Novel signaling pathways promote a paracrine wave of prostacyclin-induced vascular smooth muscle differentiation

The important athero-protective role of prostacyclin is becoming increasingly evident as recent studies have revealed adverse cardiovascular effects in mice lacking the prostacyclin receptor, in patients taking selective COX-2 inhibitors, and in patients in the presence of a dysfunctional prostacyclin receptor genetic variant. We have recently reported that this protective mechanism includes the promotion of a quiescent differentiated phenotype in human vascular smooth muscle cells (VSMC). Herein, we address the intriguing question of how localized endothelial release of the very unstable eicosanoid, prostacyclin, exerts a profound effect on the vascular media, often 30 cell layers thick. We report a novel PKA-, Akt-1- and ERK1/2-dependent prostacyclin-induced prostacyclin release that appears to play an important role in propagation of the quiescent, differentiated phenotype through adjacent arterial smooth muscle cells in the vascular media. Treating VSMC with the prostacyclin analog iloprost induced differentiation (contractile protein expression and contractile morphology), and also up-regulated COX-2 expression, leading to prostacyclin release by VSMC. This paracrine prostacyclin release, in turn, promoted differentiation and COX-2 induction in neighboring VSMC that were not exposed to iloprost. Using siRNA and pharmacologic inhibitors, we report that this positive feedback mechanism, prostacyclin-induced prostacyclin release, is mediated by cAMP/PKA signaling, ERK1/2 activation, and a novel prostacyclin receptor signaling pathway, inhibition of Akt-1. Furthermore, these pathways appear to be regulated by the prostacyclin receptor independently of one another. We conclude that prevention of de-differentiation and proliferation through a paracrine positive feedback mechanism is a major cardioprotective function of prostacyclin.     

Growth factor-induced signaling of the pancreatic epithelium

Activated signaling proteins regulate diverse processes, including the differentiation of the pancreatic islet cells during ontogeny. Here we uncover the in vivo phosphorylation status of major growth factor-activated signaling proteins in normal adult mice and during pancreatic islet regeneration. We report elevated phospho-mitogen-activated protein kinase (phospho-MAPK), phospho-c-Jun-NH2-terminal kinase (phospho-JNK), and phospho-p38 MAPK expression during pancreatic regeneration. Immunoblotting experiments demonstrated elevated phosphorylation of p52 Src-homology/collagen (SHC) in the ductal network as well, substantiating the activation of this pathway. Furthermore, protein kinase B (PKB/Akt), a key signaling protein in the anti-apoptotic pathway, was phosphorylated to a greater extent in the ductal network from regenerating pancreas. We observed fibroblast growht factor (FGF)10 and platelet-derived growth factor (PDGF)AA expression in embryonic as well as regenerating adult pancreas. Epidermal growth factor (EGF) and PDGFAA stimulated MAPK and Akt phosphorylation, while FGF10 stimulated MAPK but not Akt phosphorylation in a time-dependent manner in freshly isolated cells from the adult ductal network. These data suggest that a heightened level of expression and stimulation of key signaling proteins underlie the expansion and differentiation processes that support pancreatic ontogeny and regeneration.     

Acquired ciliary defects in bronchial epithelium of patients with chronic bronchitis

In order to quantify and classify ciliary changes of bronchial epithelium in chronic bronchitis, an ultrastructural study on biopsy specimens of 30 patients submitted to bronchoscopy was performed. Fourteen patients were affected by endoscopically and clinically confirmed chronic bronchitis, 8 showed an endoscopic picture of chronic bronchitis but without the clinical features of this pathology, 8 were free of chronic bronchitis. The mean percentage of abnormal cilia in control subjects (1.9 +/- 1.0%) was significantly lower than in patients either with only 'endoscopic' chronic bronchitis (5.7 +/- 2.6%; p less than 0.001) or with 'clinical' chronic bronchitis (14.8 +/- 15.9%; p less than 0.05). Like the ciliary body, the ciliary tip can also show some abnormalities. It is possible to subdivide them in two types: (1) ciliary membrane blebs; (2) ciliary membrane extrusions, variously shaped and sized, with homogeneous content. Bronchial cilia abnormalities become more serious as soon as the clinical picture worsens and this is likely to contribute to the impairment of the mucociliary clearance in chronic bronchitis patients.     

