Early Intervention With Dexamethasone In Reversal Of Monocrotaline-induced Pulmonary Artery Hypertension In Rats

Bacground and objectivesPulmonary arterial hypertension (PAH) is a severe condition which is mainly characterised as a disease of the small arteries of the pulmonary vasculature leading to an increased mean pulmonary artery pressure (PAP) and progressive right heart falure and ultimately death. PAH has an increased pulmonary arterial pressure, but the pulmonary venous pressure is normal. The defination of PAH is the mean pulmonary artery pressure (mPAP)≥25 mmHg through right heart catheterization, with pulmonary capillary wedge pressure (PCWP)≤15mmHg and pulmonary vascular resistance (PVR)>3 wood unit at rest at the sea leval. Histopathological changes in the various recognized forms of PAH have shown to be qualitatively similar including endothelial dysfunction, thickness of the wall of arterioles, and obstruction of small pulmonary arteries.PAH is a recognized complication of a number of systemic inflammatory conditions, such as scleroderma, systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD), which may be result from autoimmune vascularitis. Severe PAH could also be found in patients with HIV infection. Idiopathic pulmonary arterial hypertension (IPAH) is one type of PAH in which there is neither genetic mutations nor identified history of exposure to risk factors. A significant number of patients with IPAH has been diagnosed have evidence of the presence of anti-nuclear antibodies and increased serum levels of inflammatory cytokine such as interleukin (IL)-1 and IL-6. Lung histology revealed inflammatory infiltrates such as macrophages and T lymphocytes in plexiform lesion in patient with idiopathic PAH as well as in rats with PAH induced by the toxin monocrotaline (MCT). However, the mechanisms by which inflammation is related to the pathogenesis of PAH is poorly understood.The pyrrolizidine alkaloid, MCT, is bioactivated in the liver by cytochrome P450 monooxygenases to monocrotaline pyrrole (MCTP) which travel via the circulation to the lungs where they cause injury to pulmonary vascular endothelium, and then cause progressive pulmonary vascular remodeling, pulmonary hypertension, right heart hypertrophy and failure.Identification of perivascular inflammatory cell infiltrates, comprised of T-and B-lymphocytes, has supported the concept that inflammatory cells may play a role in PAH. model. How precisely the inflammatory cell infiltrates cause the process of PAH is still unknown. As a first step toward the understanding of the role of T cells involved in PAH, we sought to identify immunohistochemically the cell composition of the lesions. CD4+ and CD8+ T cells play critical roles in cell-mediated immunity, besides, the local production of cytokines could also have potential effects on the process of this vasculopathy. So we focused on Thl/Th2 subsets imbalance to clarify mechanisms of progression, remission and prognosis of PAH. Thl cells secrete interferon (IFN)-y, IL-2 and tumour necrosis factor (TNF)-βand promote mainly cellular immunity, whereas Th2 cells produce IL-4, IL-5, IL-10 and IL-13, primarily promoting humoral immunity.Leukocyte recruitment from the circulation into tissues involves two stages. The first one takes place at the interface between blood and the endothelium, and the second one involves the migration of the leukocyte from the subendothelial space deeply into the tissue. Chemokines play an important role at both stages. Fractalkine(FKN/CX3CL1), the unique member of the CX3C chemokine subfamily, is expressed by endothelial cells, both as a soluble and as a membrane bound form. Its actions are mediated by CX3CR1, a seven transmembrane receptor that is mainly expressed by monocytes and subpopulations of T-lymphocytes. We examined CX3CR1-mediated chemotaxis and adhesion in T cells and monocytes as well as the expression of FKN reflecting lesions of pulmonary endothelia.Glucocorticoids or their synthetic analogues have been used clinically as anti-in(?)ammatory and immunosuppressive drugs. Dexamethasone (Dex), a synthetic glucocorticoid, is a potent inhibitor of gene transcription of many proinflammatory cytokines, chemokines and adhesion molecules. The major anti-inflammatory effects of glucocorticoids