Resistin Pathway as Novel Mechanism of Post-lung Transplantation Bronchial Stenosis.

BACKGROUND: Bronchial stenosis remains a significant source of morbidity among lung transplant recipients. Though infection and anastomotic ischemia have been proposed etiologies of the development of bronchial stenosis, the pathophysiologic mechanism has not been well elucidated. METHODS: In this single-centered prospective study, from January 2013 through September 2015, we prospectively collected bronchoalveolar lavage (BAL) and endobronchial epithelial brushings from the direct anastomotic site of bronchial stenosis of bilateral lung transplant recipients who developed unilateral post-transplant bronchial stenosis. Endobronchial epithelial brushings from the contralateral anastomotic site without bronchial stenosis and BAL from bilateral lung transplant recipients who did not develop post-transplant bronchial stenosis were used as controls. Total RNA was isolated from the endobronchial brushings and real-time polymerase chain reaction reactions were performed. Electrochemiluminescence biomarker assay was used to measure 10 cytokines from the BAL. RESULTS: Out of 60 bilateral lung transplant recipients, 9 were found to have developed bronchial stenosis with 17 samples adequate for analysis. We observed a 1.56 to 70.8 mean-fold increase in human resistin gene expression in the anastomotic bronchial stenosis epithelial cells compared with nonstenotic airways. Furthermore, IL-1ß (21.76±10.96 pg/mL; control 0.86±0.44 pg/mL; P <0.01) and IL-8 levels (990.56±326.60 pg/mL; control 20.33±1.17 pg/mL; P <0.01) were significantly elevated in the BAL of the lung transplant patients who developed anastomotic bronchial stenosis. CONCLUSION: Our data suggest that the development of postlung transplantation bronchial stenosis may be in part mediated through the human resistin pathway by IL-1ß induced transcription factor nuclear factor-κß activation and downstream upregulation of IL-8 in alveolar macrophages. Further study is needed in the larger patient cohorts and to determine its potential therapeutic role in the management of post-transplant bronchial stenosis.

Human Resistin Induces Cardiac Dysfunction in Pulmonary Hypertension.

Background Cardiac failure is the primary cause of death in most patients with pulmonary arterial hypertension (PH). As pleiotropic cytokines, human resistin (Hresistin) and its rodent homolog, resistin-like molecule α, are mechanistically critical to pulmonary vascular remodeling in PH. However, it is still unclear whether activation of these resistin-like molecules can directly cause PH-associated cardiac dysfunction and remodeling. Methods and Results In this study, we detected Hresistin protein in right ventricular (RV) tissue of patients with PH and elevated resistin-like molecule expression in RV tissues of rodents with RV hypertrophy and failure. In a humanized mouse model, cardiac-specific Hresistin overexpression was sufficient to cause cardiac dysfunction and remodeling. Dilated hearts exhibited reduced force development and decreased intracellular Ca2+ transients. In the RV tissues overexpressing Hresistin, the impaired contractility was associated with the suppression of protein kinase A and AMP-activated protein kinase. Mechanistically, Hresistin activation triggered the inflammation mediated by signaling of the key damage-associated molecular pattern molecule high-mobility group box 1, and subsequently induced pro-proliferative Ki67 in RV tissues of the transgenic mice. Intriguingly, an anti-Hresistin human antibody that we generated protected the myocardium from hypertrophy and failure in the rodent PH models. Conclusions Our data indicate that Hresistin is expressed in heart tissues and plays a role in the development of RV dysfunction and maladaptive remodeling through its immunoregulatory activities. Targeting this signaling to modulate cardiac inflammation may offer a promising strategy to treat PH-associated RV hypertrophy and failure in humans.

The inflammatory role of dysregulated IRS2 in pulmonary vascular remodeling under hypoxic conditions.

