Hanzhong Liu

Oxidants and Antioxidants

Oxidants are an important source of injury to cells and tissues. The lung is exposed to significantly more oxidants than are most other organs. The lung is unique because of its large epithelial surface area that is directly exposed to high levels of oxygen tension, that is, oxygen pressure in inhaled air is 20 kPa (150 mm Hg). Ambient air contains additional oxidants, including cigarette smoke, asbestos fibers, mineral dust, and environmental carcinogens. A common component in most lung disease is activation of the inflammatory response, which leads to the generation of a relatively large quantity of oxidants. Even some therapeutic interventions, such as ventilation and oxygen therapy in the treatment of prematurely born neonates and acute respiratory distress syndrome, or chemotherapeutic agents, including bleomycin, carmustine, and anthracyclines, enhance oxidant burden to lung tissue.1 Thus, the lung represents a unique tissue exposed not only directly to external environmental oxidants under nor mal conditions but also to inflammation- and therapy-associated oxidants in disease state.

Sleeping Beauty-based gene therapy with indoleamine 2,3-dioxygenase inhibits lung allograft fibrosis

Sleeping Beauty (SB) transposon is a natural nonviral gene transfer system that can mediate long‐term transgene expression. Its potential utility in treating organ transplantation‐associated long‐term complications has not yet been explored. In the present study we generated an improved SB transposon encoding the human gene indoleamine‐2,3‐dioxygenase (hIDO), an enzyme that possesses both T cell‐suppressive and antioxidant properties and selectively delivered the SB transposon in combination with a hyperactive transposase plasmid to donor lung using the cationic polymer polyethylenimine (PEI) as transfection reagent. This nonviral gene therapeutic approach led to persistent and uniform transgene expression in the rat lung tissue without noticeable toxicity and inflammation. Importantly, IDO activity produced by hIDO transgene showed a remarkable therapeutic response, as evident by near normal pulmonary function (peak airway pressure and oxygenation), histological appearance, and reduced collagen content in lung allografts. In addition, we established a hIDO‐overexpressing type II cell line using the SB‐based gene transfer system and found that hIDO‐overexpressing lung cells effectively inhibited transforming growth factor–stimulated fibroblast proliferation in vitro. In summary, the SB‐based gene therapy with hIDO represents a new strategy for treating lung transplantation‐associated chronic complications, e.g., obliterative bronchiolitis.—Liu, H., Liu, L., Fletcher, B. S., Visner, G. A. Sleeping Beauty‐based gene therapy with indoleamine 2,3‐dioxygenase inhibits lung allograft fibrosis. FASEB J. 20, E1694 –E1703 (2006)

Heme oxygenase-1 mediates the protective effects of rapamycin in monocrotaline-induced pulmonary hypertension

Rapamycin inhibits the development and progression of vascular disease. We previously showed that rapamycin induces the cytoprotective protein, heme oxygenase-1 (HO-1), and more importantly, chemically inhibiting HO-1 blocked the antiproliferative actions of rapamycin. In this study, we evaluated whether HO-1 is required for the vascular protective effects of rapamycin in vivo using a rat monocrotaline-induced pulmonary hypertension model. Rats were exposed to monocrotaline with or without rapamycin and HO activity was altered using the chemical inhibitor, tin protoporphyrin or the inducer, cobalt protoporphyrin. We also evaluated possible mechanisms of rapamycin-dependent induction of HO-1, and how HO-1 mediates growth factor-dependent antiproliferative actions of rapamycin. Proliferation and cell cycle progression were examined in smooth muscle cells derived from both wild-type and HO-1 knockout (HO-1−/−) mice in response to growth factors and rapamycin. Similar to our previous findings in vitro, rapamycin induced HO-1 in rat lung. Rapamycin also inhibited the development of monocrotaline-induced pulmonary hypertension, and this protective effect was blocked with the addition of tin protoporphyrin. In addition, treatment with cobalt protoporphyrin resulted in a substantial protection in this model of pulmonary hypertension. Rapamycin induction of HO-1 was dependent upon a transcriptional event; however, it was not mediated through an altered redox state or mammalian targets of rapamycin inhibition. Unlike wild-type cells, the growth of HO-1−/− mouse aortic smooth muscle cells was not inhibited or cell cycle arrested in G1 in response to rapamycin. This study demonstrates that HO-1 is critical for the antiproliferative and vascular protective effects of rapamycin in vitro and in vivo in monocrotaline-induced pulmonary hypertension.

