Evaluation and Treatment of Endoplasmic Reticulum (ER) Stress in Right Ventricular Dysfunction during Monocrotaline-Induced Rat Pulmonary Arterial Hypertension.

PURPOSE: Endoplasmic reticulum (ER) stress contributes to pulmonary artery hypertension (PAH). However, the exact roles of ER stress in right ventricular (RV) dysfunction, which is strongly associated with PAH, are largely unknown. Here, we aimed to explore how ER stress affects RV function in a rat PAH model and evaluated the effects of an ER stress inhibitor on RV dysfunction. METHODS: We examined expression changes of an ER marker: chaperone glucose-regulated protein 78 (GRP78), three ER stress sensor proteins: activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and protein kinase RNA-like endoplasmic reticulum kinase (PERK), and a key ER stress-induced apoptosis indicator: CCAAT/enhancer-binding protein homologous protein (CHOP), with inflammation indicators: interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), and matrix metalloproteinases (MMPs) in RV at 3, 7, 14 and 28 days following a single dose of monocrotaline (MCT) injection, with or without a preventive treatment [4-phenylbutyric acid (PBA)]. RV function was evaluated by histological, molecular and echocardiographic analysis. RESULTS: 1) GRP78 protein expression started to increase (1.5 ± 0.06 fold change) at 3d post MCT injection, even before the formation of PAH. 2) ATF6, IRE1, and PERK showed distinctive expression patterns post MCT injection. 3) CHOP expression remained low at day 3 & 7, but significantly increased at day 14 (p < 0.05), along with the peak of RV cardiomyocytes apoptosis. 4) PBA inhibited ER stress and alleviated remodeling and dysfunction in the RV. CONCLUSIONS: The early phase of ER stress might benefit RV function, whereas the extended phase led to RV cardiomyocyte apoptosis and dysfunction. Inhibition of ER stress by PBA during PAH directly improved RV function.

[Clinical characteristics of 161 Chinese patients with idiopathic pulmonary arterial hypertension].

OBJECTIVE: To investigate the clinical characteristics of patients with idiopathic pulmonary arterial hypertension (IPAH) in China. METHODS: A total of 161 patients diagnosed as IPAH in Shanghai Pulmonary Hospital from June 2008 to June 2010 were retrospective analyzed. RESULTS: The mean diagnostic age was (33 ± 15) years old and 70.2% patients were female. The median duration from symptoms onset to diagnostic right heart catheterization was 12 months. Incidence of NYHA class III to IV was 56.5% at diagnosis and the mean six minutes walk distance was limited to (398 ± 108) meters. Incidence of mild obstructive, restrictive and diffusing impairment in pulmonary function test was 7.8%, 42.2% and 82.2% patients with IPAH, respectively. Right heart catheterization demonstrated severe elevated mean pulmonary arterial pressure [(63 ± 17) mm Hg (1 mm Hg = 0.133 kPa)] and pulmonary vascular resistance index [(25 ± 12) Wood U/m(2)] in this patient cohort. The response rate of acute pulmonary vasoreactivity testing was 8.7% in this cohort. Compared with non-responders, responders to acute pulmonary vasoreactivity testing were younger and with less severe pulmonary hypertension. Among non-responders, 89% patients were treated by one specific anti-pulmonary arterial hypertension drug and 27% patients received combined anti-pulmonary arterial hypertension medications. CONCLUSIONS: Young female was predominantly involved in patients with IPAH in China. The diagnosis of IPAH is often made at advanced disease stage and majority patients with IPAH received specific anti-pulmonary arterial hypertension therapies in this patient cohort.

Canis familiaris allergen Can f 6: expression, purification and analysis of B-cell epitopes in Chinese dog allergic children.