Pulmonary hypertension (PH) is a devastating disease characterized by progressive elevation of pulmonary vascular resistance, right ventricular failure, and ultimately death. We have shown previously that insulin receptor substrate 2 (IRS2), a molecule highly critical to insulin resistance and metabolism, has an anti-inflammatory role in Th2-skewed lung inflammation and pulmonary vascular remodeling. Here, we investigated the hypothesis that IRS2 has an immunomodulatory role in human and experimental PH. Expression analysis showed that IRS2 was significantly decreased in the pulmonary vasculature of patients with pulmonary arterial hypertension and in rat models of PH. In mice, genetic ablation of IRS2 enhanced the hypoxia-induced signaling pathway of Akt and Forkhead box O1 (FOXO1) in the lung tissue and increased pulmonary vascular muscularization, proliferation, and perivascular macrophage recruitment. Furthermore, mice with homozygous IRS2 gene deletion showed a significant gene dosage-dependent increase in pulmonary vascular remodeling and right ventricular hypertrophy in response to hypoxia. Functional studies with bone marrow-derived macrophages isolated from homozygous IRS2 gene-deleted mice showed that hypoxia exposure led to enhancement of the Akt and ERK signaling pathway followed by increases in the pro-PH macrophage activation markers, vascular endothelial growth factor-A and arginase 1. Our data suggest that IRS2 contributes to anti-inflammatory effects by regulating macrophage activation and recruitment, which may limit the vascular inflammation, remodeling, and right ventricular hypertrophy that are seen in PH pathology. Restoring the IRS2 pathway may be an effective therapeutic approach for the treatment of PH and right heart failure.

Systemic evaluation and localization of resistin expression in normal human tissues by a newly developed monoclonal antibody.

Resistin and resistin-like molecules are pleiotropic cytokines that are involved in inflammatory diseases. Our previous work suggested that resistin has the potential to be used as a biomarker and therapeutic target for human pulmonary arterial hypertension. However, data are limited on the distribution of resistin in healthy human organs. In this study, we used our newly developed anti-human resistin (hResistin) antibody to immunohistochemically detect the expression, localization, and intracellular/extracellular compartmentalization of hResistin in a full human tissue panel from healthy individuals. The potential cross reactivity of this monoclonal anti-hResistin IgG1 with normal human tissues also was verified. Results showed that hResistin is broadly distributed and principally localized in the cytoplasmic granules of macrophages scattered in the interstitium of most human tissues. Bone marrow hematopoietic precursor cells also exhibited hResistin signals in their cytoplasmic granules. Additionally, hResistin labeling was observed in the cytoplasm of nervous system cells. Notably, the cytokine activity of hResistin was illustrated by positively stained extracellular material in most human tissues. These data indicate that our generated antibody binds to the secreted hResistin and support its potential use for immunotherapy to reduce circulating hResistin levels in human disease. Our findings comprehensively document the basal expression patterns of hResistin protein in normal human tissues, suggest a critical role of this cytokine in normal and pathophysiologic inflammatory processes, and offer key insights for using our antibody in future pharmacokinetic studies and immunotherapeutic strategies.

Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELM alpha) recruits bone marrow-derived cells to the murine pulmonary vasculature.

BACKGROUND: Pulmonary hypertension (PH) is a disease of multiple etiologies with several common pathological features, including inflammation and pulmonary vascular remodeling. Recent evidence has suggested a potential role for the recruitment of bone marrow-derived (BMD) progenitor cells to this remodeling process. We recently demonstrated that hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELM alpha) is chemotactic to murine bone marrow cells in vitro and involved in pulmonary vascular remodeling in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We used a mouse bone marrow transplant model in which lethally irradiated mice were rescued with bone marrow transplanted from green fluorescent protein (GFP)(+) transgenic mice to determine the role of HIMF in recruiting BMD cells to the lung vasculature during PH development. Exposure to chronic hypoxia and pulmonary gene transfer of HIMF were used to induce PH. Both models resulted in markedly increased numbers of BMD cells in and around the pulmonary vasculature; in several neomuscularized small (approximately 20 microm) capillary-like vessels, an entirely new medial wall was made up of these cells. We found these GFP(+) BMD cells to be positive for stem cell antigen-1 and c-kit, but negative for CD31 and CD34. Several of the GFP(+) cells that localized to the pulmonary vasculature were alpha-smooth muscle actin(+) and localized to the media layer of the vessels. This finding suggests that these cells are of mesenchymal origin and differentiate toward myofibroblast and vascular smooth muscle. Structural location in the media of small vessels suggests a functional role in the lung vasculature. To examine a potential mechanism for HIMF-dependent recruitment of mesenchymal stem cells to the pulmonary vasculature, we performed a cell migration assay using cultured human mesenchymal stem cells (HMSCs). The addition of recombinant HIMF induced migration of HMSCs in a phosphoinosotide-3-kinase-dependent manner. CONCLUSIONS/SIGNIFICANCE: These results demonstrate HIMF-dependent recruitment of BMD mesenchymal-like cells to the remodeling pulmonary vasculature.

Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMalpha) induces the vascular and hemodynamic changes of pulmonary hypertension.

