Allan Ramirez

Vitamin D inhibition of pro-fibrotic effects of transforming growth factor β1 in lung fibroblasts and epithelial cells

The mechanisms that control fibroproliferation and matrix deposition in lung fibrosis remain unclear. We speculate that vitamin D deficiency may contribute to pulmonary fibrosis since vitamin D deficiency has been implicated in several diseases. First, we confirmed the presence of vitamin D receptors (VDRs) in cultured NIH/3T3 and lung fibroblasts. Fibroblasts transfected with a vitamin D response element-reporter construct and exposed to the active vitamin D metabolite, 1,25(OH)(2)D(3), showed increased promoter activity indicating VDR functionality in these cells. Testing the effects of 1,25(OH)(2)D(3) on fibroblasts treated with transforming growth factor beta1 (TGFbeta1), considered a driver of many fibrotic disorders, we found that 1,25(OH)(2)D(3) inhibited TGFbeta1-induced fibroblast proliferation in a dose-dependent fashion. 1,25(OH)(2)D(3) also inhibited TGFbeta1 stimulation of alpha-smooth muscle actin expression and polymerization and prevented the upregulation of fibronectin and collagen in TGFbeta1-treated fibroblasts. Finally, we examined how 1,25(OH)(2)D(3) affects epithelial-mesenchymal transformation of lung epithelial cells upon exposure to TGFbeta1. We showed that the TGFbeta1-induced upregulation of mesenchymal cell markers and abnormal expression of epithelial cell markers were blunted by 1,25(OH)(2)D(3). These observations suggest that under TGFbeta1 stimulation, 1,25(OH)(2)D(3) inhibits the pro-fibrotic phenotype of lung fibroblasts and epithelial cells.

Efficacy of Oral Ribavirin in Lung Transplant Patients With Respiratory Syncytial Virus Lower Respiratory Tract Infection

BACKGROUND Respiratory syncytial virus (RSV) can cause severe lower respiratory tract infection (LRI) and is a risk factor for the development of bronchiolitis obliterans syndrome (BOS) after lung transplantation (LTx). Currently, the most widely used therapy for RSV is inhaled ribavirin. However, this therapy is costly and cumbersome. We investigated the utility of using oral ribavirin for the treatment of RSV infection after LTx. METHODS RSV was identified in nasopharyngeal swabs (NPS) or bronchoalveolar lavage (BAL) using direct fluorescent antibody (DFA) in 5 symptomatic LTx patients diagnosed with LRI. Data were collected from December 2005 and August 2007 and included: age; gender; type of LTx; underlying disease; date of RSV; pulmonary function prior to, during and up to 565 days post-RSV infection; need for mechanical ventilation; concurrent infections; and radiographic features. Patients received oral ribavirin for 10 days with solumedrol (10 to 15 mg/kg/day intravenously) for 3 days, until repeat NPS were negative. RESULTS Five patients had their RSV-LRI diagnosis made at a median of 300 days post-LTx. Mean forced expiratory volume in 1 second (FEV(1)) fell 21% (p < 0.012) during infection. After treatment, FEV(1) returned to baseline and was maintained at follow-up of 565 days. There were no complications and no deaths with oral therapy. A 10-day course of oral ribavirin cost

Pulmonary Arterial Hypertension in Systemic Sclerosis

700 compared with

Oxidation of extracellular cysteine/cystine redox state in bleomycin-induced lung fibrosis

14,000 for nebulized ribavirin at 6 g/day. CONCLUSIONS Treatment of RSV after LTx with oral ribavirin and corticosteroids is well tolerated, effective and less costly than inhaled ribavirin. Further studies are needed to directly compare the long-term efficacy of oral vs nebulized therapy for RSV.

Myofibroblast Transdifferentiation in Obliterative Bronchiolitis: TGF-β Signaling Through Smad3-Dependent and -Independent Pathways