Dog allergy is common worldwide. However, the allergenicity of dog allergy is still unclear in China as well as in special group, such as children. In this study, we chose Can f 6, a major dog allergen which belongs to the lipocalin to study its allergenicity in Chinese dog allergic children. Can f 6 gene was subcloned into pET-28a vector and transformed into E. coli BL21 (DE3) cells for expression. The recombinant Can f 6 was purified by nickel affinity chromatography, identified by SDS-PAGE, and tested for its allergenicity by Western blot with sera and basophil activation test. Secondary structures, B cell epitopes and homology modeling of Can f 6 were predicted by using a series of bioinformatical approaches. And the verification of B cell epitopes was detected by ELISA. The recombinant allergen showed an explicit band with the molecular weight of 20 kDa by SDS-PAGE. Sera from 56.3 % (18/32) of dog-allergic children patients reacted with Can f 6. The induction of the expression of CD63 and CCR3 of dog allergic children in passively sensitized basophils was up to approximately 5.0 times higher than healthy subjects. The secondary structure of Can f 6 contains 3 α-helices, 9 ß-sheets and random coils. Five B cell epitopes of Can f 6 were predicted and were confirmed successfully by ELISA. The results showed Can f 6 is a major allergen in Chinese children, which provides a basis for further study of Can f 6 in diagnosis and treatment of symptoms in children in China. The structural information of Can f 6 will help to form a foundation for the future design of vaccines and therapies for Can f 6 related allergies.

[Effects of norepinephrine on the therapeutic effect of nitric oxide inhalation in the treatment of acute respiratory distress syndrome in goat].

OBJECTIVE: To observe effects of norepinephrine (NE) on the therapeutic effects of nitric oxide (NO) inhalation in goats with endotoxin-induced acute respiratory distress syndrome (ARDS). METHODS: A model of septic ARDS was reproduced by an intravenous infusion of low dose endotoxin in six goats, and then these animals were treated with 40 x 10(-6) NO inhalation. After 30 minutes, intravenous infusion of NE in dose of 0.5 microg x kg(-1) x h(-1) was given. The dynamic changes in gas exchange and hemodynamics were measured with the aid of Swan-Ganz catheter and arterial blood gas analysis before and after the onset of ARDS, 30 minutes after NO inhalation and administration of NE. RESULTS: Inhalation of NO rapidly reduced mean pulmonary arterial pressure (MPAP), increased PaO(2), decreased alveolar-arterial partial pressure of oxygen difference (P (A-a) O(2)) and intrapulmonary shunt fraction (Qs/Qt) in septic ARDS goats. These changes were more pronounced when NE was given compared with NO inhalation alone. The combination of NO inhalation and NE infusion resulted in an increase in mean arterial pressure. CONCLUSION: Norepinephrine enhances the beneficial effect of nitric oxide inhalation on lung gas exchange in goats with endotoxin induced acute respiratory distress syndrome.

[Effect of combination of nitric oxide inhalation and inverse ratio ventilation in endotoxin-induced acute respiratory distress syndrome in sheep].

OBJECTIVE: To observe the effect of combination of nitric oxide (NO) inhalation and inverse ratio ventilation (IRV) on oxygenation and hemodynamics in endotoxin-induced acute respiratory distress syndrome (ARDS) in sheep. METHODS: Sheep ARDS model was induced by an intravenous infusion of low dose endotoxin, and then animals were randomly divided into two groups. (1) NO group (n=6), inhalation of 40x10(-6) nitric oxide. (2) Combination group (n=6), receiving mechanical ventilation with IRV (inspiratory-to-expiratory ratio of 2:1) and inhalation of 40x10(-6) NO. The dynamic changes in gas exchange and hemodynamics were measured with the aid of Swan-Ganz catheter and arterial blood gas analysis before and after the onset of, ARDS and 30 minutes after treatment. RESULTS: The combination of IRV and 40x10(-6) NO inhalation rapidly reduced mean pulmonary arterial pressure (MPAP), increased PaO(2), decreased P((A-a))O(2), and Qs/Qt without inducing significant change in systemic hemodynamics, and it was a more effective method of correcting hypoxemia than inhalation of nitric oxide alone. NO inhalation did not change the airway pressure of the model, but the combined treatment resulted in reduction of peak inspiratory pressure and increase of mean airway pressure. CONCLUSION: The combined use of IRV and NO inhalation has an additive effect on improving oxygenation in endotoxin-induced acute respiratory distress syndrome in sheep.

