, nitrotyrosine- and 8-OHdG positive cells at PN7. NOS1 inhibition had no significant effect.

Conclusion

Our data showed that the complete absence of NOS2 and partial deficiency of NOS3 confers protection against VEGF-induced pathologic lung vascular and alveolar developmental changes, as well as injury markers. Inhibition of NOS1 does not have any modulating role on VEGF-induced changes in the NB lung. Overall, our data suggests that there is a significant differential regulation in the NOS-mediated effects of VEGF overexpression in the developing mouse lung.     

HTII-280, a Biomarker Specific to the Apical Plasma Membrane of Human Lung Alveolar Type II Cells

The pulmonary alveolar epithelium is composed of two morphologically distinct cell types, type I (TI) and type II (TII) cells. Alveolar TII cells synthesize, secrete, and recycle surfactant components; contain ion transporters; and secrete immune effector molecules. In response to alveolar injury, TII cells have the capacity to act as progenitor cells, proliferating and transdifferentiating into TI cells. Although various proteins are associated with TII cells, a plasma membrane marker specific to human TII cells that would be useful for identification in tissue and for isolating this cell type has not been described previously. We devised a strategy to produce a monoclonal antibody (MAb) specific to the apical surface of human TII cells and developed an MAb that appears to be specific for human TII cells. The antibody recognizes a 280- to 300-kDa protein, HTII-280, which has the biochemical characteristics of an integral membrane protein. HTII-280 is detected by week 11 of gestation and is developmentally regulated. HTII-280 is useful for isolating human TII cells with purities and viabilities >95%. HTII-280 is likely to be a useful morphological and biochemical marker of human TII cells that may help to advance our understanding of various lung pathological conditions, including the origin and development of various lung tumors. (J Histochem Cytochem 58:891–901, 2010)     

Repeated Activation of Lung Invariant NKT Cells Results in Chronic Obstructive Pulmonary Disease-Like Symptoms

Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation, mucus hypersecretion, and emphysema, which lead to reduced lung function and breathlessness. The pathologies of COPD are due to an abnormal immune response. Invariant natural killer T (iNKT) cells are an important population of innate lymphocytes and have been implicated in the regulation of immune responses associated with a broad range of diseases including COPD. We have here analyzed the role of iNKT cells in a model of COPD induced by repeated intranasal administration of iNKT cell agonist α-galactosylceramide (α-GalCer). Our results demonstrated that mice that received repeated intranasal administration of α-GalCer had molecular and inflammatory features of COPD including airway inflammation with significant increases in infiltration of macrophages and lymphocytes, CD8⁺ T cells, as well as proinflammatory cytokines IL-6 and TNF-α. In particular, these mice also showed the presence of pulmonary emphysema, mucus production, and pulmonary fibrosis. Furthermore, neutralization of IL-4 reduced α-GalCer induced emphysema. This study indicates the importance of iNKT cells in the pathogenesis of COPD by an IL-4 dependent mechanism.     

Surfactant Protein D Binds to Coxiella burnetii and Results in a Decrease in Interactions with Murine Alveolar Macrophages

Coxiella burnetii is a Gram-negative, obligate intracellular bacterium and the causative agent of Q fever. Infections are usually acquired after inhalation of contaminated particles, where C. burnetii infects its cellular target cells, alveolar macrophages. Respiratory pathogens encounter the C-type lectin surfactant protein D (SP-D) during the course of natural infection. SP-D is a component of the innate immune response in the lungs and other mucosal surfaces. Many Gram-negative pulmonary pathogens interact with SP-D, which can cause aggregation, bactericidal effects and aid in bacterial clearance. Here we show that SP-D binds to C. burnetii in a calcium-dependent manner with no detectable bacterial aggregation or bactericidal effects. Since SP-D interactions with bacteria often alter macrophage interactions, it was determined that SP-D treatment resulted in a significant decrease in C. burnetii interactions to a mouse alveolar macrophage model cell line MH-S indicating SP-D causes a significant decrease in phagocytosis. The ability of SP-D to modulate macrophage activation by C. burnetii was tested and it was determined that SP-D does not alter the correlates measured for macrophage activation. Taken together these studies support those demonstrating limited activation of alveolar macrophages with C. burnetii and demonstrate interactions with SP-D participate in reduction of phagocyte attachment and phagocytosis.     

Presence of Cleaved Synaptosomal-Associated Protein-25 and Decrease of Purinergic Receptors P2X3 in the Bladder Urothelium Influence Efficacy of Botulinum Toxin Treatment for Overactive Bladder Syndrome

Objectives

To evaluate whether botulinum toxin A (BoNT-A) injection and Lipotoxin (liposomes with 200 U of BoNT-A) instillation target different proteins, including P2X3, synaptic vesicle glycoprotein 2A, and SNAP-25, in the bladder mucosa, leading to different treatment outcomes.

Materials and Methods

This was a retrospective study performed in a tertiary teaching hospital. We evaluated the clinical results of 27 OAB patients treated with intravesical BoNT-A injection (n = 16) or Lipotoxin instillation (n = 11). Seven controls were treated with saline. Patients were injected with 100 U of BoNT-A or Lipotoxinin a single intravesical instillation. The patients enrolled in this study all had bladder biopsies performed at baseline and one month after BoNT-A therapy. Treatment outcome was measured by the decreases in urgency and frequency episodes at 1 month. The functional protein expressions in the urothelium were measured at baseline and after 1 month. The Wilcoxon signed-rank test and ordinal logistic regression were used to compare the treatment outcomes.