It is increasingly recognized that significant pulmonary arterial hypertension (PAH) develops in more than 15% of patients with systemic sclerosis (SSc). As this complication of SSc may occur even in the absence of overt interstitial lung disease (isolated PAH), it has been likened to primary PAH and is attributable to intrinsic vascular pathology that is the hallmark of SSc. Deregulated activity of mediators controlling vasomotor tone has been implicated, and levels of endothelin-1 (ET-1) are elevated in the circulation and in the lungs. By causing enhanced vasoconstriction, vascular endothelial cell proliferation, smooth muscle hypertrophy, and irreversible vascular remodeling in the lungs, ET-1 appears to play a significant role in the pathogenesis of SSc-associated PAH.Although patients with the limited cutaneous form of SSc are more likely to develop PAH than those with the diffuse form, the true prevalence of PAH in SSc, and the risk factors for its development, are not yet known. Because the prognosis of patients with SSc-associated PAH is substantially worse than that of patients without this complication, intensive efforts are underway to develop sensitive screening strategies and effective treatments. Serial evaluation of SSc patients with Doppler echocardiography appears to be prudent. Antibodies against the centromere or fibrillarin proteins may be useful in identifying those patients with SSc at highest risk for developing PAH.The US FDA has approved a number of novel treatments, including long-acting oral ET-1 receptor antagonists such as bosentan and short-acting parenteral prostacyclin analogs, such as epoprostenol, for PAH. In particular, bosentan appear to be well tolerated, and short-term therapy results in improved exercise tolerance, improved hemodynamics, and possibly improved survival in patients with advanced PAH. These agents may be used alone, or possibly in combination with prostacyclin analogs. Therapeutic agents that modulate the synthesis of nitric oxide, and additional agents targeting the ET-1 signaling system are under preclinical development. Although the large-scale clinical trials that resulted in obtaining FDA approval for these agents were generally carried out in patients with primary PAH, it appears that patients with SSc-associated PAH respond similarly. Therefore, it is reasonable to conclude that ET-1 receptor antagonists and parenteral prostacyclin analogs should be used in SSc patients with moderate to severe PAH. The efficacy of these agents for treating patients with PAH who also experience significant interstitial lung disease, as occurs in many SSc patients, remains unknown. Additional important unresolved issues relate to the long-term efficacy of ET-1 receptor antagonists, and their effects on survival and progression of PAH. Additionally, it is not yet clear if early intervention for SSc patients with mild PAH is beneficial.

Smad3 Deficiency Ameliorates Experimental Obliterative Bronchiolitis in a Heterotopic Tracheal Transplantation Model

Several lines of evidence indicate that depletion of glutathione (GSH), a critical thiol antioxidant, is associated with the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, GSH synthesis depends on the amino acid cysteine (Cys), and relatively little is known about the regulation of Cys in fibrosis. Cys and its disulfide, cystine (CySS), constitute the most abundant low-molecular weight thiol/disulfide redox couple in the plasma, and the Cys/CySS redox state (E(h) Cys/CySS) is oxidized in association with age and smoking, known risk factors for IPF. Furthermore, oxidized E(h) Cys/CySS in the culture media of lung fibroblasts stimulates proliferation and expression of transitional matrix components. The present study was undertaken to determine whether bleomycin-induced lung fibrosis is associated with a decrease in Cys and/or an oxidation of the Cys/CySS redox state and to determine whether these changes were associated with changes in E(h) GSH/glutathione disulfide (GSSG). We observed distinct effects on plasma GSH and Cys redox systems during the progression of bleomycin-induced lung injury. Plasma E(h) GSH/GSSG was selectively oxidized during the proinflammatory phase, whereas oxidation of E(h) Cys/CySS occurred at the fibrotic phase. In the epithelial lining fluid, oxidation of E(h) Cys/CySS was due to decreased food intake. Thus the data show that decreased precursor availability and enhanced oxidation of Cys each contribute to the oxidation of extracellular Cys/CySS redox state in bleomycin-induced lung fibrosis.

Receptor for Advanced Glycation End Products in Donor Lungs Is Associated with Primary Graft Dysfunction After Lung Transplantation

We have shown that Smad3, an intracellular signal transducer for transforming growth factor‐β1 (TGF‐β1), is required to elicit the full histological manifestations of obliterative airway disease in a tracheal transplant model. This suggests that chronic allograft rejection results in TGF‐β1‐induced Smad3 activation that leads to airway obliteration through fibroproliferation and increased matrix deposition. In other systems, these latter events are causally related to the transdifferentiation of fibroblasts into myofibroblasts, but their role in obliterative bronchiolitis (OB) after lung transplantation is unknown. We confirmed the presence of myofibroblasts inside affected airways associated with experimental OB using immunohistochemistry. Studying airway fibroblasts in vitro, we observed increased myofibroblast transdifferentiation in response to TGF‐β1, evidenced by increased α‐smooth muscle actin mRNA and protein expression. In Smad3‐null fibroblasts, TGF‐β1 induction of myofibroblast transdifferentiation was greatly diminished but not abolished, suggesting the presence of Smad3‐independent pathways. Further studies revealed that small molecule inhibitors of p38 (SB203580) and MEK/ERK (U1026) further reduced the remaining effect of TGF‐β1 in Smad3‐deficient fibroblasts. Together, these studies suggest that in chronic allograft rejection, TGF‐β1 stimulates myofibroblast transdifferentiation through Smad3‐dependent and ‐independent signals, contributing to the excessive matrix deposition that characterizes obliterative bronchiolitis.