Adenosine-mediated cyclic AMP-dependent inhibition of ciliary activity in rabbit tracheal epithelium

We wished to determine whether adenosine, a purine nucleotide, modulates activity of respiratory cilia and, to this end, we studied cultured rabbit tracheal epithelium in response to adenosine and related substances in vitro. Ciliary beat frequency (CBF) as determined by a photoelectric method was depressed by adenosine (10(-3) M), the maximal decrease from the baseline value (965 +/- 29 beats/min, mean +/- SE) being 31.6 +/- 5.0% (p less than 0.001). The adenosine A2-receptor agonist N-ethylcarboxamide adenosine had only a small effect on ciliary activity, whereas other adenosine analogs elicited decreases in CBF in a dose-dependent fashion. The order of potency of cilia-inhibitory action was N-cyclohexyladenosine (an agonist for adenosine A1-receptor) greater than phenylisopropyladenosine greater than adenosine greater than N-ethylcarboxamide adenosine. Intracellular cyclic AMP (cAMP) levels were decreased by 10(-3) M adenosine from 39.2 +/- 6.5 to 25.3 +/- 4.8 pM/mg protein (p less than 0.05). The effect of adenosine on CBF was enhanced by dipyridamole, an adenosine uptake inhibitor, and by deoxycoformycin, an adenosine deaminase inhibitor. The adenosine-induced decreases in CBF and cAMP content were reversed by 8-phenyltheophylline, an adenosine receptor antagonist. These results suggest that there is an adenosine A1-receptor on rabbit tracheal epithelium that inhibits adenylate cyclase, which may result in the impairment of respiratory ciliary activity, and that adenosine-induced ciliary inhibition may be modulated by adenosine uptake and its catabolism by airway epithelial cells.     

p63RhoGEF regulates auto- and paracrine signaling in cardiac fibroblasts

Cardiac remodeling, a hallmark of heart disease, is associated with intense auto- and paracrine signaling leading to cardiac fibrosis. We hypothesized that the specific mediator of G_q/11-dependent RhoA activation p63RhoGEF, which is expressed in cardiac fibroblasts, plays a role in the underlying processes. We could show that p63RhoGEF is up-regulated in mouse hearts subjected to transverse aortic constriction (TAC). In an engineered heart muscle model (EHM), p63RhoGEF expression in cardiac fibroblasts increased resting and twitch tensions, and the dominant negative p63ΔN decreased both. In an engineered connective tissue model (ECT), p63RhoGEF increased tissue stiffness and its knockdown as well as p63ΔN reduced stiffness. In 2D cultures of neonatal rat cardiac fibroblasts, p63RhoGEF regulated the angiotensin II (Ang II)-dependent RhoA activation, the activation of the serum response factor, and the expression and secretion of the connective tissue growth factor (CTGF). All these processes were inhibited by the knockdown of p63RhoGEF or by p63ΔN likely based on their negative influence on the actin cytoskeleton. Moreover, we show that p63RhoGEF also regulates CTGF in engineered tissues and correlates with it in the TAC model. Finally, confocal studies revealed a closely related localization of p63RhoGEF and CTGF in the trans-Golgi network.     

Somatostatin modulates PI3K-Akt, eNOS and NHE activity in the ciliary epithelium

Somatostatin (SST) is a biologically active peptide produced in neuroendocrine cells. In the present study, we provide evidence of pro-SST and SST receptor (SSTR1 and 2A) mRNA expression in ocular ciliary epithelium (CE). SST or SST-like immunoreactivity was detected by radioimmunoassay in tissue extract from ciliary processes and in aqueous humor. The distinct immunolabeling of CE with SST and proprotein convertases PC1 and PC2 antibodies suggested a tissue and cell-specific processing of pro-SST. SST (10(-8) to 10(-4)M) added exogenously to the CE, elicited the following effects: (i) a dose-dependent attenuation of Na+/H+-exchanger (NHE) activity; (ii) up to a two-fold increase phosphorylation of p-Akt-Ser473 and of p-eNOS-Ser617, and (iii) lack of response on intracellular cyclic GMP production. LY294002, a PI3K-inhibitor, blocked SST-induced p-Akt-Ser473 and partially p-eNOS-Ser617, however, it did not reverse SST-induced NHE attenuation. Collectively, these results suggested involvement of SST in multiple intracellular signaling pathways in the CE.     