appear to be due largely to interaction between the activated glucocorticoid receptor and transcription factors, notably nuclear factor-KB (NF-κB) and activator protein-1 (AP-1). A NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) was reported to ameliorate PAH in rats. In spite of much progress have been made on the treatment of pulmonary hypertension, the effective method is still pending considering about therapeutic effect, cost and safety.The current treatment of pulmonary hypertension has changed significantly. Except for oxygen, diuretics, warfarin and digoxin and other conventional treatment, calcium channel blocker(CCB) has been used to dilate pulmonary vascular. Other new drugs, such as prostacyclin, endothelin receptor antagonists, phosphodiesterase inhibitors have also been used to improve the function of endothelial and to inhibit the remodeling of pulmonary arterials. Modern treatment has extended survival, and some data showed that the 5-year mortality rate is 50%. However, because of the complexity of the disease's diagnosis and treatment, and limitations of the expensive drug use, a new therapeutic strategy is urgely desirable.Thus, does Dex have the same effect to attenuate the PAP? Could Dex act as a combined treatment of PAH? Whether Dex play a protective role on pulmonary vascular endothelia? In this study, with comparison to PDTC, we observe the theraputic effect of dexamethasone on pulmonary hypertension as well as perivascular T-lymphocyte infiltration and Th cell cytokine gene expression in order to sought for evidence of inflammatory and immune response in PAH. Methods1. Animal models:64 male Wistar rats (150 to 180g) were randomly divided into following groups:①Model groups of PAH:32 rats were randomly divided into 4 groups(each group n=8), Normal control (NC) group (saline injection),1wk-MCT group (1wk after MCT injection),2wk-MCT group (2wk after MCT injection) and 3wk-MCT group (3wk after MCT injection);②Treatment groups of PAH:32 rats were randomly divided into 4 groups(each group n=8), Control/vehicle group that received 0.9% saline daily 2 days after saline exposure(the same volume as MCT); MCT/vehicle group that received 0.9% saline daily 2 days after the same MCT exposure as Control group; MCT/PDTC group that received PDTC 100mg/kg daily from 2 days after the same MCT exposure as Control group; MCT/Dex group that received Dex 1.0mg/kg daily from 2 days after the same MCT exposure as Control group; all groups were killed on day 21.2. Measurements of PAP and Assessment of Right Ventricular Hypertrophy:On the last day of treatment, rats were anesthetized with an injection of pentobarbital (45mg/kg). After an incision in the neck, the external jugular vein was exposed and cannulated with a 3F tubing. Right ventricular systolic pressure (RVSP) was used as PAP.Each heart was trimmed of the atrial appendages, and the free wall of the RV was separated from the left ventricle (LV) and the septum (S). The ratio of RV/(LV+S) was calculated to assess right ventricular hypertrophy (RVH).3. The perivascular numbers of CD4+ and CD8+ T cells were counted by immunohistochemical stain.4. mRNA expression of IL-4, IFN-γ, Fkn and CX3CR1 were detected by real-time quatitative PCR (RT-qPCR).5. The protein level of FKN was detected by Western Blot.6. The serum levels of IL-4 and IFN-γwere measured by enzyme-linked immunosorb-ent assay (ELISA) method.7. Statistical analysis was performed using SPSS 16.0 for windows with a significance level of 0.05 and presented as mean±SD. The differences between groups were analysed using ANOVA with post hoc testing. Comparisons between two groups were performed by Student's t test for parametric data. Results1. Pathological changes of pulmonary arterials:Light microscopy indicated that, the control group, continuity of pulmonary vascular endothelial cells were well with non-alveolar edema, congestion and exudation; while endothelial cells of the PAH group appeared swollen, degenerated and necrotic. Lumen area was decreased. Smooth muscle cells in the media proliferated and became thicken apparently. We can also find congested capillaries of alveolar, thickened lung interstitial, and infiltration of inflammatory cells around the vascular wall.2. RVSP and RVH:Rats in PAH Model group developed