Shamimunisa B. Mustafa

Endothelin stimulates platelet‐activating factor synthesis by cultured rat Kupffer cells

Endothelins are potent peptide mediators that elicit glycogenolytic and vasoconstrictor actions in the liver. Endothelins were found to stimulate the synthesis and release of the lipid mediator platelet-activating factor in cultured rat Kupffer cells. Endothelin-mediated synthesis of platelet-activating factor required extracellular calcium in that the calcium chelator, EGTA and nifedipine, a calcium ion channel blocker, inhibited platelet-activating factor synthesis. The phospholipase A2 inhibitor, 4-bromophenacyl bromide, strongly inhibited endothelin-induced platelet activating factor synthesis. Endothelin-stimulated platelet activating factor synthesis was inhibited after treatment of Kupffer cells with cholera toxin, whereas pertussis toxin inhibited only this response to endothelin-1. Agents that elevate intracellular cyclic AMP levels were found to inhibit endothelin-induced platelet-activating factor synthesis in Kupffer cells. Staurosporine, a protein kinase C inhibitor minimized endothelin-induced platelet-activating factor synthesis but phorbol myristate acetate, an activator of protein kinase C, did not affect endothelin-induced platelet activating factor synthesis. Thus, the current study demonstrates that activation of an endothelin receptor in cultured rat Kupffer cells results in the synthesis and release of platelet-activating factor. The importance of endothelin-mediated platelet-activating factor synthesis relates to the mechanism of intercellular signaling occurring between endothelial cells (i.e., the site of endothelin synthesis) and Kupffer cells (i.e., the site of formation of secondary mediators such as platelet-activating factor and eicosanoids) within the rat liver exposed to various types of pathophysiological episodes.

Effects of calcium channel antagonists on LPS-induced hepatic iNOS expression

The onset of liver injury is a pivotal event during endotoxemia. Lipopolysaccharide (LPS) activates the Kupffer cells (KC), the resident macrophages of the liver, to generate an abundance of inflammatory substances, including nitric oxide (NO). Elevated levels of NO are thought to contribute to the propagation of liver injury during sepsis. Calcium, a major second messenger in several cellular signaling events, is required by the KC for the generation of inducible nitric oxide synthase (iNOS). The purpose of this study was to determine whether calcium channel antagonists limit hepatic injury and iNOS expression in vivo following LPS exposure and to evaluate their effects on the regulation of iNOS expression in cultured KC. In rats subjected to LPS for 6 h, the serum alanine aminotransferase (ALT) level was elevated significantly; this response was accompanied by an increase in iNOS mRNA formation in the intact liver. Pretreatment of rats with calcium channel antagonists (i.e., diltiazem, nifedipine, or verapamil) before LPS exposure attenuated the serum ALT level and iNOS mRNA expression in the liver. Pretreatment of cultured KC with calcium channel antagonists for 1 h followed by the addition of LPS markedly repressed iNOS protein and mRNA expression. Time-course studies revealed that calcium channel antagonists were most effective at inhibiting LPS-induced iNOS mRNA formation by KC when added before LPS. Treatment of KC with calcium channel antagonists prior to the addition of LPS decreased nuclear levels of the p65 subunit of nuclear factor-κB and prevented the LPS-dependent degradation of the inhibitory protein IκBα. Thus our findings indicate that under endotoxemic conditions calcium channel antagonists limit hepatocellular injury that is accompanied by an inhibition of LPS-mediated iNOS expression in rat liver KC.