Results

Both BoNT-A injection and Lipotoxin instillation treatments effectively decreased the frequency of urgency episodes in OAB patients. Lipotoxin instillation did not increase post-void residual volume. BoNT-A injection effectively cleaved SNAP-25 (p < 0.01). Liposome encapsulated BoNT-A decreased urothelial P2X3 expression in the five responders (p = 0.04), while SNAP-25 was not significantly cleaved.

Conclusions

The results of this study provide a possible mechanism for the therapeutic effects of BoNT-A for the treatment of OAB via different treatment forms. BoNT-A and Lipotoxin treatments effectively decreased the frequency of urgency episodes in patients with OAB.     

尿路上皮癌胚抗原1（UCA1）在人膀胱癌组织及膀胱癌细胞株中特异表达

UCA1（urothelial carcinoma antigen 1）为自主研发的1个尿路上皮癌基因.应用 实时荧光定量PCR检测UCA1 mRNA在2种膀胱癌细胞系、11种非膀胱癌细胞系、18对膀胱 癌组织和配对癌旁正常膀胱组织中的表达,对表达差异进行统计学分析.结果显示, UCA1在2种膀胱癌细胞系中显著高表达,而在其他11种非膀胱癌细胞系中表达水平很低 或者不表达,二者表达差异达14~24 812倍,差异有统计学意义（P<0.001）;在18例膀 胱癌组织中,UCA1的平均表达水平是癌旁正常膀胱组织的12.4倍,表达差异有统计学意 义（P<0.001）. 实时荧光定量PCR使UCA1在膀胱癌细胞系及组织中的特异性高表达得以 量化.实验结果明确了UCA1作为潜在的肿瘤标记物在膀胱癌临床诊断中的意义,为定量 检测尿液UCA1表达并确定诊断膀胱癌的参考值打下基础.     

Examination of the Role of ADP-Ribosylation Factor and Phospholipase D Activation in Regulated Exocytosis in Chromaffin and PC12 Cells

Abstract: The possible role of ADP-ribosylation factor (ARF)-activated and constitutive phospholipase D (PLD) activity in regulated exocytosis of preformed secretory granules in adrenal chromaffin and PC12 cells was examined. With use of digitonin-permeabilised cells, the effect of GTP analogues and exogenous ARF1 on PLD activity was determined. No evidence was seen for ARF-stimulated PLD activity in these cell types. Exocytosis from cytosol-depleted permeabilised chromaffin cells was not increased by adding recombinant nonmyristoylated or myristoylated ARF1, and exocytosis from both cell types was resistant to brefeldin A (BFA). Addition of bacterial PLD with demonstrably high activity in permeabilised chromaffin cells did not increase exocytosis in cytosol-depleted chromaffin cells. Diversion of PLD activity from production of phosphatidic acid (PA) due to the presence of 4% ethanol did not inhibit exocytosis triggered by Ca²⁺ or poorly hydrolysable GTP analogues in permeabilised chromaffin or PC12 cells. These results indicate that exocytosis in these cell types does not appear to require a BFA-sensitive ARF and the triggering of exocytosis does not require PLD activity and formation of PA. These findings rule out a general requirement for PLD activity during regulated exocytosis.     

Environmentally Determined Differences in the Murine Lung Microbiota and Their Relation to Alveolar Architecture

Commensal bacteria control the micro-ecology of metazoan epithelial surfaces with pivotal effect on tissue homeostasis and host defense. In contrast to the upper respiratory tract, the lower respiratory tract of healthy individuals has largely been considered free of microorganisms. To understand airway micro-ecology we studied microbiota of sterilely excised lungs from mice of different origin including outbred wild mice caught in the natural environment or kept under non-specific-pathogen-free (SPF) conditions as well as inbred mice maintained in non-SPF, SPF or germ-free (GF) facilities. High-throughput pyrosequencing of reverse transcribed 16S rRNA revealed metabolically active murine lung microbiota in all but GF mice. The overall composition across samples was similar at the phylum and family level. However, species richness was significantly different between lung microbiota from SPF and non-SPF mice. Non-cultivatable Betaproteobacteria such as Ralstonia spp. made up the major constituents and were also confirmed by 16S rRNA gene cloning analysis. Additionally, Pasteurellaceae, Enterobacteria and Firmicutes were isolated from lungs of non-SPF mice. Bacterial communities were detectable by fluorescent in situ hybridization (FISH) at alveolar epithelia in the absence of inflammation. Notably, higher bacterial abundance in non-SPF mice correlated with more and smaller size alveolae, which was corroborated by transplanting Lactobacillus spp. lung isolates into GF mice. Our data indicate a common microbial composition of murine lungs, which is diversified through different environmental conditions and affects lung architecture. Identification of the microbiota of murine lungs will pave the path to study their influence on pulmonary immunity to infection and allergens using mouse models.     

Frequency of a Specific Cytochrome P4502D6B (CYP2D6B) Mutant Allele in Clinically Differentiated Groups of Patients with Parkinson Disease

The frequency of the cytochrome P4502D6B CYP2D6B (29B) mutant allele has been determined in three clinically distinct groups of patients with Parkinson disease. No differences in mutation frequency among the patients with the rigidity-akinetic and monosymptomatic tremor forms has been observed compared to the healthy control group, while in the group with akinetic-rigidity tremor symptoms the frequency of heterozygous wt/29B individuals was significantly increased. Therefore, individuals bearing the CYP2D6B mutation appear to be predisposed to the development of this particular form of Parkinson disease, and the presence of the 29B mutation in the genotype may serve as an additional diagnostic criteriaum for the clinical differentiation of patients with Parkinson disease.