Measurement of frequency of ciliary beats of human respiratory epithelium

A method for accurate measurement of beats on ciliated respiratory epithelial cells in vitro is described. The technique allows the frequency of ciliary beats to be recorded directly on minute specimens of human respiratory epithelium obtained by bronchoscopic brushing. A "hanging-drop" preparation of the scraped material is placed on a thin, flat welled slide and is viewed with a phase-contrast microscope at a magnification of x450. Light transmitted through the specimen is deflected in varying intensities due to the beating action of the cilia and is detected by a photomultiplier cell, amplified and transduced as peaks on an oscilloscope. Due to the relatively high magnification, a small group of cilia may be focused upon and their activity measured. Specimens from two groups totaling 53 patients were measured; in the first group the frequency of ciliary beats was measured at 23 degrees C and ranged from 9.1 to 12.9 beats per second with a mean and standard deviation of 11.0 +/- 1.3 beats per second. The second group was measured at 37 degrees C, and values ranged from 10.3 to 16.8 beats per second, with a mean and standard deviation of 13.8 +/- 1.8 beats per second. These values are interpreted to reflect the autonomous frequency of ciliary beats of the isolated respiratory cells. This simple method may be applied to screen for abnormalities of ciliary beating in patients with altered respiratory mucous clearance, as well as to examine the specific effects in vitro of chemicals, drugs, or pollutants on human ciliary activity.     

Morphometric aspects of ciliary distribution and ciliogenesis in human nasal epithelium.

Observations of freeze-fracture preparations of human nasal epithelium have provided a unique perspective of the spatial distribution of epithelial cell cilia unattainable by more conventional ultrastructural techniques. The initial stages of ciliogenesis were characterized ultrastructurally in these preparations by differentiation of the lumenal aspect of the epithelial cell membrane prior to the emergence and maturation of new cilia. Morphometric analyses of the resultant electron micrographs indicate that the development of an optimal ciliary population during differentiation of ciliated cells may be integral to the adequate functioning of respiratory mucociliary mechanisms. The frequency with which such ciliogenic structures are observed indicates that ciliogenesis is a common feature of the nasal epithelium and suggests that epithelial cell turnover in the nasal cavities is relatively rapid.     

Effect of roxithromycin on ciliary motility of rabbit tracheal epithelium in culture]

Mucociliary transport plays an important role in the lung defense mechanism by clearing inhaled particles and bacteria from the respiratory tract. In the present study, we tried to determine whether roxithromycin (RXM), a new semisynthetic macrolide, can affect airway mucociliary functions and, if so, what the mechanism of its action is. To do so we measured ciliary beat frequency (CBF) of rabbit cultured tracheal epithelium by a photoelectric method. Addition of RXM (10(-4) M) to a Rose chamber containing epithelial cells caused a rapid increase in CBF from 12.1 +/- 0.6 to 16.2 +/- 0.8 Hz (mean +/- SE, p less than 0.001) within 5 min. This effect was dose-dependent, the maximal increase from the baseline CBF and EC50 being 34.6 +/- 5.0% and 3 x 10(-6)M, respectively. The increase in CBF produced by RXM (3 x 10(-6)M) was not influenced by pretreatment of cells with the beta-adrenergic blocker propranolol, the lipoxygenase inhibitor AA-861, or the Ca(2+)-channel blocker verapamil, but it was partially reduced by the cyclooxygenase inhibitor indomethacin (p less than 0.05). Intracellular cyclic AMP levels were increased by 10(-4)M RXM from 42.3 +/- 8.7 to 72.7 +/- 7.0 pmoles/mg protein (p less than 0.05). These results indicate that RXM stimulates ciliary motility; an effect that may be attributed at least in part to the synthesis of cyclooxygenase products and cyclic AMP, and suggest that this macrolide could be useful in treating patients with impaired mucociliary transport in the airway.     

Novel role of Rac1/WAVE signaling mechanism in regulation of the epithelial Na+ channel

The epithelial Na(+) channel (ENaC) is an essential channel responsible for Na(+) reabsorption in the aldosterone-sensitive distal nephron. Consequently, ENaC is a major effector impacting systemic blood volume and pressure. We have shown recently that Rac1 increases ENaC activity, whereas Cdc42 fails to change channel activity. Here we tested whether Rac1 signaling plays a physiological role in modulating ENaC in native tissue and polarized epithelial cells. We found that Rac1 inhibitor NSC23766 markedly decreased ENaC activity in freshly isolated collecting ducts. Knockdown of Rac1 in native principal cells decreased ENaC-mediated sodium reabsorption and the number of channels at the apical plasma membrane. Members of the Wiskott-Aldrich syndrome protein (WASP) family play a central role in the control of the actin cytoskeleton. N-WASP functions downstream of Cdc42, whereas WAVEs are effectors of Rac1 activity. N-WASP and all 3 isoforms of WAVE significantly increased ENaC activity when coexpressed in Chinese hamster ovary cells. However, wiskostatin, an inhibitor of N-WASP, had no effect on ENaC activity. Immunoblotting demonstrated the presence of WAVE1 and WAVE2 and absence of N-WASP and WAVE3 in mpkCCD(c14) and M-1 principal cells. Immunohistochemistry analysis also revealed localization of WAVE1 and WAVE2 but not N-WASP in the cortical collecting duct of Sprague-Dawley rat kidneys. Moreover, patch clamp analysis revealed that Rac1 and WAVE1/2 are parts of the same signaling pathway with respect to activation of ENaC. Thus, our findings suggest that Rac1 is essential for ENaC activity and regulates the channel via WAVE proteins.     

