Hanbo Hu

Uric acid decreases NO production and increases arginase activity in cultured pulmonary artery endothelial cells

Elevated levels of serum uric acid (UA) are commonly associated with primary pulmonary hypertension but have generally not been thought to have any causal role. Recent experimental studies, however, have suggested that UA may affect various vasoactive mediators. We therefore tested the hypothesis that UA might alter nitric oxide (NO) levels in pulmonary arterial endothelial cells (PAEC). In isolated porcine pulmonary artery segments (PAS), UA (7.5 mg/dl) inhibits acetylcholine-induced vasodilation. The incubation of PAEC with UA caused a dose-dependent decrease in NO and cGMP production stimulated by bradykinin or Ca(2+)-ionophore A23187. We explored cellular mechanisms by which UA might cause reduced NO production focusing on the effects of UA on the l-arginine-endothelial NO synthase (eNOS) and l-arginine-arginase pathways. Incubation of PAEC with different concentrations of UA (2.5-15 mg/dl) for 24 h did not affect l-[(3)H]arginine uptake or activity/expression of eNOS. However, PAEC incubated with UA (7.5 mg/dl; 24 h) released more urea in culture media than control PAEC, suggesting that arginase activation might be involved in the UA effect. Kinetic analysis of arginase activity in PAEC lysates and rat liver and kidney homogenates demonstrated that UA activated arginase by increasing its affinity for l-arginine. An inhibitor of arginase (S)-(2-boronoethyl)-l-cysteine prevented UA-induced reduction of A23187-stimulated cGMP production by PAEC and abolished UA-induced inhibition of acetylcholine-stimulated vasodilation in PAS. We conclude that UA-induced arginase activation is a potential mechanism for reduction of NO production in PAEC.

Peptides modified by myristoylation activate eNOS in endothelial cells through Akt phosphorylation

1 Myristoylated pseudosubstrate of PKCζ (mPS) – a synthetic myristoylated peptide with a sequence (13 amino acids) mimicking the endogenous PKCζ pseudosubstrate region – is considered a selective cell‐permeable inhibitor of PKCζ. We present strong evidence that in endothelial cells the action of mPS is not limited to inhibition of PKC activity and that myristoylation of certain peptides can activate eNOS (endothelial nitric oxide synthase) through Akt phosphorylation. 2 mPS at micromolar concentrations (1–10 μM) induced profound phosphorylation of eNOS, Akt, ERK 1/2, and p38 MAPK in cultured pulmonary artery endothelial cells (PAEC). The same changes were observed after treatment of PAEC with a myristoylated scrambled version of mPS (mScr), whereas a cell‐permeable version of PKCζ pseudosubstrate fused to the HIV‐TAT membrane‐translocating peptide did not induce analogous changes, suggesting that myristoylation confers new properties on the peptides consisting of activation of different signaling pathways in endothelial cells. 3 In addition to mPS and mScr, a number of other myristoylated peptides induced phosphorylation of eNOS suggesting that myristoylation of peptides can activate eNOS by mechanisms unrelated to inhibition of PKC. All active myristoylated peptides contained basic amino acids motif and were longer than six amino acids. 4 Activation of eNOS by myristoylated peptides was dependent on the PI3K/Akt pathway and the rise of intracellular calcium and was associated with an elevation of cGMP levels in PAEC and with relaxation of precontracted isolated pulmonary artery segments. 5 Myristoylated peptides can be considered a new class of activators of NO production in endothelial cells and that using mPS as a specific inhibitor of PKCζ should be done with caution, especially in endothelial cells.

Hypoxia-induced endothelial CX3CL1 triggers lung smooth muscle cell phenotypic switching and proliferative expansion

Distal arterioles with limited smooth muscles help maintain the high blood flow and low pressure in the lung circulation. Chronic hypoxia induces lung distal vessel muscularization. However, the molecular events that trigger alveolar hypoxia-induced peripheral endothelium modulation of vessel wall smooth muscle cell (SMC) proliferation and filling of nonmuscular areas are unclear. Here, we investigated the role of CX3CL1/CX3CR1 system in endothelial-SMC cross talk in response to hypoxia. Human lung microvascular endothelial cells responded to alveolar oxygen deficiency by overproduction of the chemokine CX3CL1. The CX3CL1 receptor CX3CR1 is expressed by SMCs that are adjacent to the distal endothelium. Hypoxic release of endothelial CX3CL1 induced SMC phenotypic switching from the contractile to the proliferative state. Inhibition of CX3CR1 prevented CX3CL1 stimulation of SMC proliferation and monolayer expansion. Furthermore, CX3CR1 deficiency attenuated spiral muscle expansion, distal vessel muscularization, and pressure elevation in response to hypoxia. Our findings indicate that the capillary endothelium relies on the CX3CL1-CX3CR1 axis to sense alveolar hypoxia and promote peripheral vessel muscularization. These results have clinical significance in the development of novel therapeutics that target mechanisms of distal arterial remodeling associated with pulmonary hypertension induced by oxygen deficiency that is present in people living at high altitudes and patients with obstructive lung diseases.

