Stimulation of ß2-adrenergic receptor increases CFTR function and decreases ATP levels in murine hematopoietic stem/progenitor cells.

BACKGROUND: The chloride channel CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) is expressed by many cell types, including hematopoietic stem/progenitor cells (HSPCs). In this study, we sought to better comprehend the regulation of CFTR activity in HSPCs, namely by beta-adrenergic stimuli. METHODS: The expression of ß2-adrenergic receptor (ß2-AR) in murine Sca-1(+) HSPCs was investigated by immunofluorescence/confocal microscopy and flow-cytometric analysis. Association with CFTR was assessed by immunoprecipitation. HSPCs were evaluated for ATP content and CFTR activity by means of luminometric and spectrofluorometric methods, respectively, upon stimulation with salbutamol. RESULTS: HSPCs express ß2-AR over the whole plasma membrane and are associated in cellula with both the immature and mature forms of CFTR. ß2-AR was predominantly expressed by HSPCs with bigger size. CFTR channel activity was increased by salbutamol treatment and this activation was inhibited by either a specific CFTR inhibitor (CFTRinh172) or a ß2-AR receptor inhibitor (ICI 118,551). Intracellular ATP levels were reduced by salbutamol stimulation and this effect was reversed when ICI 118,551 or CFTR inhibitors were present. A trend in the increase of extracellular ATP upon salbutamol stimulation was observed. CONCLUSIONS: In HSPCs, CFTR is regulated by ß2-adrenergic receptor stimulation determining intracellular ATP depletion.

Hematopoietic and mesenchymal stem cells for the treatment of chronic respiratory diseases: role of plasticity and heterogeneity.

Chronic lung diseases, such as cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease (COPD) are incurable and represent a very high social burden. Stem cell-based treatment may represent a hope for the cure of these diseases. In this paper, we revise the overall knowledge about the plasticity and engraftment of exogenous marrow-derived stem cells into the lung, as well as their usefulness in lung repair and therapy of chronic lung diseases. The lung is easily accessible and the pathophysiology of these diseases is characterized by injury, inflammation, and eventually by remodeling of the airways. Bone marrow-derived stem cells, including hematopoietic stem/progenitor cells (HSPCs) and mesenchymal stromal (stem) cells (MSCs), encompass a wide array of cell subsets with different capacities of engraftment and injured tissue regenerating potential. Proof-of-principle that marrow cells administered locally may engraft and give rise to specialized epithelial cells has been given, but the efficiency of this conversion is too limited to give a therapeutic effect. Besides the identification of plasticity mechanisms, the characterization/isolation of the stem cell subpopulations represents a major challenge to improving the efficacy of transplantation protocols used in regenerative medicine for lung diseases.

Effect of acute lung injury on VLA-4 and CXCR4 expression in resident and circulating hematopoietic stem/progenitor cells.

BACKGROUND: The effect of acute lung injury on adhesion molecule expression in hematopoietic stem/progenitor cells (HSPCs) is poorly understood. OBJECTIVES: The aim of this study was to determine whether there is a relationship -between pulmonary inflammation, expression of VLA-4 (CD49d), LFA-1 (CD11a), L-selectin (CD62L), CXCR4, and chemotaxis in resident HSPCs, as well as the level of circulating HSPCs. METHODS: Following intratracheal administration of a single LPS bolus in C57Bl/6 mice, the number of inflammatory cells, differential counts, and amounts of cytokines/ chemokines were studied in cytospins and bronchoalveolar lavage fluid (BALF) specimens. Expressions of adhesion -molecules and CXCR4 were analyzed in HSPCs by flow cytometry, as well as SDF-1-directed chemotaxis. Levels of HSPCs in the blood were studied in ungated and circulating subpopulations. RESULTS: In coincidence with a peak of airway neutrophils, cytokine (IL-1ß, TNF-α, and IL-6), chemokine (KC, MIP-2, and SDF-1) levels in BALF and the number of marrow HSPCs expressing CD49d and CXCR4 significantly increased at 48 h. The number of CD49d- and CXCR4-positive HSPCs dropped at 72 h. The HSPC subset comprising bigger cells behaved the same for CD49d. Chemotaxis of the marrow HSPC subset of bigger cells was higher in LPS-treated animals than in controls at 72 h. Finally, we could detect a significant decrease in circulating Sca-1(+) cells in the mononuclear population at 72 h in LPS-treated mice. CONCLUSIONS: Our data provide evidence for a temporal relationship between pulmonary inflammation, CD49d and CXCR4 expression fluctuation in resident HSPCs, and the level of circulating HSPCs.