Effect of cyclic AMP on ciliary activity of human respiratory epithelium.

In order to investigate the effect of cyclic adenosine monophosphate (cAMP) on ciliary beat frequency (CBF) in human respiratory epithelium, cells were brushed from the inferior nasal turbinates of three groups of ten subjects: awake adults (aged 20-34 yrs), anaesthetized children (2-15 yrs) and anaesthetized adults (19-61 yrs). Cells from the awake adults were also studied after storage for 24 h in tissue culture medium. CBF was measured in vitro with a photometric technique at room temperature (22.0 +/- 1.5 degrees C). Samples were mounted in a perfusion chamber and challenged with either control solutions, dibutyryl cAMP (10(-4) or 10(-3) M), or the cyclic nucleotide-dependent protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H-7). Dibutyryl cAMP (10(-3) M) caused a significant increase in CBF in all groups studied: awake adults (+1.1 Hz; p less than 0.01), anaesthetized children (+1.2 Hz; p less than 0.01), anaesthetized adults (+1.2 Hz; p less than 0.01), stored cells (+1.1 Hz; p less than 0.01). This response was inhibited by preincubation with H-7 (10(-4) and 10(-3) M). It is concluded that cAMP is a regulator of ciliary activity in human respiratory epithelium.     

Effect of antitubercular preparations on the function of the ciliary epithelium of the respiratory tract mucosa

The effect of various concentrations of streptomycin, isoniazid, rifampicin and ethambutol on the function of the siliated epithelium of the frog oral mucosa was studied (110 experiments with 50 frogs). The level of inhibition of the siliated epithelium function depended on the properties of the drugs and their concentration in solution. More pronounced inhibition was induced by 15 per cent ethambutol solution and 10 per cent isoniazid solution. The least inhibition of the siliated epithelium function was observed with the use of 6.25 per cent streptomycin solution, 5 per cent isoniazid solution, 7.5 per cent ethambutol solution and 3.75 per cent rifampicin solution.     

Excitation-contraction coupling in ventricular myocytes is enhanced by paracrine signaling from mesenchymal stem cells

In clinical trials mesenchymal stem cells (MSCs) are transplanted into cardiac ischemic regions to decrease infarct size and improve contractility. However, the mechanism and time course of MSC-mediated cardioprotection are incompletely understood. We tested the hypothesis that paracrine signaling by MSCs promotes changes in cardiac excitation–contraction (EC) coupling that protects myocytes from cell death and enhances contractility. Isolated mouse ventricular myocytes (VMs) were treated with control tyrode, MSC conditioned-tyrode (ConT) or co-cultured with MSCs. The Ca handling properties of VMs were monitored by laser scanning confocal microscopy and whole cell voltage clamp. ConT superfusion of VMs resulted in a time dependent increase of the Ca transient amplitude (ConT_15min: ΔF/F₀ = 3.52 ± 0.38, n = 14; Ctrl_15min:? ΔF/F₀ = 2.41 ± 0.35, n = 14) and acceleration of the Ca transient decay (τ: ConT: 269 ± 18 ms n = 14; vs. Ctrl: 315 ± 57 ms, n = 14). Voltage clamp recordings confirmed a ConT induced increase in I_Ca,L (ConT: ? 5.9 ± 0.5 pA/pF n = 11; vs. Ctrl: ? 4.04 ± 0.3 pA/pF, n = 12). The change of τ resulted from increased SERCA activity. Changes in the Ca transient amplitude and τ were prevented by the PI3K inhibitors Wortmannin (100 nmol/L) and LY294002 (10 μmol/L) and the Akt inhibitor V (20 μmol/L) indicating regulation through PI3K signal transduction and Akt activation which was confirmed by western blotting. A change in τ was also prevented in eNOS^?/? myocytes or by inhibition of eNOS suggesting an NO mediated regulation of SERCA activity. Since paracrine signaling further resulted in increased survival of VMs we propose that the Akt induced change in Ca signaling is also a mechanism by which MSCs mediate an anti-apoptotic effect.     

