Phosphate induces inflammation and exacerbates injury from cigarette smoke in the bronchial epithelium.

An elevation in serum phosphate-also called hyperphosphatemia-is associated with reduced kidney function in chronic kidney disease (CKD). Reports show CKD patients are more likely to develop lung disease and have poorer kidney function that positively correlates with pulmonary obstruction. However, the underlying mechanisms are not well understood. Here, we report that two murine models of CKD, which both exhibit increased serum levels of phosphate and fibroblast growth factor (FGF) 23, a regulator of phosphate homeostasis, develop concomitant airway inflammation. Our in vitro studies point towards a similar increase of phosphate-induced inflammatory markers in human bronchial epithelial cells. FGF23 stimulation alone does not induce a proinflammatory response in the non-COPD bronchial epithelium and phosphate does not cause endogenous FGF23 release. Upregulation of the phosphate-induced proinflammatory cytokines is accompanied by activation of the extracellular-signal regulated kinase (ERK) pathway. Moreover, the addition of cigarette smoke extract (CSE) during phosphate treatments exacerbates inflammation as well as ERK activation, whereas co-treatment with FGF23 attenuates both the phosphate as well as the combined phosphate- and CS-induced inflammatory response, independent of ERK activation. Together, these data demonstrate a novel pathway that potentially explains pathological kidney-lung crosstalk with phosphate as a key mediator.

ST6GAL1 and α2-6 Sialylation Regulates IL-6 Expression and Secretion in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a systemic disease strongly associated with cigarette smoking, airway inflammation, and acute disease exacerbations. Changes in terminal sialylation and fucosylation of asparagine (N)-linked glycans have been documented in COPD, but the role that glycosyltransferases may play in the regulation of N-linked glycans in COPD has not been fully elucidated. Recent studies suggest that modulation of ST6GAL1 (ST6 beta-galactoside alpha-2,6-sialyltransferase-1), which catalyzes terminal α2-6 sialylation of cellular proteins, may regulate inflammation and contribute to COPD phenotype(s). Interestingly, it has been previously demonstrated that ST6GAL1, a Golgi resident protein, can be proteolytically processed by BACE1 (beta-site amyloid precursor protein cleaving enzyme-1) to a circulating form that retains activity. In this study, we showed that loss of ST6GAL1 expression increased interleukin (IL)-6 expression and secretion in human bronchial epithelial cells (HBECs). Furthermore, exposure to cigarette smoke medium/extract (CSE) or BACE1 inhibition resulted in decreased ST6GAL1 secretion, reduced α2-6 sialylation, and increased IL-6 production in HBECs. Analysis of plasma ST6GAL1 levels in a small COPD patient cohort demonstrated an inverse association with prospective acute exacerbations of COPD (AECOPD), while IL-6 was positively associated. Altogether, these results suggest that reduced ST6GAL1 and α2-6 sialylation augments IL-6 expression/secretion in HBECs and is associated with poor clinical outcomes in COPD.

O-GlcNAc Transferase Regulates Angiogenesis in Idiopathic Pulmonary Arterial Hypertension.