Monocrotaline-induced pulmonary arterial hypertension is attenuated by TNF-α antagonists via the suppression of TNF-α expression and NF-κB pathway in rats.

Inflammation is involved in various types of human pulmonary arterial hypertension (PAH), especially in PAH-associated connective tissue diseases. Although the pathogenesis of pulmonary hypertension has still remained largely unclear, TNF-α has been reported as a key pro-inflammatory cytokine in severe pulmonary hypertension and emphysema. The aim of this study was to investigate the effect of a TNF-α antagonist, recombinant TNF-α receptor II:IgG Fc fusion protein (rhTNFRFc), on the development of monocrotaline (MCT)-induced PAH in rats. Our results revealed that treatment of rhTNFRFc in these rats had favorable effects on mPAP levels, hemodynamics and pulmonary vascular remodeling, preventing PAH development at 3weeks following MCT. Furthermore, rhTNFRFc treatment resulted in markedly reduced expression of TNF-α via the inhibition of NF-κB activity in rat lungs. These results demonstrated that rhTNFRFc attenuated the process of MCT-induced PAH through its anti-inflammatory property. Although further studies are needed to define the appropriate treatment regimen, our findings suggest that rhTNFRFc might provide therapeutic benefits for PAH patients.

Ruscogenin attenuates monocrotaline-induced pulmonary hypertension in rats.

Inflammation, endothelial dysfunction, and thrombosis contribute to the pathogenesis and development of human pulmonary arterial hypertension (PAH). The aim of this study was to investigate the effects of ruscogenin, a natural anti-inflammatory and anti-thrombotic agent, on the development of monocrotaline (MCT)-induced PAH in rats. Our results revealed that ruscogenin had favorable effects on hemodynamics and pulmonary vascular remodeling, preventing the development of PAH 3 weeks after MCT. In addition, ruscogenin resulted in markedly reduced expression of inflammatory cytokine and leukocyte infiltration via the inhibition of nuclear factor (NF)-κB activity in rat lungs. Ruscogenin also attenuated MCT-induced endothelial cell apoptosis in the remodeled pulmonary arterioles and rescued destruction of endothelial cell membrane proteins such as eNOS, caveolin-1, and CD31. Our findings suggest that ruscogenin might have therapeutic benefits for PAH patients.

Usefulness of intravenous adenosine in idiopathic pulmonary arterial hypertension as a screening agent for identifying long-term responders to calcium channel blockers.

Although intravenous adenosine is recommended for acute vasodilator testing in patients with pulmonary hypertension, long-term outcomes in acute responders treated with calcium channel blockers (CCBs) who are identified by adenosine remain unknown. In this study, the value of adenosine for identifying long-term responders to CCBs was investigated in patients with idiopathic pulmonary arterial hypertension (IPAH). All acute responders were subsequently treated with high-dose CCB monotherapy, and 6-minute walk distances, hemodynamic data, and World Health Organization functional classifications were followed. Nine of 104 patients exhibited an acute response with intravenous adenosine (8.7%, 95% confidence interval 3.2 to 14.2). After 12 months of follow-up, all acute responders were still alive; however, only 6 patients showed sustained hemodynamic improvement (5.8%, 95% confidence interval 2 to 13). Three patients had failed CCB monotherapy and bosentan was added to their treatment. Mean tolerated dose of intravenous adenosine was 142 ± 49 µg/kg/min. No life-threatening adverse events were observed and only 2 patients of the nonresponders exhibited a 20% decrease in mean systemic arterial pressure. In nonresponders, 1- and 3-year survival rates were 89% and 75%, respectively. In conclusion, acute vasodilator testing with intravenous adenosine was safe and able to screen responders to CCB therapy in patients with IPAH. Long-term CCB responders accounted for about 5.8% of patients with IPAH.

Iptakalim rescues human pulmonary artery endothelial cells from hypoxia-induced nitric oxide system dysfunction.