Hypoxia downregulates PPARγ via an ERK1/2–NF-κB–Nox4-dependent mechanism in human pulmonary artery smooth muscle cells

Chronic allograft rejection manifested as obliterative bronchiolitis (OB) remains the single greatest impediment to long-term survival after lung transplantation. Transforming growth factor-beta1 (TGF-beta1) has been implicated in the tissue remodeling response associated with OB. Therefore, its intracellular signal transducer, Smad3, is a prime target of investigation. Herein, we examine the role of TGF-beta1, through Smad3, in the development of OB using heterotopic tracheal transplantation in wild-type and Smad3-null mice. TGF-beta1 was detectable within infiltrating mononuclear cells early after transplantation. Later it was detected in fibroblasts and in the connective tissue accumulating within the lumen and the airway wall of the transplanted allografts. Connective tissue growth factor had a similar time and tissue distribution. Nuclear detection of Smad3 and phosphorylated Smads within intraluminal fibroblasts coincided with increased intraluminal deposition of fibronectin and collagen. When transplanted into Smad3-null mice, allografts failed to organize the intraluminal exudates despite fibroblast accumulation and showed reduced fibronectin and collagen deposition. In culture, Smad3-deficient fibroblasts expressed reduced fibronectin in response to TGF-beta1 compared to wild-type cells. Together, these studies suggest that the TGF-beta signal transducer, Smad3, is required for the development of experimental OB in transplanted tracheas.

Gastroesophageal Reflux Disease Is Associated With an Increased Rate of Acute Rejection in Lung Transplant Allografts

Development of primary graft dysfunction (PGD) is associated with poor outcomes after transplantation. We hypothesized that Receptor for Advanced Glycation End‐products (RAGE) levels in donor lungs is associated with the development of PGD. Furthermore, we hypothesized that RAGE levels would be increased with PGD in recipients after transplantation. We measured RAGE in bronchoalveolar lavage fluid (BALf) from 25 donors and 34 recipients. RAGE was also detected in biopsies (transbronchial biopsy) from recipients with and without PGD. RAGE levels were significantly higher in donor lungs that subsequently developed sustained PGD versus transplanted lungs that did not display PGD. Donor RAGE level was a predictor of recipient PGD (odds ratio = 1.768 per 0.25 ng/mL increase in donor RAGE level). In addition, RAGE levels remained high for 14 days in those recipients that developed severe graft dysfunction. Recipients may be at higher risk for developing PGD if they receive transplanted organs that have higher levels of soluble RAGE prior to explantation. Moreover, the clinical and pathologic abnormalities associated with PGD posttransplantation are associated with increased RAGE expression. These findings also raise the possibility that targeting the RAGE signaling pathway could be a novel strategy for treatment and/or prevention of PGD.

Dynamics of Human Regulatory T Cells in Lung Lavages of Lung Transplant Recipients

The ligand-activated transcription factor peroxisome proliferator-activated receptor γ (PPARγ) regulates metabolism, cell proliferation, and inflammation. Pulmonary hypertension (PH) is associated with reduced PPARγ expression, and hypoxia exposure regimens that cause PH reduce PPARγ expression. This study examines mechanisms of hypoxia-induced PPARγ downregulation in vitro and in vivo. Hypoxia reduced PPARγ mRNA and protein levels, PPARγ activity, and the expression of PPARγ-regulated genes in human pulmonary artery smooth muscle cells (HPASMCs) exposed to 1% oxygen for 72 h. Similarly, exposure of mice to hypoxia (10% O₂) for 3 weeks reduced PPARγ mRNA and protein in mouse lung. Inhibiting ERK1/2 with PD98059 or treatment with siRNA directed against either NF-κB p65 or Nox4 attenuated hypoxic reductions in PPARγ expression and activity. Furthermore, degradation of H₂O₂ using PEG-catalase prevented hypoxia-induced ERK1/2 phosphorylation and Nox4 expression, suggesting sustained ERK1/2-mediated signaling and Nox4 expression in this response. Mammalian two-hybrid assays demonstrated that PPARγ and p65 bind directly to each other in a mutually repressive fashion. We conclude from these results that hypoxic regimens that promote PH pathogenesis and HPASMC proliferation reduce PPARγ expression and activity through ERK1/2-, p65-, and Nox4-dependent pathways. These findings provide novel insights into mechanisms by which pathophysiological stimuli such as hypoxia cause loss of PPARγ activity and pulmonary vascular cell proliferation, pulmonary vascular remodeling, and PH. These results also indicate that restoration of PPARγ activity with pharmacological ligands may provide a novel therapeutic approach in selected forms of PH.