Ana L. Mora

PINK1 deficiency impairs mitochondrial homeostasis and promotes lung fibrosis

Although aging is a known risk factor for idiopathic pulmonary fibrosis (IPF), the pathogenic mechanisms that underlie the effects of advancing age remain largely unexplained. Some age-related neurodegenerative diseases have an etiology that is related to mitochondrial dysfunction. Here, we found that alveolar type II cells (AECIIs) in the lungs of IPF patients exhibit marked accumulation of dysmorphic and dysfunctional mitochondria. These mitochondrial abnormalities in AECIIs of IPF lungs were associated with upregulation of ER stress markers and were recapitulated in normal mice with advancing age in response to stimulation of ER stress. We found that impaired mitochondria in IPF and aging lungs were associated with low expression of PTEN-induced putative kinase 1 (PINK1). Knockdown of PINK1 expression in lung epithelial cells resulted in mitochondria depolarization and expression of profibrotic factors. Moreover, young PINK1-deficient mice developed similarly dysmorphic, dysfunctional mitochondria in the AECIIs and were vulnerable to apoptosis and development of lung fibrosis. Our data indicate that PINK1 deficiency results in swollen, dysfunctional mitochondria and defective mitophagy, and promotes fibrosis in the aging lung.

Twist: a regulator of epithelial-mesenchymal transition in lung fibrosis.

Background Several studies have implicated viral infection as an important factor in the pathogenesis of IPF and related fibrotic lung disorders. Viruses are thought to cause epithelial cell injury and promote epithelial-mesenchymal transition (EMT), a process whereby differentiated epithelial cells undergo transition to a mesenchymal phenotype, and considered a source of fibroblasts in the setting of lung injury. We have demonstrated an association between the epithelial injury caused by chronic herpes virus infection with the murine γ-herpes virus, MHV68, and lung fibrosis. We hypothesize that EMT in this model of virus-induced pulmonary fibrosis is driven by the expression of the transcription factor Twist. Methods/Findings In vitro MHV68 infection of murine lung epithelial cells induced expression of Twist, and mesenchymal markers. Stable overexpression of Twist promoted EMT in MLE15 lung epithelial cells. Transient knockdown expression of Twist resulted in preservation of epithelial phenotype after in vitro MHV68 infection. In concordance, high expression of Twist was found in lung epithelial cells of MHV68 infected mice, but not in mock infected mice. Alveolar epithelial cells from lung tissue of idiopathic pulmonary fibrosis (IPF) patients were strongly positive for Twist. These cells demonstrated features of EMT with low expression of E-cadherin and upregulation of the mesenchymal marker N-cadherin. Finally, IPF tissue with high Twist protein levels was also positive for the herpesvirus, EBV. Conclusions/Significance We conclude that Twist contributes to EMT in the model of virus-induced pulmonary fibrosis. We speculate that in some IPF cases, γ-herpes virus infection with EBV might be a source of injury precipitating EMT through the expression of Twist.

Pulmonary Arterial Hypertension The Clinical Syndrome

Pulmonary arterial hypertension is a progressive disorder in which endothelial dysfunction and vascular remodeling obstruct small pulmonary arteries, resulting in increased pulmonary vascular resistance and pulmonary pressures. This leads to reduced cardiac output, right heart failure, and ultimately death. In this review, we attempt to answer some important questions commonly asked by patients diagnosed with pulmonary arterial hypertension pertaining to the disease, and aim to provide an explanation in terms of classification, diagnosis, pathophysiology, genetic causes, demographics, and prognostic factors. Furthermore, important molecular pathways that are central to the pathogenesis of pulmonary arterial hypertension are reviewed, including nitric oxide, prostacyclin, endothelin-1, reactive oxygen species, and endothelial and smooth muscle proliferation.