Idiopathic pulmonary arterial hypertension (IPAH) is considered a vasculopathy characterized by elevated pulmonary vascular resistance due to vasoconstriction and/or lung remodeling such as plexiform lesions, the hallmark of the PAH, as well as cell proliferation and vascular and angiogenic dysfunction. The serine/threonine hydroxyl-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT) has been shown to drive pulmonary arterial smooth muscle cell (PASMC) proliferation in IPAH. OGT is a cellular nutrient sensor that is essential in maintaining proper cell function through the regulation of cell signaling, proliferation, and metabolism. The aim of this study was to determine the role of OGT and O-GlcNAc in vascular and angiogenic dysfunction in IPAH. Primary isolated human control and IPAH patient PASMCs and pulmonary arterial endothelial cells (PAECs) were grown in the presence or absence of OGT inhibitors and subjected to biochemical assessments in monolayer cultures and tube formation assays, in vitro vascular sprouting 3D spheroid co-culture models, and de novo vascularization models in NODSCID mice. We showed that knockdown of OGT resulted in reduced vascular endothelial growth factor (VEGF) expression in IPAH primary isolated vascular cells. In addition, specificity protein 1 (SP1), a known stimulator of VEGF expression, was shown to have higher O-GlcNAc levels in IPAH compared to control at physiological (5 mM) and high (25 mM) glucose concentrations, and knockdown resulted in decreased VEGF protein levels. Furthermore, human IPAH PAECs demonstrated a significantly higher degree of capillary tube-like structures and increased length compared to control PAECs. Addition of an OGT inhibitor, OSMI-1, significantly reduced the number of tube-like structures and tube length similar to control levels. Assessment of vascular sprouting from an in vitro 3D spheroid co-culture model using IPAH and control PAEC/PASMCs and an in vivo vascularization model using control and PAEC-embedded collagen implants demonstrated higher vascularization in IPAH compared to control. Blocking OGT activity in these experiments, however, altered the vascular sprouting and de novo vascularization in IPAH similar to control levels when compared to controls. Our findings in this report are the first to describe a role for the OGT/O-GlcNAc axis in modulating VEGF expression and vascularization in IPAH. These findings provide greater insight into the potential role that altered glucose uptake and metabolism may have on the angiogenic process and the development of plexiform lesions. Therefore, we believe that the OGT/O-GlcNAc axis may be a potential therapeutic target for treating the angiogenic dysregulation that is present in IPAH.

Fibronectin connecting segment-1 peptide inhibits pathogenic leukocyte trafficking and inflammatory demyelination in experimental models of chronic inflammatory demyelinating polyradiculoneuropathy.

The molecular determinants of pathogenic leukocyte migration across the blood-nerve barrier (BNB) in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) are unknown. Specific disease modifying therapies for CIDP are also lacking. Fibronectin connecting segment-1 (FNCS1), an alternatively spliced fibronectin variant expressed by microvascular endothelial cells at sites of inflammation in vitro and in situ, is a counterligand for leukocyte α4 integrin (also known as CD49d) implicated in pathogenic leukocyte trafficking in multiple sclerosis and inflammatory bowel disease. We sought to determine the role of FNCS1 in CIDP patient leukocyte trafficking across the BNB in vitro and in severe chronic demyelinating neuritis in vivo using a representative spontaneous murine CIDP model. Peripheral blood mononuclear leukocytes from 7 untreated CIDP patients were independently infused into a cytokine-treated, flow-dependent in vitro BNB model system. Time-lapse digital video microscopy was performed to visualize and quantify leukocyte trafficking, comparing FNCS1 peptide blockade to relevant controls. Fifty 24-week old female B7-2 deficient non-obese diabetic mice with spontaneous autoimmune peripheral polyneuropathy (SAPP) were treated daily with 2mg/kg FNCS1 peptide for 5days via intraperitoneal injection with appropriate controls. Neurobehavioral measures of disease severity, motor nerve electrophysiology assessments and histopathological quantification of inflammation and morphometric assessment of demyelination were performed to determine in vivo efficacy. The biological relevance of FNCS1 and CD49d in CIDP was evaluated by immunohistochemical detection in affected patient sural nerve biopsies. 25µM FNCS1 peptide maximally inhibited CIDP leukocyte trafficking at the human BNB in vitro. FNCS1 peptide treatment resulted in significant improvements in disease severity, motor electrophysiological parameters of demyelination and histological measures of inflammatory demyelination. Microvessels demonstrating FNCS1 expression and CD49d+ leukocytes were seen within the endoneurium of patient nerve biopsies. Taken together, these results imply a role for FNCS1 in pathogenic leukocyte trafficking in CIDP, providing a potential target for therapeutic modulation.

A novel method for measuring hydraulic conductivity at the human blood-nerve barrier in vitro.