Hematopoietic stem/progenitor cells express functional mitochondrial energy-dependent cystic fibrosis transmembrane conductance regulator.

Bone marrow-derived hematopoietic stem/progenitor cells (HSPCs) encompass a wide array of cell subsets with different capacities of engraftment and injured tissue-regenerating potential. The characterization/isolation of the stem cell subpopulations represents a major challenge to improve the efficacy of transplantation protocols used in regenerative medicine. Cystic fibrosis (CF) is one of the diseases whose hope of cure relies on the successful application of cell-based gene therapy. This study was aimed at characterizing murine HSPCs on the basis of their bioenergetic competence and CF transmembrane conductance regulator (CFTR) expression. Positively immunoselected Sca-1(+) HSPCs encompassed 2 populations distinguished by their different size, Sca-1 expression and mitochondrial content. The smaller were the cells, the higher was Sca-1 expression and the lower was the intracellular density of functional mitochondria. Reverse transcription-polymerase chain reaction and western blotting revealed that HSPCs expressed CFTR mRNA and protein, which was also functional, as assessed by spectrofluorimetric and patch-clamp techniques. Inhibition of mitochondrial oxidative phosphorylation by oligomycin resulted in a 70% decrease of both the intracelluar adenosine triphosphate content and CFTR-mediated channel activity. Finally, HSPCs with lower Sca-1 expression and higher mitochondrial content displayed higher CFTR levels. Our findings identify 2 subpopulations in HSPCs and unveil a so-far unappreciated relationship between bioenergetic metabolism and CFTR in HSPC biology.

Stem cell therapy for cystic fibrosis: current status and future prospects.

Although cystic fibrosis (CF), an autosomal recessive disease caused by mutations in the gene encoding for the CF transmembrane conductance regulator (CFTR), seems a good candidate for gene therapy, 15 years of intense investigation and a number of clinical trials have not yet produced a viable clinical gene-therapy strategy. In addition, the duration of gene expression has been shown to be limited, only lasting 1-4 weeks. Therefore, alternative approaches involve the search for, and use of, stem cell populations. Bone marrow contains different stem cell types, including hematopoietic stem cells and multipotent mesenchymal stromal cells. Numerous studies have now demonstrated the ability of hematopoietic stem cells and mesenchymal stromal cells to home to the lung and differentiate into epithelial cells of both the conducting airways and the alveolar region. However, engraftment of bone marrow-derived stem cells into the airways is a very inefficient process. Detailed knowledge of the cellular and molecular determinants governing homing to the lung and transformation of marrow cells into lung epithelial cells would benefit this process. Despite a very low level of engraftment of donor cells into the nose and gut, significant CFTR mRNA expression and a measurable level of correction of the electrophysiological defect were observed after transplantation of wild-type marrow cells into CF mice. It is uncertain whether this effect is due to the presence of CFTR-expressing epithelial cells derived from donor cells or to the immunomodulatory role of transplanted cells. Finally, initial studies on the usefulness of umbilical cord blood and embryonic stem cells in the generation of airway epithelial cells will be discussed in this review.

Profibrinolytic activity of the direct thrombin inhibitor melagatran and unfractionated heparin in platelet-poor and platelet-rich clots.