SIRT3–AMP-Activated Protein Kinase Activation by Nitrite and Metformin Improves Hyperglycemia and Normalizes Pulmonary Hypertension Associated With Heart Failure With Preserved Ejection Fraction

Background— Pulmonary hypertension associated with heart failure with preserved ejection fraction (PH-HFpEF) is an increasingly recognized clinical complication of metabolic syndrome. No adequate animal model of PH-HFpEF is available, and no effective therapies have been identified to date. A recent study suggested that dietary nitrate improves insulin resistance in endothelial nitric oxide synthase null mice, and multiple studies have reported that both nitrate and its active metabolite, nitrite, have therapeutic activity in preclinical models of pulmonary hypertension. Methods and Results— To evaluate the efficacy and mechanism of nitrite in metabolic syndrome associated with PH-HFpEF, we developed a 2-hit PH-HFpEF model in rats with multiple features of metabolic syndrome attributable to double-leptin receptor defect (obese ZSF1) with the combined treatment of vascular endothelial growth factor receptor blocker SU5416. Chronic oral nitrite treatment improved hyperglycemia in obese ZSF1 rats by a process that requires skeletal muscle SIRT3-AMPK-GLUT4 signaling. The glucose-lowering effect of nitrite was abolished in SIRT3-deficient human skeletal muscle cells, and in SIRT3 knockout mice fed a high-fat diet, as well. Skeletal muscle biopsies from humans with metabolic syndrome after 12 weeks of oral sodium nitrite and nitrate treatment (IND#115926) displayed increased activation of SIRT3 and AMP-activated protein kinase. Finally, early treatments with nitrite and metformin at the time of SU5416 injection reduced pulmonary pressures and vascular remodeling in the PH-HFpEF model with robust activation of skeletal muscle SIRT3 and AMP-activated protein kinase. Conclusions— These studies validate a rodent model of metabolic syndrome and PH-HFpEF, suggesting a potential role of nitrite and metformin as a preventative treatment for this disease.

Role of Endoplasmic Reticulum Stress in Age-Related Susceptibility to Lung Fibrosis

The incidence of idiopathic pulmonary fibrosis (IPF) increases with age. The mechanisms that underlie the age-dependent risk for IPF are unknown. Based on studies that suggest an association of IPF and γherpesvirus infection, we infected young (2-3 mo) and old (≥18 mo) C57BL/6 mice with the murine γherpesvirus 68. Acute murine γherpesvirus 68 infection in aging mice resulted in severe pneumonitis and fibrosis compared with young animals. Progressive clinical deterioration and lung fibrosis in the late chronic phase of infection was observed exclusively in old mice with diminution of tidal volume. Infected aging mice showed higher expression of transforming growth factor-β during the acute phase of infection. In addition, aging, infected mice showed elevation of proinflammatory cytokines and the fibrocyte recruitment chemokine, CXCL12, in bronchoalveolar lavage. Analyses of lytic virus infection and virus reactivation indicate that old mice were able to control chronic infection and elicit antivirus immune responses. However, old, infected mice showed a significant increase in apoptotic responses determined by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling assay, levels of caspase-3, and expression of the proapoptotitc molecule, Bcl-2 interacting mediator. Apoptosis of type II lung epithelial cells in aging lungs was accompanied by up-regulation of endoplasmic reticulum stress marker, binding immunoglobulin protein, and splicing of X-box-binding protein 1. These results indicate that the aging lung is more susceptible to injury and fibrosis associated with endoplasmic reticulum stress, apoptosis of type II lung epithelial cells, and activation of profibrotic pathways.

Aging Mesenchymal Stem Cells Fail to Protect Because of Impaired Migration and Antiinflammatory Response