Microvascular barrier permeability to water is an essential biophysical property required for the homeostatic maintenance of unique tissue microenvironments. This is of particular importance in peripheral nerves where strict control of ionic concentrations is needed for axonal signal transduction. Previous studies have associated inflammation, trauma, toxin exposure and metabolic disease with increases in water influx and hydrostatic pressure in peripheral nerves with resultant endoneurial edema that may impair axonal function. The regulation of water permeability across endoneurial microvessels that form the blood-nerve barrier (BNB) is poorly understood. Variations exist in apparatus and methods used to measure hydraulic conductivity. The objective of the study was to develop a simplified hydraulic conductivity system using commercially available components to evaluate the BNB. We determined the mean hydraulic conductivity of cultured confluent primary and immortalized human endoneurial endothelial cell layers as 2.00×10-7 and 2.17×10-7cm/s/cm H2O respectively, consistent with restrictive microvascular endothelial cells in vitro. We also determined the mean hydraulic conductivity of immortalized human brain microvascular endothelial cell layers, a commonly used blood-brain barrier (BBB) cell line, as 0.20×10-7cm/s/cm H2O, implying a mean 10-fold higher resistance to transendothelial water flux in the brain compared to peripheral nerves. To our knowledge, this is the first reported measurement of human BNB and BBB hydraulic conductivities. This model represents an important tool to further characterize the human BNB and deduce the molecular determinants and signaling mechanisms responsible for BNB hydraulic conductivity in normal and disease states in vitro.

p53 modulation of the DNA damage response.

The tumor suppressor p53 plays a central role in the DNA damage response. After exposure to genotoxic stress, p53 can both positively and negatively regulate cell fate. Initially, p53 promotes cell survival by inducing cell cycle arrest, DNA repair, and other pro-survival pathways. However, when cells accumulate DNA damage or demonstrate aberrant growth, p53 can direct the elimination of damaged cells. In this review, we will discuss the transcriptional-dependent and -independent roles of p53 in regulating the DNA damage response.

The unique NH2-terminally deleted (DeltaN) residues, the PXXP motif, and the PPXY motif are required for the transcriptional activity of the DeltaN variant of p63.

p63, a member of the p53 family of transcription factors, is known to be involved in epithelial development. However, its role in tumorigenesis is unclear. Contributing to this uncertainty, the TP63 locus can express multiple gene products from two different promoters. Utilization of the upstream promoter results in expression of the TAp63 variant with an activation domain similar to p53. In contrast, the NH2-terminally deleted (DeltaN) p63 variant, transcribed from a cryptic promoter in intron 3, lacks such an activation domain. Thus, the TAp63 and DeltaNp63 variants possess a wide ranging ability to up-regulate p53 target genes. Consequentially, the disparity in transactivation potential between p63 variants has given rise to the hypothesis that the DeltaNp63 variant can serve as oncoprotein by opposing the activity of the TAp63 variant and p53. However, recent studies have revealed a transcriptional activity for DeltaNp63. This study was undertaken to address the transcriptional activity of the DeltaNp63 variant. Here, we showed that all NH2-terminally deleted p63 isoforms retain a potential in transactivation and growth suppression. Interestingly, DeltaNp63beta possesses a remarkable ability to suppress cell proliferation and transactivate target genes, which is consistently higher than that seen with DeltaNp63alpha. In contrast, DeltaNp63gamma has a weak or undetectable activity dependent upon the cell lines used. We also demonstrate that an intact DNA-binding domain is required for DeltaNp63 function. In addition, we found that the novel activation domain for the DeltaNp63 variant is composed of the 14 unique DeltaN residues along with the adjacent region, including a PXXP motif. Finally, we demonstrated that a PPXY motif shared by DeltaNp63alpha and DeltaNp63beta is required for optimal transactivation of target gene promoters, suggesting that the PPXY motif is requisite for DeltaNp63 function.