The aim of this study was to assess whether hypoxia inhibits endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production, and whether iptakalim may rescue human pulmonary artery endothelial cells (HPAECs) from hypoxia-induced NO system dysfunction. HPAECs were cultured under hypoxic conditions in the absence or presence of 0.1, 10 and 1,000 µM iptakalim or the combination of 10 µM iptakalim and 1, 10 and 100 µM glibenclamide for 24 h, and the eNOS activity and NO levels were measured in the conditioned medium from the HPAEC cultures. The eNOS activity and NO levels were reduced significantly in the conditioned medium from HPAEC cultures under hypoxic conditions. Pre-treatment with 10 µM iptakalim normalized the reduction of the eNOS activity and NO levels caused by hypoxia in the conditioned medium from HPAEC cultures. Iptakalim raised the eNOS activity and NO levels under hypoxic conditions, but was blocked by the K(ATP) channel blocker, glibenclamide. Our results indicate that hypoxia impairs NO system function, whereas the ATP-sensitive K(+) channel opener, iptakalim, may rescue HPAECs from hypoxia-induced NO system dysfunction.

Nicorandil prevents right ventricular remodeling by inhibiting apoptosis and lowering pressure overload in rats with pulmonary arterial hypertension.

BACKGROUND: Most of the deaths among patients with severe pulmonary arterial hypertension (PAH) are caused by progressive right ventricular (RV) pathological remodeling, dysfunction, and failure. Nicorandil can inhibit the development of PAH by reducing pulmonary artery pressure and RV hypertrophy. However, whether nicorandil can inhibit apoptosis in RV cardiomyocytes and prevent RV remodeling has been unclear. METHODOLOGY/PRINCIPAL FINDINGS: RV remodeling was induced in rats by intraperitoneal injection of monocrotaline (MCT). RV systolic pressure (RVSP) was measured at the end of each week after MCT injection. Blood samples were drawn for brain natriuretic peptide (BNP) ELISA analysis. The hearts were excised for histopathological, ultrastructural, immunohistochemical, and Western blotting analyses. The MCT-injected rats exhibited greater mortality and less weight gain and showed significantly increased RVSP and RV hypertrophy during the second week. These worsened during the third week. MCT injection for three weeks caused pathological RV remodeling, characterized by hypertrophy, fibrosis, dysfunction, and RV mitochondrial impairment, as indicated by increased levels of apoptosis. Nicorandil improved survival, weight gain, and RV function, ameliorated RV pressure overload, and prevented maladaptive RV remodeling in PAH rats. Nicorandil also reduced the number of apoptotic cardiomyocytes, with a concomitant increase in Bcl-2/Bax ratio. 5-hydroxydecanoate (5-HD) reversed these beneficial effects of nicorandil in MCT-injected rats. CONCLUSIONS/SIGNIFICANCE: Nicorandil inhibits PAH-induced RV remodeling in rats not only by reducing RV pressure overload but also by inhibiting apoptosis in cardiomyocytes through the activation of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels. The use of a mitoK(ATP) channel opener such as nicorandil for PAH-associated RV remodeling and dysfunction may represent a new therapeutic strategy for the amelioration of RV remodeling during the early stages of PAH.

The effects of siRNA against RPL22 on ET-1-induced proliferation of human pulmonary arterial smooth muscle cells.

The aim of this study was to investigate the effects of small interference RNA (siRNA) against ribosomal protein L22 (RPL22) on ET-1-induced proliferation of human pulmonary arterial smooth muscle cells (HPASMCs). HPASMCs were transfected with siRNA against RPL22, incubated in smooth muscle cell culture medium (SMCM) and ET-1. RPL22 gene expression and protein levels of HPASMCs were measured by real-time PCR and western blotting, respectively. PCNA, CCK-8 immunohistochemistry and flow cytometry analysis were used to evaluate HPASMC proliferation. Cyclin D1 (CCND1) expression was also assayed. Following transfection with siRNA against RPL22, RPL22 expression was significantly inhibited in the control and ET-1 groups. HPASMC proliferation was significantly suppressed by transfection with siRNA against RPL22. Moreover, CCND1 was also downregulated by inhibiting RPL22 expression. In conclusion, these data suggest that inhibition of RPL22 expression can suppress HPASMC proliferation and CCND1 expression. The effects of siRNA against RPL22 on HPASMC proliferation are considered to be mediated through inhibition of CCND1 expression.