Daniel Boveda-Ruiz

Differential role of regulatory T cells in early and late stages of pulmonary fibrosis

Regulatory T cells (Tregs) are a specific subset of T lymphocytes that regulate the function of other subsets of lymphocytes. Contradictory results have been reported regarding the role of Tregs in lung fibrosis. We wished to clarify the role of Tregs in the early and late stages of bleomycin-induced lung fibrosis in mice by depleting them with anti-CD25+ antibody (PC61). Mice treated with PC61 in early stages had significantly decreased number of CD4+CD25+ T cells compared to mice treated with the isotype control. The number of inflammatory cells, the concentrations of collagen, TGFβ1, the content of collagen and hydroxyproline in lung tissue were significantly reduced in PC61-treated mice compared to mice treated with the isotype control group. Pathological examination of the lung also disclosed reduced fibrotic changes and decreased fibrosis score in the PC61 group compared to control group. By contrast, mice treated with PC61 in late stages of the disease showed more infiltration of inflammatory cells and higher fibrotic score and hydroxyproline content in the lungs than mice treated with the isotype control. Our results suggest that Tregs play a detrimental role in early stages but protective role in late stages of pulmonary fibrosis in mice.

Efficacy of a novel class of RNA interference therapeutic agents.

RNA interference (RNAi) is being widely used in functional gene research and is an important tool for drug discovery. However, canonical double-stranded short interfering RNAs are unstable and induce undesirable adverse effects, and thus there is no currently RNAi-based therapy in the clinic. We have developed a novel class of RNAi agents, and evaluated their effectiveness in vitro and in mouse models of acute lung injury (ALI) and pulmonary fibrosis. The novel class of RNAi agents (nkRNA®, PnkRNA™) were synthesized on solid phase as single-stranded RNAs that, following synthesis, self-anneal into a unique helical structure containing a central stem and two loops. They are resistant to degradation and suppress their target genes. nkRNA and PnkRNA directed against TGF-β1mRNA ameliorate outcomes and induce no off-target effects in three animal models of lung disease. The results of this study support the pathological relevance of TGF-β1 in lung diseases, and suggest the potential usefulness of these novel RNAi agents for therapeutic application.

Eosinophils Promote Epithelial to Mesenchymal Transition of Bronchial Epithelial Cells

Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling.

Development and Preclinical Efficacy of Novel Transforming Growth Factor-β1 Short Interfering RNAs for Pulmonary Fibrosis

Idiopathic pulmonary fibrosis is a chronic devastating disease of unknown etiology. No therapy is currently available. A growing body of evidence supports the role of transforming growth factor (TGF)-β1 as the major player in the pathogenesis of the disease. However, attempts to control its expression and to improve the outcome of pulmonary fibrosis have been disappointing. We tested the hypothesis that TGF-β1 is the dominant factor in the acute and chronic phases of pulmonary fibrosis and developed short interfering (si)RNAs directed toward molecules implicated in the disease. This study developed novel sequences of siRNAs targeting the TGF-β1 gene and evaluated their therapeutic efficacy in two models of pulmonary fibrosis: a model induced by bleomycin and a novel model of the disease developed spontaneously in mice overexpressing the full length of human TGF-β1 in the lungs. Intrapulmonary delivery of aerosolized siRNAs of TGF-β1 with sequences common to humans and rodents significantly inhibited bleomycin-induced pulmonary fibrosis in the acute and chronic phases of the disease and in a dose-dependent manner. Aerosolized human-specific siRNA also efficiently inhibited pulmonary fibrosis, improved lung function, and prolonged survival in human TGF-β1 transgenic mice. Mice showed no off-target effects after intratracheal administration of siRNA. These results suggest the applicability of these novel siRNAs as tools for treating pulmonary fibrosis in humans.

Direct effects of protein S in ameliorating acute lung injury

Summary.  Objective: Protein S may exert an anticoagulant activity by enhancing the anticoagulant activity of activated protein C and/or by directly inhibiting the prothrombinase complex. Protein S itself may also directly regulate inflammatory responses and apoptosis. The role of protein S in acute lung injury (ALI) was unknown. This study evaluated the effect of protein S on ALI in the mouse. Methods: Animal ALI was induced in C57/BL6 mice by intratracheal instillation of lipopolysaccharide (LPS). Mice were treated with protein S or saline by intraperitoneal injection 1 h before LPS instillation. Results: Activated protein or protein S alone and combined activated protein C + protein S therapy decreased inflammatory markers and cytokines in mice with acute lung injury. In LPS‐treated mice compared with controls ALI was induced as shown by significantly increased levels of total protein, tumor necrosis factor‐α, interleukin‐6 and monocyte chemoattractant protein‐1 in the bronchoalveolar lavage fluid. Mice with ALI treated with protein S had significantly decreased concentrations of tumor necrosis factor‐α and interleukin‐6 in the lung compared with untreated animals. Thrombin‐antithrombin III, a marker of the activity of the coagulation cascade, was unchanged. Protein S inhibited the expression of cytokines in vitro and increased activation of the Axl tyrosine kinase pathway in A549 epithelial cells. Conclusion: Protein S protects against LPS‐induced ALI, possibly by directly inhibiting the local expression of inflammatory cytokines without affecting coagulation.