Anticoagulants have been shown to stimulate fibrinolysis principally via inhibition of thrombin-mediated activation of TAFI (thrombin activatable fibrinolysis inhibitor). Their profibrinolytic effect, however, may vary according to their mechanism of action and to the clot composition. We compared the fibrinolytic activity of the direct thrombin inhibitor melagatran with that of unfractionated heparin in platelet-poor (PPP) and platelet-rich (PRP) models consisting of tissue-factor-induced clots exposed to exogenous t-PA (25 ng/ml). In the PPP clot model, both heparin (0.1-0.6 U/ml) and melagatran (20-320 ng/ml) caused a concentration-dependent shortening of lysis time. However, when drug profibrinolytic activity (lysis ratio) was expressed in function of the aPTT prolongation (aPTT ratio), melagatran was more efficient than heparin. In the PRP clot model, melagatran displayed a fibrinolytic activity fairly comparable to that observed in PPP whilst heparin caused a modest reduction of lysis time only at the highest concentrations. Assay of thrombin and TAFIa generation in defibrinated plasma showed that the presence of platelets markedly reduced the ability of heparin, but not that of melagatran, to inhibit the formation of these enzymes. Altogether these data indicate that melagatran is more efficient than heparin in promoting fibrinolysis, particularly in plateletrich clots, and may thus grant a greater antithrombotic activity by enhancing thrombus dissolution.

An antifibrinolytic effect associated with an anti-factor V antibody in a patient with severe thrombophilia.

BACKGROUND AND OBJECTIVES: The case of a patient with thrombotic manifestations and severe activated protein C resistance due to an anti-factor V antibody has recently been described. Since activated protein C (APC) is also profibrinolytic we wanted to determine whether the presence of antibodies interfering with the anticoagulant activity of APC also inhibits its profibrinolytic effect. DESIGN AND METHODS: Plasma clots were formed in the presence of tissue plasminogen activator, thrombin, phospholipids, Ca++, and various concentrations of APC, and the rate of lysis was monitored over time by the reduction in turbidity. Generation of endogenous thrombin and activation of thrombin activatable fibrinolysis inhibitor (TAFI) were also determined during fibrinolysis by clotting and spectrophotometric assays, respectively. RESULTS: Addition of APC to the patient's plasma failed to stimulate fibrinolysis even at a concentration 4 times higher than that needed to produce the maximal effect in control plasma. Removal of IgG from the patient's plasma fully restored the fibrinolytic response to APC. Accordingly, addition of the patient's IgG to control plasma caused a concentration-dependent inhibition of APC-dependent fibrinolysis. The patient's IgG did not, however, inhibit the profibrinolytic effect of heparin. Determination of thrombin and activated TAFI generation during clot lysis showed that APC inhibited the generation of these enzymes by less than 20% in plasma supplemented with the patient's IgG as opposed to >80% in a control sample. INTERPRETATION AND CONCLUSIONS: Our data suggest that the anti-factor V antibody inhibits fibrinolysis by antagonizing the anticoagulant effect of APC thereby favoring thrombin generation and TAFI activation. Impaired fibrinolysis may represent an additional mechanism contributing to thrombosis in patients with severe APC resistance phenotype.

Effect of heparin on TAFI-dependent inhibition of fibrinolysis: relative importance of TAFIa generated by clot-bound and fluid phase thrombin.

Heparin has been proposed to enhance thrombolysis by inhibiting thrombin-dependent generation of activated TAFI (thrombin activatable fibrinolysis inhibitor), a carboxypeptidase that inhibits fibrinolysis. We evaluated the effect of heparin in an in vitro thrombolysis model consisting of a radiolabelled blood clot submerged in defibrinated plasma. Fibrinolysis was induced by adding t-PA (250 ng/ml) and calcium to the plasma bath. Control experiments indicated that thrombin generation induced by recalcification caused significant TAFI activation and inhibited clot lysis. Heparin (up to 1 U/ml), added to the plasma bath, failed to enhance clot lysis. Thrombin generation in the fluid phase was totally inhibited by heparin at concentrations > 0.5 U/ml. In contrast, thrombin generation on the clot surface was not inhibited by heparin (1 U/ml). TAFIa generation did occur in heparin-containing samples (1 U/ml) and amounted to about 10% of TAFIa formed in control samples. This low amount of TAFIa did exert antifibrinolytic activity as indicated by the observation that the addition of a specific TAFIa inhibitor (PTI) along with heparin enhanced clot lysis. Hirudin (10 micrograms/ml), at variance with heparin, inhibited clot-bound thrombin and enhanced clot lysis. These data show that heparin is unable to stimulate fibrinolysis through a TAFI-dependent mechanism, most likely because of its inefficiency in inhibiting thrombin generation on the clot surface. Moreover, they suggest that clot-bound thrombin plays a major role in TAFI-mediated inhibition of fibrinolysis through "localized" TAFIa generation.