Effect of Ca2+ on the ciliary beat frequency of skinned dog tracheal epithelium

Beat frequency of dog tracheal ciliated epithelium was measured using a profile projector and a photomultiplier. The preparation, treated with 50 micrograms/ml of saponin for 15 min, lost osmotic behavior and the ciliary beat came to depend on externally applied MgATP2- indicating that ciliated epithelium is skinned with saponin. Beat frequency of skinned cilia did not increase with Ca2+ in 0.1 mM MgATP2- with no ATP-regenerating system. Under 4 mM Mg ATP2- the beat frequency increased with an increase in Ca2+ from 0.3 to 10 microM, although a marked beat continued in the virtual absence of Ca2+ sensitivity and maximum beat frequency increased with the addition of 2.4 microM calmodulin. The effect of calmodulin inhibitor (W-7) on skinned preparations was somewhat weaker than that on intact ones. We concluded that Ca2+, within the physiological range of concentrations, directly activated the ciliary proteins and increased the ciliary beat frequency. The addition of calmodulin augments the effect of Ca2+ but the basal beat frequency is not Ca2+ dependent.     

Lymphotoxin beta receptor signaling induces the chemokine CCL20 in intestinal epithelium

BACKGROUND & AIMS: The follicle-associated epithelium (FAE) that overlies Peyer's patches (PPs) exhibits distinct features compared with the adjacent villus epithelium. Besides the presence of antigen-sampling membranous M cells and the down-regulation of digestive functions, it constitutively expresses the chemokine CCL20. The mechanisms that induce FAE differentiation and CCL20 expression are poorly understood. The aim of this work was to test whether lymphotoxin beta receptor signaling (LTbetaR), which plays a central role in PPs' organogenesis, mediates CCL20 gene expression in intestinal epithelial cells. METHODS: CCL20, lymphotoxin beta (LTbeta) and LTbetaR expression were monitored during embryonic development by in situ hybridization of mouse intestine. The human intestinal epithelial cell line T84 was used to study CCL20 expression following LTalpha(1)/beta(2) stimulation. In vivo CCL20 expression following agonistic anti-LTbetaR antibody treatment was studied by laser microdissection and quantitative RT-PCR. RESULTS: CCL20 was expressed in the FAE before birth at the time when the first hematopoietic CD4(+)CD3(-) appeared in the PP anlage. LTbetaR was expressed in the epithelium during PP organogenesis, making it a putative target for LTalpha(1)beta(2)signals. In vitro, CCL20 was induced in T84 cells upon LTbetaR signaling, either using an agonistic ligand or anti-LTbeta receptor agonistic antibody. LTalpha(1)beta(2)-induced CCL20 expression was found to be NF-kappaB dependent. LTbetaR signaling up-regulated CCL20 expression in the small intestinal epithelium in vivo. CONCLUSIONS: Our results show that LTbetaR signaling induces CCL20 expression in intestinal epithelial cells, suggesting that this pathway triggers constitutive production of CCL20 in the FAE.     

Vacuolar H+-ATPase in ocular ciliary epithelium

The mechanisms controlling the production of aqueous humor and the regulation of intraocular pressure are poorly understood. Here, we provide evidence that a vacuolar H⁺-ATPase (V-ATPase) in the ocular ciliary epithelium is a key component of this process. In intracellular pH (pH_i) measurements of isolated ciliary epithelium performed with 2′,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF), the selective V-ATPase inhibitor bafilomycin A₁ slowed the recovery of pH_i in response to acute intracellular acidification, demonstrating the presence of V-ATPase in the plasma membrane. In isolated rabbit ciliary body preparations examined under voltage-clamped conditions, bafilomycin A₁ produced a concentration-dependent decrease in short-circuit current, and topical application of bafilomycin A₁ reduced intraocular pressure in rabbits, indicating an essential role of the V-ATPase in ciliary epithelial ion transport. Immunocytochemistry utilizing antibodies specific for the B1 isoform of the V-ATPase 56-kDa subunit revealed localization of V-ATPase in both the plasma membrane and cytoplasm of the native ciliary epithelium in both rabbit and rat eye. The regional and subcellular distribution of V-ATPase in specific regions of the ciliary process was altered profoundly by isoproterenol and phorbol esters, suggesting that change in the intracellular distribution of the enzyme is a mechanism by which drugs, hormones, and neurotransmitters modify aqueous humor production.