PAH at 2wk and with a further progress at 3wk after exposure to MCT. In PDTC intervention group, RVSP was significantly attenuated, but there was still some difference with NC group, while Dex restored PAP. The ratio of RVH was also consistent with this trend.3. mRNA expression of IFN-y and IL-4 in lung:Results detected by RT-qPCR indicated that:①Model groups of PAH:The expression of IL-4 mRNA was down-regulated in the lung of rat at first 2 weeks after MCT injection. But there is no significence between 3wk and NC group. The gene expression of IFN-y didn't change obviously during the first 2 weeks compared to NC group after MCT injection, but it increased at the 3wk (P<0.05 vs. NC).②Treatment groups of PAH:The expression of IFN-y in rats of both PDTC and Dex group was much lower than that in PAH group; while the expression of IL-4 of Dex group was much higher than that in PAH group and PDTC group(P<0.05).4. mRNA expression of Fkn and CX3CR1 in lung:Results detected by RT-qPCR indicated that:①Model groups of PAH:The expression of Fkn in rat of NC group was maximum, while all lower in the other groups and lowest at 3wk. More CX3CR1 gene was expressed in 2wk and 3wk groups compared to control group.②Treatment groups of PAH:Rats of PDTC group and Dex group both expressed more Fkn than rats of 3wk did. Minimal CX3CR1 expression was shown in Dex group.5. Serum levels of IL-4 and IFN-y:①Model groups of PAH:serum levels of IFN-y and IL-4 did not differ from controls at 1-,2-, or 3-week PAH.;②Treatment groups of PAH:serum levels of IFN-γhave no significant difference among the four grops。So did the serum levels of IL-4 except for the group of PDTC。6. CD8+ T cells around the pulmonary artery:The number of CD8+T cells increased significantly with MCT-induced PAH at 3 weeks, but treatment with PDTC and Dex prevented the increase in number of cells.7. CD4+ T cells around the pulmonary artery:The number of CD4+T cells gradually increased after MCT injection during the 3 weeks, with no significant difference in number of cells with PDTC and Dex treatment. Conclusions:1. RVSP and right-ventricular hypertrophy index (RVHI) began to increase at 2 weeks after MCT-exposure and data were significant at 3 weeks, indicating that MCT-induced pulmonary hypertension model was successfully created.2.With the progress of pulmonary hypertension, alterations of Th1 and Th2-type cytokines mRNA expression were detected, suggesting that cytokines play an important role in the pathogenesis of pulmonary hypertension.3. mRNA expression of Fkn was lowest at 3 weeks coincident with the severe pulmonary hypertension, implying serious damage of pulmonary vascular endothelia, while PDTC and Dex could improve the situation. mRNA expression of CX3CR1 increased with the progression of disease, while Dex significantly reduce the expression, indicating that it could inhibit the infiltration of inflammatory cells in lung.4. The rising CD8+ T cell count of severe pulmonary hypertension and reduced ones of PDTC-and Dex-treatment suggested that CD8+ T played an important role cells in the pathogenesis of PAH. Changes of CD4+ T cells may indicate their protective effect on pulmonary arteries.5. No significant changes of serum level of IL-4 and IFN-γprobably mean that Dex did not influence much of the systemic immune system.6. Dexamethasone can significantly reduce the pulmonary artery pressure, protect the pulmonary vascular endothelium, attenuate pulmonary vascular remodeling, and all these properties indicate a good prospect of clinical application. Innovations and meanings1. This study inveatigated the relationship between inflammation and immune and pulmonary vascular remodeling in models of MCT-induced PAH, elaborated the potential mechanisms from different ways. Thus potent theoretical evidences were offered for as a new therapeutic target in PAH.2. In vivo tests confirmed the gene expression of Thl/Th2 type cytokines and subtypes of T-cell during PAH, provides a new perspectivefor cytokine therapy and antibody therapy.3. This study observed the effect of Dex on PAH in rats. Dex could inhibit the progress of pulmonary hypertension through its anti-inflammation and anti-immune properties. This provide a new theoretical and experimental basis for clinical treatment of glucocorticoids for PAH.