ABSTRACT Idiopathic pulmonary fibrosis (IPF), one of the most common forms of interstitial lung disease, is a poorly understood, chronic, and often fatal fibroproliferative condition with only two FDA-approved medications. Understanding the pathobiology of the fibroblast in IPF is critical to evaluating and discovering novel therapeutics. Unfortunately, our ability to interrogate this biology in vitro is greatly limited by the well-documented effects of tissue culture plastic on the fibroblast phenotype. Using a decellularized lung matrix derived from IPF patients, we generate three-dimensional (3D) hydrogels as in vitro models of lung physiology and characterize the phenotype of fibroblasts seeded into the hydrogels. When cultured in our hydrogels, IPF fibroblasts display differential contractility compared to their normal counterparts, lose the classical myofibroblast marker α-smooth muscle actin, and increase expression of proinflammatory cytokines compared to fibroblasts seeded two-dimensionally (2D) on tissue culture dishes. We validate this proinflammatory state in fibroblast conditioned media studies with monocytes and monocyte-derived macrophages. These findings add to a growing understanding of the lung microenvironment effect on fibroblast phenotypes, shed light on the potential role of fibroblasts as immune signaling hubs during lung fibrosis, and suggest intervention in fibroblast-immune cell crosstalk as a possible novel therapeutic avenue.
Idiopathic Pulmonary Fibrosis (IPF) is a progressive disorder that is marked by an over accumulation of activated fibroblast populations. Despite the improved understanding of many mechanisms within this disease, global gene expression analysis has few focused studies on the fibroblast, the central effector cell of progressive fibrosis. We present a unique analysis of IPF pulmonary fibroblasts as they transition through cell culture and identify in vitro altered cellular processes. Fibroblasts were isolated from diseased (n = 8) and non-diseased (n = 4) lungs. Global gene expression analysis was carried out at the initial point of isolation and after 3 weeks of culture. We identify several genes that are altered by removal of the fibroblast from the IPF environment. Comparison of this subset of genes to four previously published whole lung analyses refined our list to a small subset of key fibroblast specific genes important in IPF. Application of STRING database analysis and confirmation via in-vitro and histological assay highlights the CXCL14/CXCR4 chemokine axis with a possible role in the progression and/or activation of fibroblasts within the IPF lung. Our findings, present a possible therapeutic target for IPF and a model for the study and discovery of novel protein and processes in this terrible disease.
Pulmonary fibrosis is a progressive, fatal manifestation of Hermansky-Pudlak syndrome (HPS). Some patients with advanced HPS pulmonary fibrosis undergo lung transplantation despite their disease-associated bleeding tendency; others die while awaiting donor organs. The objective of this study is to determine the clinical management and outcomes of a cohort with advanced HPS pulmonary fibrosis who were evaluated for lung transplantation. Six patients with HPS-1 pulmonary fibrosis were evaluated at the National Institutes of Health Clinical Center and one of two regional lung transplant centers. Their median age was 41.5 years pre-transplant. Three of six patients died without receiving a lung transplant. One of these was referred with end-stage pulmonary fibrosis and died before a donor organ became available, and donor organs were not identified for two other patients sensitized from prior blood product transfusions. Three of six patients received bilateral lung transplants; they did not have a history of excessive bleeding. One patient received peri-operative desmopressin, one was transfused with intra-operative platelets, and one received extracorporeal membrane oxygenation and intra-operative prothrombin complex concentrate, platelet transfusion, and desmopressin. One transplant recipient experienced acute rejection that responded to pulsed steroids. No evidence of chronic lung allograft dysfunction or recurrence of HPS pulmonary fibrosis was detected up to 6 years post-transplant in these three lung transplant recipients. In conclusion, lung transplantation and extracorporeal membrane oxygenation are viable options for patients with HPS pulmonary fibrosis. Alloimmunization in HPS patients is an important and potentially preventable barrier to lung transplantation; interventions to limit alloimmunization should be implemented in HPS patients at risk of pulmonary fibrosis to optimize their candidacy for future lung transplants.
Idiopathic Pulmonary Fibrosis (IPF) is a fatal lung disease of unknown etiology with only two federally approved drug options. Given the complex molecular pathogenesis of IPF involving multiple cell types and multiple pathways, we explore the effects of a potential antifibrotic and antioxidant drug combination. Curcumin is a polyphenolic compound derived from turmeric with significant biological activity including a potential antifibrotic capacity. N-acetylcysteine (NAC) is a precursor to the antioxidant glutathione. To advance our understanding of these molecules, and to identify a clinical application, we present a small number of focused experiments that interrogates the effect of curcumin and NAC on pathways relevant to IPF in both fibroblasts and epithelial cells. Primary epithelial cell and fibroblasts isolated from patients with IPF were challenged with a combination treatment of NAC and curcumin. Evaluation of the antifibrotic potential and effect on oxidative stress was performed through QPCR gene expression analysis and functional assays including scratch tests, viability assays, and measurement of induced reactive oxygen species. We demonstrate that curcumin alone does have antifibrotic potential, but that effect is accompanied by proapoptotic increases in oxidative stress. Coupled with this, we find that NAC alone can reduce oxidative stress, but that epithelial cell viability is decreased through this treatment. However, co-administration of these two molecules decreases oxidative stress and maintains high cell viability in both cell types. In addition, this co-treatment maintains an antifibrotic potential. These findings suggest a novel application for these molecules in IPF and encourage further exploration of this potential therapeutic approach.