Pirfenidone Inhibits Lung Allograft Fibrosis through L-Arginine-Arginase Pathway

Transplant‐related lung fibrosis is characterized by excessive fibro‐collagenous deposition. Induction of arginase, an enzyme that metabolizes L‐arginine to urea and L‐ornithine, is vital for collagen synthesis. Pirfenidone is an investigational anti‐fibrotic agent shown to be effective in blocking pulmonary fibrosis. The purpose of this study was to determine if pirfenidone was protective against the development of fibro‐collagenous injury in rat lung orthotopic transplants through altering L‐arginine–arginase metabolic pathways. Lung transplants were performed using Lewis donors and Sprague‐Dawley recipients (allografts) or the same strain (isografts). Recipients were given pirfenidone (0.5% chow) 1–21‐day post‐transplantation. A significantly increased peak airway pressure (PawP) with excessive collagen deposition was found in untreated lung allografts. Pirfenidone treatment decreased PawP and collagen content in lung allografts. The beneficial effects were associated with downregulation of arginase protein expression and activity. In addition, pirfenidone decreased endogenous transforming growth factor (TGF)‐β level in lung allografts, and TGF‐β stimulated arginase activity in a dose‐dependent manner in both lung tissue and fibroblasts. These results suggest that pirfenidone inhibits local arginase activity possibly through suppression of endogenous TGF‐β, hence, limiting the development of fibrosis in lung allografts.

Reduced Cytotoxic Function of Effector CD8+ T Cells Is Responsible for Indoleamine 2,3-Dioxygenase-Dependent Immune Suppression

Indoleamine 2,3-dioxygenase (IDO), a potent immunosuppressive enzyme, contributes to tumoral escape, immune tolerance, and protection against allograft injury. In this paper, we report that inhibition of CD8+ T cell-mediated cytotoxic function is an important mechanism behind IDO’s immune-modulating property. The experimental rat lung allograft proved attractive for evaluating effector CD8+ T cells. Enhanced IDO activity achieved by using a lung-tissue-targeted nonviral human IDO gene transfer approach reduced, but did not eliminate, infiltrating CD8+ T cells. Although CD8+ T cells existed in the IDO-high lung allografts, CD8+ T cells remained viable and could proliferate for an extended period. However, cells lost their ability to attack allogeneic donor lung cells in vivo and allogeneic target cells in vitro. The impaired cytotoxic function seen in the IDO-treated CD8+ T cells was accompanied by defects in production of granule cytotoxic proteins, including perforin and granzyme A and B. Furthermore, we discovered that IDO leads to an impaired bioenergetic condition in active CD8+ T cells via selective inhibition of complex I in the mitochondrial electron transfer chain. These intriguing findings provide a base for establishing a novel mode of IDO’s immune-suppressing action. Additionally, donor lung IDO delivery, a direct and/or leukocyte passenger effect, impaired CD8+ effector cell function.

Sleeping Beauty-mediated eNOS gene therapy attenuates monocrotaline-induced pulmonary hypertension in rats