Priming Donor Lungs With Thioredoxin-1 Attenuates Acute Allograft Injury in a Rat Model of Lung Transplantation

BACKGROUND Lung graft dysfunction and rejection are significant causes of morbidity and mortality in transplant recipients. Thioredoxin-1, a redox-regulatory protein, functions as an antioxidant in multiple organs, including lungs. We examined whether priming of the donor lungs with thioredoxin-1 before transplantation attenuates acute lung injury. METHODS Orthotopic left lung transplantation was performed from Lewis (donor) to Sprague-Dawley (recipient) rats. Donor lungs were perfused and stored in Perfadex solution (Vitrolife, Uppsala, Sweden), with or without purified thioredoxin-1. Changes in bronchoalveolar lavage (BAL) analysis, allograft oxygen exchange function, nuclear factor kappaB (NF-kappaB)/DNA binding, myeloperoxidase activities, and immunohistologic evaluation of neutrophils, macrophages, and cytotoxic T-cells (CD8(+)) infiltration were examined in post-transplant allograft (left) and native (right) lungs at Days 1 and 5. RESULTS BAL cell differential analysis showed significant increases in macrophages and neutrophils in allografts at Day 1 post-transplant. At Days 1 and 5, lymphocyte infiltration was significantly increased and myeloperoxidase and NF-kappaB/DNA binding activities were increased vs basal activities. Immunohistology staining revealed increased infiltration of macrophages, neutrophils, and CD8(+) T cell sub-sets. Pre-transplant priming of donor lungs with thioredoxin-1 improved oxygen exchange and attenuated NF-kappaB/DNA binding activity, and infiltration of macrophages, neutrophils, and CD8(+) T cell sub-sets in allografts at Days 1 and 5 post-transplant. CONCLUSIONS Priming of donor lungs with thioredoxin-1 before transplant attenuates acute allograft injury in a rat model of lung transplantation, and appears to be associated with the antioxidant function of thioredoxin-1 that limits early ischemia-reperfusion injury, NF-kappaB activation, and progressive infiltration of inflammatory and immune cells in allografts.

Thioredoxin as a biomarker for graft rejection in lung transplant recipients

Abstract Primary graft dysfunction and rejection are common complications in lung transplant recipients. Increased expression of thioredoxin-1 (Trx), a 12-kDa redox-regulatory protein, has been reported in multiple lung pathophysiological conditions involving oxidative and inflammatory mediated injury including graft rejection in canine and rat models of lung transplantation. Our objective was to determine whether increased Trx expression is associated with progression of rejection pathophysiology in human lung transplant recipients. Bronchoalveolar lavage (BAL) fluid and transbronchial biopsy samples were collected as a routine part of post-transplant clinical care from 18 lung transplant patients from our adult lung transplant programme. Lung transplant recipient profile included age/sex, ethnic background, days on ventilator, total ischaemic time, and cytomegalovirus (CMV) status. Based on histopathological grading criteria, patients were divided into two groups, rejecting (A1/A2 or B1) and non-rejecting (A0/B0). Rejecting and non-rejecting group total BAL cell counts and differential cell counts for neutrophils, macrophages, lymphocytes and eosinophils as well as total BAL cell Trx levels were analysed. Total BAL cell counts were significantly (p<0.05) elevated in graft rejecting versus non-rejecting patients. Differential BAL macrophage counts were comparable in rejection and non-rejection groups, whereas there were significant increases in neutrophils and lymphocytes but not eosinophils in patients with rejection versus non-rejection pathology (p<0.05). Total ischaemic time and days on ventilator in rejection and non-rejection groups were comparable. However, Trx levels were significantly elevated in BAL cells from graft-rejecting patients compared with non-rejecting patients (p<0.05). These data suggest that surveillance monitoring of BAL Trx levels after lung transplantation can serve as a biomarker to assess severity of graft rejection.

Administration of recombinant human thioredoxin‐1 significantly delays and prevents autoimmune diabetes in nonobese diabetic mice through modulation of autoimmunity

Thioredoxin as a biological antioxidant plays an important role in regulating the redox system. The administration of recombinant thioredoxin has been demonstrated to be anti‐inflammatory. In this study, the effect of recombinant human thioredoxin‐1 (rhTrx‐1) in preventing type 1 diabetes (T1D) in nonobese diabetic (NOD) mice was evaluated.

Thioredoxin priming prolongs lung allograft survival by promoting immune tolerance.

Tolerance to allograft antigen is the major challenge and final goal of transplant medicine. Our previous study demonstrated that thioredoxin-1 (Trx) priming of donor lung significantly protected allogeneic lung graft. To determine whether Trx priming of donor lung inhibits allograft rejection, extends allograft survival and induces immune tolerance, orthotopic left lung transplantation was performed from Lewis to Sprague-Dawley rats without immunosuppression. Donor lungs were primed with Trx at 4°C for 4 hr prior to transplantation. After up to 37 days post-transplantation, allograft lung morphology, recipient T cell and humoral alloantigen-specific immune responses were examined. We found that Trx-primed lungs exhibited much reduced acute rejection and associated lung injuries resulting in loss of graft functional area at 5-37 days post-transplant in contrast to the control groups. CD4+ T cells from the recipients with Trx-primed grafts responded to the stimulation of dendritic cells (DCs) of donor origin, in contrast to DCs from the third party, with significantly reduced proliferation. Consistent with above findings, we observed that CD4+Foxp3+ regulatory T cells in spleen cells from the recipients with Trx-primed grafts were significantly increased compared to controls, and CD4+ T cells from the recipients with Trx-primed grafts produced much higher levels of immunosuppressive cytokine, IL-10 when stimulated with allogeneic donor DCs. In addition, humoral immune tolerance was also induced as there was no significant increase levels of serum antibodies against donor antigens in Trx-lung recipients when re-challenged with allogeneic donor antigens. Our results demonstrate that one-time Trx-priming of donor lung grafts prior to transplantation significantly prolongs the survival of the grafts through inducing or promoting cellular and humoral alloantigen-specific immune tolerance, which might be associated with the induction of immunosuppressive regulatory T cells.