Thrombin-Activatable Fibrinolysis Inhibitor Protects against Acute Lung Injury by Inhibiting the Complement System

Acute lung injury (ALI) is a devastating disease with an overall mortality rate of 30 to 40%. The coagulation/fibrinolysis system is implicated in the pathogenesis of ALI. Thrombin-activatable fibronolysis inhibitor (TAFI) is an important component of the fibrinolysis system. Recent studies have shown that the active form of TAFI can also regulate inflammatory responses by its ability to inhibit complement C3a, C5a, and osteopontin. We hypothesized that TAFI might have a protective role in ALI. To demonstrate this hypothesis, the development of ALI was compared between wild-type (WT) and TAFI-deficient mice. ALI was induced by intratracheal instillation of LPS. Control mice were treated with saline. Animals were killed 24 hours after LPS. The number of inflammatory cells and the concentration of total protein and inflammatory cytokines were significantly increased in bronchoalveolar lavage fluid from LPS-treated, TAFI-deficient mice compared with their WT counterparts. Significantly higher concentrations of C5a were found in bronchoalveolar lavage fluid and plasma in LPS-treated TAFI knockout mice compared with WT mice. Pretreatment with inhaled C5a receptor antagonist blocked the detrimental effects of TAFI deficiency to levels found in WT mice. Our results show that TAFI protects against ALI, at least in part, by inhibiting the complement system.

Role of Thrombin-Activatable Fibrinolysis Inhibitor in Allergic Bronchial Asthma

BackgroundBronchial asthma is an inflammatory disease of the airways. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase that besides inhibiting fibrinolysis, also regulates inflammatory processes. The only validated substrate known for TAFI is fibrin. In the present study we evaluated the role of TAFI in bronchial asthma by comparing the development of allergic bronchial asthma between wild-type (WT) and TAFI-deficient mice (KO).MethodsAsthmatic inflammation was induced by sensitization and challenge with ovalbumin in WT (WT/OVA) and TAFI KO (KO/OVA) mice. WT mice (WT/SAL) and TAFI KO (KO/SAL) were used as controls. Cytokines, markers of inflammation, and coagulation were measured in bronchoalveolar lavage fluid (BALF).ResultsAirway hyperresponsiveness was worse in KO/OVA mice than in WT/OVA mice or control mice. Markers of lung injury were significantly increased in BALF from KO/OVA mice compared to WT/OVA mice. Airway hyperresponsiveness and the BALF concentrations of IL-5 and osteopontin were significantly increased in KO/OVA mice compared to WT/OVA mice. Treatment of WT/OVA and KO/OVA mice with a C5a receptor antagonist significantly decreased hyperresponsiveness along with the BALF concentrations of total protein and C5a compared to untreated asthmatic mice.ConclusionThe results of this study suggest that TAFI plays a protective role in the pathogenesis of allergic inflammation probably by inhibiting the complement system.

Pulmonary hypertension is ameliorated in mice deficient in thrombin-activatable fibrinolysis inhibitor.

Summary.  Background: The fibrinolytic system has been implicated in the pathogenesis of pulmonary hypertension (PH). Thrombin‐activatable fibrinolysis inhibitor (TAFI) inhibits fibrinolysis and therefore its absence would be expected to increase fibrinolysis and ameliorate PH. Objective: The objective of the present study was to evaluate the effect of TAFI deficiency on pulmonary hypertension in the mouse. Methods and results: PH was induced in C57/Bl6 wild‐type (WT) or TAFI‐deficient (KO) mice by weekly subcutaneous treatment with 600 mg kg−1 monocrotaline (MCT) for 8 weeks. PH was inferred from right heart hypertrophy measured using the ratio of right ventricle‐to‐left ventricle‐plus‐septum weight [RV/(LV+S)]. Pulmonary vascular remodeling was analyzed by morphometry. TAFI‐deficient MCT‐treated and wild‐type MCT‐treated mice suffered similar weight loss. TAFI‐deficient MCT‐treated mice had reduced levels of total protein and tumor necrosis factor‐alpha (TNF‐α), interleukin‐6 (IL‐6), transforming growth factor‐β (TGF‐β) and monocyte chemoattractant protein‐1 (MCP‐1) in bronchial alveolar lavage compared with wild‐type MCT‐treated mice. The ratio of RV to (LV+S) weight was significantly higher in WT/MCT than in KO/MCT mice. The pulmonary artery wall area and vascular stenosis were both greater in MCT‐treated WT mice compared with MCT‐treated TAFI‐deficient mice. Conclusions: TAFI‐deficient MCT‐treated mice had less pulmonary hypertension, vascular remodeling and reduced levels of cytokines compared with MCT‐treated WT animals, possibly as a result of reduced coagulation activation.

High incidence of tumors in diabetic thrombin activatable fibrinolysis inhibitor and apolipoprotein E double-deficient mice

Summary.  Background: Activation of the complement system has been implicated in tumor growth. The antifibrinolytic protein, activated thrombin‐activatable fibrinolysis inhibitor (TAFIa), can modulate the activation of the complement system by inactivating the anaphylatoxins C3a and C5a. The apolipoprotein‐E (ApoE) genotype has been associated with carcinogenesis. Objective: The aim of this study was to evaluate whether TAFIa can affect the development of cancer in the ApoE‐deficient mouse model. Methods: TAFI and ApoE double‐knockout mice were generated. A group of mice was treated with the diabetogenic and carcinogenic compound streptozotocin (stz). Mice treated with saline, single knockout mice and wild‐type (wt) mice served as controls. Results: Six months after treatment with stz, mice were sacrificed. Hepatic tumors were found in male double‐knockout mice treated with stz but none was found in control animals that were not treated with stz or in single knockout of ApoE or wt animals. There was no significant difference in coagulation system activation between the groups of mice. The plasma concentrations of C5a, factor D and transforming growth factor‐β1 were increased in TAFI/ApoE double‐deficient mice treated with stz compared with the mice of the same genotype treated with saline. Conclusion: Apo‐E deficiency alone was not associated with tumors but the lack of TAFI appears to make the mice permissive for tumor formation in ApoE mice.