Several trials have just demonstrated the therapeutic efficacy of gabexate mesylate (Foy). For this drug numerous mechanisms of action have been postulated; protease inhibition, direct or indirect lysosomal membrane stabilization, etc. The aim of this study has been to verify if Foy would express antioxidant properties against free radical overgeneration, responsible for the increase in microvascular permeability which represents one of the most important pathogenetic phenomenon of any condition of shock. A mixture of hypoxanthine-xanthine oxidase has been used to obtain a generation of oxygen free radicals in vivo.Experiments were performed on the mesocecum of male Wistar rats; fluorescent labelled bovine albumin has been injected intra-arterially to evaluate the capillary permeability and the mesocecum microcirculation has been observed by fluorescent microscopy. The control group received saline i.v., the second group received topically applied on the mesocecum a mixture of 0.96 mM hypoxanthine and 0.05 UI/ml xanthine oxidase; the third and the fourth groups were pretreated respectively with a topical application and continuous infusion of a Foy 1% solution (50 ml/kg/min). To evaluate capillary permeability and to quantify the degree of extravasation by counting the number of leaky sites, labelled bovine albumin was injected i.a. and mesocecum was observed with fluorescent microscopy for 2 hours.Gabexate mesilate (Foy) exerts its efficacy preventing the increase in capillary permeability provoked by an overgeneration of free radicals; it could express antioxidant properties.
Endotoxin administration increases capillary permeability in experimental animals and this is one of the most relevant events in the pathophysiology of endotoxin shock; the aim of the present experiment was to demonstrate that the action of endotoxin on capillary permeability is due to oxygen-free radicals generation. Experiments were performed on the mesocecum of male Wistar rats; fluorescent labeled bovine albumin (FITC-BSA) was injected intra-arterially to evaluate the capillary permeability; the mesocecum microcirculation was observed by fluorescent light. Permeability was quantified by changes in the number of leaky sites at 10 minute intervals for an hour. The effects of Endotoxin (DIFCO 0111:B4, 30 mg/kg i.a.) were assessed in rats receiving 1) saline, 2) reduced glutathione (250 or 500 mg/kg i.p.), 3) three different nitrones (PBN, DMPO, POBN) (6.25 mg/kg i.p.) whose action is to "trap" oxygen radicals. Capillary permeability largely increased in a few minutes in control rats but it was quite unaffected in rats receiving glutathione or nitrones. As a conclusion the increased capillary permeability observed after endotoxin injection in rats may be due to an oxygen-radicals generation.
In this study are considered the short-middle term results of anterior mitral leaflet prolapse repair obtained by means of a personal operative technique: chordal shortening and free edge remodeling. In our institution since 1993 34 consecutive patients with degenerative myxomatous mitral regurgitation, (mean age 63.3 years, range 25 to 83 years), underwent surgery. Before the operation 22 patients (64.7%) were in NYHA functional class III or IV. Mitral insufficiency, evaluated by echocardiogram, was severe in all patients; a prolapse of only anterior leaflet was present in 10 patients, both leaflets prolapsed in the others. Patients with chordal rupture of anterior mitral leaflet were excluded. Anterior mitral leaflet prolapse repair was performed with two continuous sutures including the free edge as well as the chordae for a variable length (2 mm up to 5 mm) depending on the degree of the elongation. A concomitant posterior leaflet quadrangular resection was performed in 24 patients (70.5%), and the procedure was almost always completed by a posterior suture annuloplasty reinforced by a glutaraldehyde-tanned strip of autologous pericardium. There were no perioperative deaths. The postoperative course was uneventful in all cases, and there were no hospital deaths. Postoperative echocardiographic evaluation showed satisfactory valve function. The mean valvular regurgitation before surgical procedure was 3.67 +/- 0.4, after repair 0.30 +/- 0.5 (p < 0.01). Follow-up was completed in all patients (mean 16.5 months) with no late deaths. One patient required early reoperation for recurrent mitral regurgitation resulting for a recurring anterior leaflet prolapse. We conclude that this technique is a safe, effective and easy procedure for the repair of anterior mitral leaflet prolapse without rupture. Nevertheless, a larger number of patients and a longer follow-up are required to confirm our results.
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