Immunosuppressive properties of surfactant in alveolar macrophage NR8383

Abstract.Objective:To evaluate the anti-inflammatory effects of exogenous surfactants and surfactant phospholipid without surfactant proteins (SP-A and SP-D) on the lipopolysaccharide- (LPS) stimulated rat alveolar macrophage (AM) cell line NR8383.Methods:Exogenous surfactants (beractant, calfactant or colfosceril) and surfactant phospholipid (dipalmitoyl phosphatidylcholine, DPPC), standardized to phospholipid content of 25–1,000 μg/ml were incubated with LPS- (1 μg/ml) stimulated NR8383 AMs.Results:TNF-α and IL-1β secretion and nitric oxide (NO) formation following LPS stimulation were inhibited by treatment with surfactants or DPPC. Furthermore, LPS-dependent NO production and iNOS protein levels were significantly suppressed in cells pretreated for one hour with beractant compared to beractant added simultaneously with or following LPS. Additionally, LPS-stimulated oxidative burst, measured by flow cytometry, was significantly decreased by beractant. Finally, beractant inhibited the translocation of NF-κB from cytoplasmic into nuclear extract in LPS-stimulated NR8383 AMs.Conclusions:Exogenous surfactants and surfactant phospholipid inhibit secretion of proinflammatory cytokines and NO in NR8383 AMs. The inhibitory effects of beractant on oxygen radical and LPS-induced NO formation may result from unique mechanisms of decreasing cell signaling. The anti-inflammatory activity of surfactant products used in the treatment of neonatal respiratory distress syndrome (RDS) may depend upon the specific preparation or dose used.

Protein Kinase A and Mitogen-Activated Protein Kinase Pathways Mediate cAMP Induction of α-Epithelial Na+ Channels (α-ENaC)

A major mechanism for Na+ transport across epithelia occurs through epithelial Na+ channels (ENaC). ENaC is a multimeric channel consisting of three subunits (α, β, and γ). The α‐subunit is critical for ENaC function. In specific culture conditions, the rat submandibular gland epithelial cell line (SMG‐C6) demonstrates minimal Na+ transport properties and exposure to dibutyryl cAMP (DbcAMP) for up to 48 h caused an elevation of α‐ENaC mRNA and protein expression and amiloride‐sensitive short‐circuit current (ISC). Here we examined the early signaling pathways evoked by DbcAMP which contribute to the eventual increase in Na+ transport is present. Treatment with either of the protein kinase A (PKA) inhibitors KT5720 or H‐89 followed by exposure to 1 mM DbcAMP for 24 h markedly attenuated DbcAMP‐induced α‐ENaC protein formation and ISC. Exposure of SMG‐C6 cells to 1 mM DbcAMP induced a rapid, transient phosphorylation of the cAMP response element binding protein (CREB). This response was attenuated in the presence of either KT5720 or H‐89. Dominant‐negative CREB decreased DbcAMP‐induced α‐ENaC expression. Suppression of the extracellular signal‐regulated protein kinase (ERK 1,2) with PD98059 or the p38 mitogen‐activated protein kinase (MAPK) pathway with SB203580 reduced DbcAMP‐induced α‐ENaC protein levels in SMG‐C6 cells. DbcAMP‐induced phosphorylation of CREB was markedly attenuated by PD98059 or SB203580. DbcAMP‐induced activation of the either the p38 or the ERK 1,2 MAPK pathways was abolished by either of the PKA inhibitors, H‐89 or KT5720. Cross talk between these signaling pathways induced by DbcAMP via the activation of CREB appears to contribute to increased levels of α‐ENaC observed after 24 h of treatment in SMG‐C6 epithelial cells. J. Cell. Physiol. 215: 101–110, 2008.