RATIONALE Aging is characterized by functional impairment and reduced capacity to respond appropriately to environmental stimuli and injury. With age, there is an increase in the incidence and severity of chronic and acute lung diseases. However, the relationship between age and the lungs reduced ability to repair is far from established and necessitates further research in the field. OBJECTIVES Little is currently known about age-related phenomena in mesenchymal stem cells (MSCs). On account of their ability to protect the endothelium and the alveolar epithelium through multiple paracrine mechanisms, we looked for adverse effects that aging might cause in MSC biology. Such age-related changes might partly account for the increased susceptibility of the aging lung to injury. MEASUREMENTS AND MAIN RESULTS We demonstrated that old mice have more inflammation in response to acute lung injury. To investigate the causes, we compared the global gene expression of aged and young bone marrow-derived MSCs (B-MSCs). Our results revealed that the expression levels of inflammatory response genes depended on the age of the B-MSCs. We demonstrated that the age-dependent decrease in expression of several cytokine and chemokine receptors is important for the migration and activation of B-MSCs. Finally, we showed by adoptive transfer of aged B-MSCs to young endotoxemic mice that aged cells lacked the antiinflammatory protective effect of their young counterparts. CONCLUSIONS Taken together, the decreased expression of cytokine and chemokine receptors in aged B-MSCs compromises their protective role by perturbing the potential of B-MSCs to become activated and mobilize to the site of injury.

Use of Senescence-Accelerated Mouse Model in Bleomycin-Induced Lung Injury Suggests That Bone Marrow–Derived Cells Can Alter the Outcome of Lung Injury in Aged Mice

The incidence of pulmonary fibrosis increases with age. Studies from our group have implicated circulating progenitor cells, termed fibrocytes, in lung fibrosis. In this study, we investigate whether the preceding determinants of inflammation and fibrosis were augmented with aging. We compared responses to intratracheal bleomycin in senescence-accelerated prone mice (SAMP), with responses in age-matched control senescence-accelerated resistant mice (SAMR). SAMP mice demonstrated an exaggerated inflammatory response as evidenced by lung histology. Bleomycin-induced fibrosis was significantly higher in SAMP mice compared with SAMR controls. Consistent with fibrotic changes in the lung, SAMP mice expressed higher levels of transforming growth factor-beta1 in the lung. Furthermore, SAMP mice showed higher numbers of fibrocytes and higher levels of stromal cell-derived factor-1 in the peripheral blood. This study provides the novel observation that apart from increases in inflammatory and fibrotic factors in response to injury, the increased mobilization of fibrocytes may be involved in age-related susceptibility to lung fibrosis.

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

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.

A gammaherpesvirus-secreted activator of Vβ4+ CD8+ T cells regulates chronic infection and immunopathology

Little is known about herpesvirus modulation of T cell activation in latently infected individuals or the implications of such for chronic immune disorders. Murine gammaherpesvirus 68 (MHV68) elicits persistent activation of CD8+ T cells bearing a Vβ4+ T cell receptor (TCR) by a completely unknown mechanism. We show that a novel MHV68 protein encoded by the M1 gene is responsible for Vβ4+ CD8+ T cell stimulation in a manner reminiscent of a viral superantigen. During infection, M1 expression induces a Vβ4+ effector T cell response that resists functional exhaustion and appears to suppress virus reactivation from peritoneal cells by means of long-term interferon-γ (IFNγ) production. Mice lacking an IFNγ receptor (IFNγR−/−) fail to control MHV68 replication, and Vβ4+ and CD8+ T cell activation by M1 instead contributes to severe inflammation and multiorgan fibrotic disease. Thus, M1 manipulates the host CD8+ T cell response in a manner that facilitates latent infection in an immunocompetent setting, but promotes disease during a dysregulated immune response. Identification of a viral pathogenecity determinant with superantigen-like activity for CD8+ T cells broadens the known repertoire of viral immunomodulatory molecules, and its function illustrates the delicate balance achieved between persistent viruses and the host immune response.

Aging and lung injury repair: A role for bone marrow derived mesenchymal stem cells

The incidence of lung fibrosis increases with age. Aging is associated with modifications in the intracellular and extracellular environment including alteration of the extracellular matrix, imbalance of the redox state, accumulation of senescent cells and potential alteration of the recruitment of bone marrow mesenchymal stem cells. The combination of these senescence‐related alterations in the lung and in bone marrow progenitor cells might be responsible of the higher susceptibility to lung fibrosis in elderly individuals. The understanding of these age related changes must be considered in the rationale for the development of therapeutic interventions to control lung injury and fibrosis. J. Cell. Biochem. 105: 641–647, 2008.