Pulmonary hypertension (PH) is a life‐threatening disorder with high mortality rates and limited treatment options. Gene therapy is an alternative treatment strategy, yet viral vectors have inherent disadvantages including immune activation. The Sleeping Beauty (SB) transposon is a nonviral method of gene delivery that overcomes some of these drawbacks. A SB‐based transposon harboring a constitutively active endothelial nitric oxide synthase (eNOS) gene was administered to Sprague‐Dawley rats via tail vein injection using the carrier polyethylenimine. Two days after transposon delivery, monocrotaline (MCT) was administered to induce PH. Hemodynamic, histological, and molecular measurements were performed four weeks later. Animals coinjected with transposase showed a significant reduction in pulmonary arterial pressure (PABP, 31.67±6.03 mmHg, P<0.01), an attenuation of right ventricle (RV) to whole heart (WH) wt ratios (0.227±0.0252, P<0.05) and a decrease in the pulmonary vessel wall thickness index (36.87%, P<0.001), compared with those animals receiving the eNOS transposon and a nonfunctional transposase (PABP 44.33±4.04 mmHg; RV/WH ratio 0.280±0.01; wall thickness index 62.14%) or control animals receiving MCT injection alone (PABP 49.67±3.22 mmHg; RV/WH ratio 0.290±0.0265; wall thickness index 71.99%). The physiological improvements correlated with therapeutic gene expression, suggesting that transposon‐based genetic approaches have utility in the treatment of PH.—Liu, L., Liu, H., Visner, G., Fletcher, B. S. Sleeping Beauty‐mediated eNOS gene therapy attenuates monocrotaline‐induced pulmonary hypertension in rats. FASEB J. 20, E2068–E2076 (2006)

Pirfenidone Inhibits T-Cell Activation, Proliferation, Cytokine and Chemokine Production, and Host Alloresponses

Background. We previously showed that pirfenidone, an anti-fibrotic agent, reduces lung allograft injury or rejection. In this study, we tested the hypothesis that pirfenidone has immune modulating activities and evaluated its effects on the function of T-cell subsets, which play important roles in allograft rejection. Method. We first evaluated whether pirfenidone alters T-cell proliferation and cytokine release in response to T-cell receptor (TCR) activation, and whether pirfenidone alters regulatory T cells (CD4+CD25+) suppressive effects using an in vitro assay. Additionally, pirfenidone effects on alloantigen-induced T-cell proliferation in vivo were assessed by adoptive transfer of carboxyfluorescein diacetate succinimidyl ester-labeled T cells across a parent->F1 major histocompatibility complex mismatch, as well as using a murine heterotopic cardiac allograft model (BALB/c->C57BL/6). Results. Pirfenidone was found to inhibit the responder frequency of TCR-stimulated CD4+ cell total proliferation in vitro and in vivo, whereas both CD4 and CD8 proliferation index were reduced by pirfenidone. Additionally, pirfenidone inhibited TCR-induced production of multiple pro-inflammatory cytokines and chemokines. Interestingly, there was no change on transforming growth factor-&bgr; production by purified T cells, and pirfenidone had no effect on the suppressive properties of naturally occurring regulatory T cells. Pirfenidone alone showed a small but significant (P<0.05) effect on the in vivo allogeneic response, whereas the combination of pirfenidone and low dose rapamycin had more remarkable effect in reducing the alloantigen response with prolonged graft survival. Conclusion. Pirfenidone may be an important new agent in transplantation, with particular relevance to combating chronic rejection by inhibiting both fibroproliferative and alloimmune responses.

Nonviral Gene Delivery With Indoleamine 2,3‐Dioxygenase Targeting Pulmonary Endothelium Protects Against Ischemia‐Reperfusion Injury

Pulmonary endothelial dysfunction induced by inflammation and inflammation‐associated reactive oxygen species is a central component in the pathophysiology of lung transplant ischemia‐reperfusion (IR) injury. Indoleamine‐2,3‐dioxygenase (IDO) is a unique cytosolic enzyme possessing both immune modulating and antioxidant properties. This study investigated whether enhanced pulmonary endothelial IDO activity by a targeted nonviral gene transfer approach ameliorates lung IR injury. Orthotopic syngeneic lung transplants were performed in Lewis rats. A human IDO (hIDO)‐expressing plasmid driven by an endothelial cell‐specific endothelin‐1 promoter was generated and intravenously delivered to donor lung using cationic polymer polyethylenimine. This nonviral gene transfer approach augmented hIDO expression specifically in endothelial cells within lung grafts. Importantly, enhanced IDO activity induced by the hIDO transgene prevented endothelial cell apoptosis, reduced vascular permeability and leukocyte extravasation, and consequently improved graft function and histologic appearance. Furthermore, our in vitro studies showed that increased IDO activity in endothelial cells protected its mitochondrial function and ultrastructure from oxidative stress through stabilization of intracellular redox status. The approach used in these experiments has properties that could eliminate the inherent side effects associated with viral vectors and/or antibody‐directed targeted therapy, and thus may represent a potential therapeutic strategy against lung IR injury in patients.