Neonatal chlamydial pneumonia induces altered respiratory structure and function lasting into adult life

Respiratory dysfunction in adults has been correlated with neonatal Chlamydia trachomatis pneumonia in several studies, but a causal association has not been clearly demonstrated. In this study, we examined radial alveolar counts (RACs) by microscopy, and airway and parenchymal lung function using a small animal ventilator in juvenile (5 weeks age) and adult (8 weeks age) BALB/c mice challenged as neonates with Chlamydia muridarum (C. mur) on day 1 or day 7 after birth, representing saccular (human pre-term neonates) and alveolar (human term neonates) stages of lung development, respectively. Pups challenged with C. mur on either day 1 or 7 after birth demonstrated significantly enhanced airway hyperreactivity and lung compliance, both as juveniles (5 weeks age) and adults (8 weeks age), compared with mock-challenged mice. Moreover, mice challenged neonatally with Chlamydia displayed significantly reduced RACs, suggesting emphysematous changes. Antimicrobial treatment during the neonatal infection induced early bacterial clearance and partially ameliorated the Chlamydia-induced lung dysfunction as adults. These results suggest that neonatal chlamydial pneumonia, especially in pre-term neonates, is a cause of respiratory dysfunction continuing into adulthood, and that antimicrobial administration may be partially effective in preventing the adverse respiratory sequelae in adulthood. The results of our studies also emphasize the importance of prenatal screening and treatment of pregnant women for C. trachomatis in order to prevent the infection of neonates.

Induction of serum- and glucocorticoid-induced kinase-1 (SGK1) by cAMP regulates increases in α-ENaC

α‐ENaC expression and activity is regulated by a variety of hormones including β‐adrenergic agonists via the second messenger cAMP. We evaluated the early intermediate pathways involved in the up‐regulation of SGK1 by DbcAMP and whether SGK1 is a prerequisite for induction of α‐ENaC expression. Submandibular gland epithelial (SMG‐C6) cells treated with DbcAMP (1 mM) induced both SGK1 mRNA and protein expression. DbcAMP‐stimulated SGK1 mRNA expression was decreased by actinomycin D and mRNA and protein expressions were attenuated by PKA inhibitors (H‐89 and KT5720). Inhibition of PI3‐K with either LY294002 or dominant negative PI3‐K reduced DbcAMP‐stimulated SGK1 protein and mRNA levels, attenuated the phosphorylation of CREB (a cAMP‐activated transcription factor) and decreased α‐ENaC protein levels and Na+ transport. In addition, the combination of PKA inhibitors with dominant negative PI3‐K synergistically inhibited DbcAMP‐induced Na+ transport. Inhibition of SGK1 expression by siRNA decreased but did not obliterate DbcAMP‐induced α‐ENaC expression. Thus, in a cell line which endogenously exhibits minimal α‐ENaC expression, induction of SGK1 by DbcAMP occurs via the PI3‐K and PKA pathways. Increased α‐ENaC levels and function are partly dependent upon the early induction of SGK1 expression. J. Cell. Physiol. 217: 632–642, 2008.

Mechanical ventilation down-regulates surfactant protein a and keratinocyte growth factor expression in premature rabbits

Surfactant-associated proteins (SP-A, SP-B, and SP-C) are critical for the endogenous function of surfactant. Keratinocyte growth factor (KGF) and vascular endothelial growth factor (VEGF) are key regulators of lung development. The objective of this study was to evaluate the effects of early mechanical ventilation on the expression of these important regulatory proteins in a preterm rabbit model. Premature fetuses were delivered at 29 d of gestation and randomized to necropsy at birth, i.e. no ventilation (NV), spontaneous breathing (SB), or mechanical ventilation (MV) for 16 h. MV animals were further randomized to treatment with dexamethasone (dex). Our findings showed that SB rabbits increased their expression of SP-A mRNA and protein after birth compared with NV controls. MV significantly attenuated this response in the absence of dex. Exposure to dex elevated SP-B mRNA expression in both SB and MV rabbits. KGF protein levels were markedly increased in SB animals compared with MV counterparts. VEGF levels were similar in SB and MV animals, but were significantly increased compared with NV controls. These data suggest that MV alters surfactant-associated protein and growth factor expression, which may contribute to injury in the developing lung.

Regulation of epithelial Na+ channel (ENaC) in the salivary cell line SMG-C6.