Pulmonary hypertension (PH) is a serious disease of multiple etiologies mediated by hypoxia, immune stimuli, and elevated pulmonary pressure that leads to vascular thickening and eventual right heart failure. In a chronic hypoxia model of PH, we previously reported the induction of a novel pleiotropic cytokine, hypoxia-induced mitogenic factor (HIMF), that exhibits mitogenic, vasculogenic, contractile, and chemokine properties during PH-associated vascular remodeling. To examine the role of HIMF in hypoxia-induced vascular remodeling, we performed in vivo knockdown of HIMF using short hairpin RNA directed at rat HIMF in the chronic hypoxia model of PH. Knockdown of HIMF partially blocked increases in mean pulmonary artery pressure, pulmonary vascular resistance, right heart hypertrophy, and vascular remodeling caused by chronic hypoxia. To demonstrate a direct role for HIMF in the mechanism of PH development, we performed HIMF-gene transfer into the lungs of rats using a HIMF-expressing adeno-associated virus (AAV). AAV-HIMF alone caused development of PH similar to that of chronic hypoxia with increased mean pulmonary artery pressure and pulmonary vascular resistance, right heart hypertrophy, and neomuscularization and thickening of small pulmonary arterioles. The findings suggest that HIMF represents a critical cytokine-like growth factor in the development of PH.

Cocaine induces a differential dose-dependent alteration in the expression profile of immediate early genes, transcription factors, and caspases in PC12 cells: a possible mechanism of neurotoxic damage in cocaine addiction.

Cocaine is a widely used drug of abuse and psychostimulant that acts on the central nervous system by blocking the dopamine reuptake sites. PC12 cells, a rat pheochromocytoma clonal line, in the presence of nerve growth factor (NGF), multiply and differentiate into competent neurons that can synthesize, store, and secrete the neurotransmitter dopamine (DA). In the present study, we evaluated the effect of increasing doses of cocaine on the expression of immediate early genes (IEGs), c-fos and c-jun, and closely related transcription factors, SP-1 and NF-kbeta, at 24 h after the exposure to cocaine (50, 100, 200, 500, 1000, 2500 microM) in NGF-differentiated PC12 cells. Cocaine (50-500 microM) resulted in significant induction of the expression of c-fos, c-jun, SP-1, and NF-kbeta. However, higher concentrations of cocaine (1000 and 2500 microM) resulted in the downregulation of these expressions after 24 h. To further understand the role of dose-dependent changes in the mechanisms of cell death, we evaluated the protein expression of apoptotic markers. A concentration-dependent increase in the expression of caspase-9 and -3 was observed up to 500 microM cocaine. However, the higher dose did not show any expression. We also evaluated the effect of increasing doses of cocaine on DA concentration and the expression of dopamine transporter (DAT). A significant dose-dependent decrease in the concentration of DA as well as the expression of DAT was observed 24 h after the exposure of PC12 cells to cocaine. Therefore, in the present study, we reported that cocaine has both upstream and downstream regulatory actions on some IEGs and transcription factors that can regulate the mechanism of cell death, and these effects on gene expression are independent of its action on the dopaminergic system.

Nitric oxide mediates increased susceptibility to dopaminergic damage in Nurr1 heterozygous mice.

Knocking out of Nurr1 gene, a member of nuclear receptor superfamily, causes selective agenesis of dopaminergic neurons in midbrain. Reduced expression of Nurr1 increases the vulnerability of mesencephalic dopamine neurons to dopaminergic toxins. We evaluated the role of nitric oxide as a possible mechanism for this increased susceptibility. Increased expression of neuronal nitric oxide synthase and increased 3-nitrotyrosine were observed in striatum of Nurr1 heterozygous (Nurr1 +/-) mice as compared with wild-type. Increased cytochrome C activation and consecutive release of Smac/DIABLO were also observed in Nurr1 +/- mice. An induction of active Caspase-3 and p53, cleavage of poly-ADP (RNase) polymerase and reduced expression of bcl-2 were observed in Nurr1 +/- mice. Methamphetamine significantly increased these markers in Nurr1 +/- mice as compared with wild-type. The present data therefore suggest that nitric oxide plays a role as a modulating factor for the increased susceptibility, but not the potentiation, of the dopaminergic terminals in Nurr1 +/- mice. We also report that this increased neuronal nitric oxide synthase expression and increased nitration in Nurr1 +/- mice led to the activation of apoptotic cascade via the differential alterations in the DNA binding activity of transcription factors responsible for the propagation of growth arrest as well as apoptosis.