Endothelial indoleamine 2,3-dioxygenase protects against development of pulmonary hypertension.

RATIONALE A proliferative and apoptosis-resistant phenotype in pulmonary arterial smooth muscle cells (PASMCs) is key to pathologic vascular remodeling in pulmonary hypertension (PH). Expression of indoleamine-2,3-dioxygenase (IDO) by vascular endothelium is a newly identified vasomotor-regulatory mechanism also involved in molecular signaling cascades governing vascular smooth muscle cell (vSMC) plasticity. OBJECTIVES To investigate the therapeutic potential of enhanced endothelial IDO in development of PH and its associated vascular remodeling. METHODS We used loss and gain of function in vivo studies to establish the role and determine the therapeutic effect of endothelial IDO in hypoxia-induced PH in mice and monocrotaline-induced PH in rats. We also studied PASMC phenotype in an IDO-high in vivo and in vitro tissue microenvironment. MEASUREMENTS AND MAIN RESULTS The endothelium was the primary site for endogenous IDO production within mouse lung, and the mice lacking this gene had exaggerated hypoxia-induced PH. Conversely, augmented pulmonary endothelial IDO expression, through a human IDO-encoding Sleeping Beauty (SB)-based nonviral gene-integrating approach, halted and attenuated the development of PH, right ventricular hypertrophy, and vascular remodeling in both preclinical models of PH. IDO derived from endothelial cells promoted apoptosis in PH-PASMCs through depolarization of mitochondrial transmembrane potential and down-regulated PH-PASMC proliferative/synthetic capacity through enhanced binding of myocardin to CArG box DNA sequences present within the promoters of vSMC differentiation-specific genes. CONCLUSIONS Enhanced endothelial IDO ameliorates PH and its associated vascular structural remodeling through paracrine phenotypic modulation of PH-PASMCs toward a proapoptotic and less proliferative/synthetic state.

Indoleamine 2,3-Dioxygenase and Metabolites Protect Murine Lung Allografts and Impair the Calcium Mobilization of T Cells

The enzyme indoleamine 2,3-dioxygenase (IDO) converts tryptophan into kynurenine metabolites that suppress effector T-cell function. In this study, we investigated IDO and its metabolite, 3-hydroxyanthranilic acid (3HAA), in regulating lung allograft rejection, using a murine orthotopic lung transplant model with a major mismatch (BALB/c donor and C57BL6 recipient). IDO was overexpressed in murine donor lungs, using an established nonviral (polyethylenimine carrier)-based gene transfer approach, whereas 3HAA was delivered daily via intraperitoneal injection. Increased IDO expression or its metabolite, 3HAA, resulted in a remarkable therapeutic effect with near normal lung function and little acute rejection, approximately A1, compared with A3 in untreated allografts (grading based on International Society for Heart and Lung Transplantation guidelines). We found that a high IDO environment for 7 days in lung allografts resulted in impaired T-cell activation, the production of multiple effector cytokines (IL-2, IL-4, IL-5, IL-6, IFN-γ, TNF-α, IL-12, and IL-13), and the generation of effector memory T cells (CD62L(lo)CD44(hi) phenotype). In isolated murine splenocytes, we observed that IDO/3HAA impaired T-cell receptor (TCR)-mediated T-cell activation, and more importantly, a decrease of intracellular calcium, phospholipase C-γ1 phosphorylation, and mitochondrial mass was evident. This work further illustrates the potential role of a high IDO environment in lung transplantation, and that the high IDO environment directly impairs TCR activation via the disruption of calcium signaling.