Glucocorticoids and mineralocorticoids modulate Na+ transport via epithelial Na+ channels (ENaC). The rat submandibular epithelial cell line, SMG-C6, expresses α-ENaC mRNA and protein and exhibits amiloride-sensitive Na+ transport when grown in low-serum (2.5%) defined medium, therefore, we examined the effects of altering the composition of the SMG-C6 cell growth medium on ENaC expression and function. No differences in basal or amiloride-sensitive short-circuit current (Isc) were measured across SMG-C6 monolayers grown in the absence of thyroid hormone, insulin, transferrin, or EGF. In the absence of hydrocortisone, basal and amiloride-sensitive Isc significantly decreased. Similarly, monolayers grown in 10% serum-supplemented medium had lower basal Isc and no response to amiloride. Adding hydrocortisone (1.1 μM) to either the low or 10% serum medium increased basal and amiloride-sensitive Isc, which was blocked by RU486, the glucocorticoid and progesterone receptor antagonist. Aldosterone also induced an increase in α-ENaC expression and Na+ transport, which was also blocked by RU486 but not by the mineralocorticoid receptor antagonist spironolactone. Thus, in the SMG-C6 cell line, hydrocortisone and aldosterone increased ENaC expression and basal epithelial Na+ transport. The absence of endogenous ENaC expression in culture conditions devoid of steroids makes the properties of this cell line an excellent model for investigating pathways regulating ENaC expression and Na+ transport.

Upcycling umbilical cords: bridging regenerative medicine with neonatology

Abstract Preterm birth is a major health concern that affects 10% of all worldwide deliveries. Many preterm infants are discharged from the hospital with morbidities that lead to an increased risk for neurodevelopmental impairment, recurrent hospitalizations, and life-long conditions. Unfortunately, the treatment of these conditions is palliative rather than curative, which calls for novel and innovative strategies. Progress in regenerative medicine has offered therapeutic options for many of these conditions. Specifically, human umbilical cord mesenchymal stem cells (MSCs) and cord blood (UCB) cells have shown promise in treating adult-onset diseases. Unlike bone-marrow and embryonic derived stem cells, umbilical cord-derived cells are easily and humanely obtained, have low immunogenicity, and offer the potential of autologous therapy. While there are several studies to uphold the efficacy of umbilical cord MSCs in adult therapies, there remains an unmet need for the investigation of its use in treating neonates. The purpose of this review is to provide a summary of current information on the potential therapeutic benefits and clinical applicability of umbilical cord MSCs and UCB cells. Promising preclinical studies have now led to a research movement that is focusing on cell-based therapies for preterm infants.

IgA modulates respiratory dysfunction as a sequela to pulmonary chlamydial infection as neonates

Neonatal Chlamydia lung infections are associated with serious sequelae such as asthma and airway hyper-reactivity in children and adults. Our previous studies demonstrated the importance of Th-1 type cytokines, IL-12 and IFN-γ in protection against neonatal pulmonary chlamydial challenge; however, the role of the humoral arm of defense has not been elucidated. We hypothesized that B-cells and IgA, the major mucosal antibody, play a protective role in newborns against development of later life respiratory sequelae to Chlamydia infection. Our studies using neonatal mice revealed that all WT and IgA-deficient (IgA(-/-)) animals survived a sublethal pulmonary Chlamydia muridarum challenge at one day after birth with similar reduction in bacterial burdens over time. In contrast, all B-cell-deficient (μMT) mice succumbed to infection at the same challenge dose correlating to failure to control bacterial burdens in the lungs. Although IgA may not be important for bacterial clearance, we observed IgA(-/-) mice displayed greater respiratory dysfunction 5 weeks post challenge. Specifically, comparative respiratory functional analyses revealed a significant shift upward in P-V loops, and higher dynamic resistance in IgA(-/-) animals. This study provides insight(s) into the protective role of IgA in neonates against pulmonary chlamydial infection induced respiratory pathological sequelae observed later in life.