Thong Hua-Huy

Serum CC chemokine ligand-18 predicts lung disease worsening in systemic sclerosis

Elevated serum CC chemokine ligand (CCL)18 reflects lung fibrosis activity in systemic sclerosis (SSc) and could be an early marker of lung function worsening. Therefore, we sought to evaluate whether serum CCL18 levels at baseline could predict worsening of lung disease in SSc. In this prospective study, 83 SSc patients were analysed longitudinally over a 4-yr observation period for the risk of occurrence of combined deleterious events, defined as a 10% decrease from baseline of total lung capacity or forced vital capacity % predicted, or death, according to serum CCL18 at inclusion. Receiver operating characteristic (ROC) curve analysis was performed for prediction of events during the first year after inclusion. The best cut-off level of serum CCL18 for prediction of a combined event within the follow-up period was 187 ng·mL−1, with 53% sensitivity and 96% specificity (area under the ROC curve 0.86; p<0.001). After a mean±sd follow-up of 33.7±10.8 months, a higher rate of disease progression occurred in the group with serum CCL18 levels >187 ng·mL−1. The adjusted hazard ratio was 5.36 (95% CI 2.44–11.75; p<0.001). In summary, serum CCL18 is an accurate predictive biomarker for the identification of patients with a higher risk of subsequent scleroderma lung disease worsening.

Alveolar concentration of nitric oxide predicts pulmonary function deterioration in scleroderma

Background Respiratory failure is a life-threatening and unpredictable complication of systemic sclerosis (SSc). A study was undertaken to assess the value of alveolar nitric oxide (NO) in predicting the risk of lung function deterioration leading to respiratory failure or death in patients with SSc. Methods 105 patients with SSc were enrolled in this prospective cohort and were followed longitudinally over a 3-year period during which the risk of occurrence of deleterious events was analysed according to alveolar concentration (CAno), conducting airway output (J′awno) and fractional concentration (FEno0.05) of exhaled NO measured at inclusion. Comparison was made between each NO parameter to predict the occurrence of deleterious events, defined as a 10% decrease in total lung capacity or forced vital capacity from baseline, or death. Results The area under the receiver operating characteristic curve of CAno to predict the occurrence of the combined events was 0.84 (95% CI 0.76 to 0.92; p<0.001), which was significantly higher than those of J′awno and FEno0.05 (p<0.001). A cut-off of CAno of 5.3 ppb had a sensitivity of 88% and a specificity of 62% for the prediction of the occurrence of combined events during follow-up, and was validated in an independent cohort of patients with SSc. Combined events occurred more frequently in patients whose CAno was >5.3 ppb. The adjusted HR for patients with CAno >5.3 ppb was 6.06 (95% CI 2.36 to 15.53; p<0.001). CAno accurately predicted the occurrence of combined events irrespective of forced vital capacity values or the presence of interstitial lung disease at baseline. Conclusions Increased CAno accurately identifies patients with SSc with a high risk of developing lung function deterioration and may help to initiate early appropriate treatment.

Long-term treatment with fasudil improves bleomycin-induced pulmonary fibrosis and pulmonary hypertension via inhibition of Smad2/3 phosphorylation

Pulmonary hypertension (PH) associated with pulmonary fibrosis (PF) considerably worsens prognosis of interstitial lung diseases (ILD). RhoA/Rho-kinases (ROCK) pathway is implicated in high pulmonary vascular tone and pulmonary fibrosis but the effect of ROCK inhibitors on PH associated with PF is not known. We therefore aimed to determine whether long-term treatment with fasudil, a selective ROCK inhibitor, could attenuate PF and PH induced by bleomycin in mice. Male C57BL/6 mice received a single dose of intratracheal bleomycin (3.3 U/kg) to induce PF. Treatment with fasudil (30 mg kg(-1) day(-1)) was given intraperitoneally for 7, 14 or 21 days until mice underwent hemodynamic measurements. Right ventricular systolic pressure (RVSP) and RV/(LV + S) ratio were assessed. Lung inflammatory cells profiles, including macrophages, neutrophils, lymphocytes B and lymphocytes T were assessed by immunohistochemistry. Lung fibrosis was evaluated by histological and biochemical methods. Pulmonary arteriole muscularization and medial wall thickness (MWT) were evaluated by immunohistochemical staining for α-SMA. Bleomycin induced severe PF and PH in mice, associated with an increased RhoA/ROCK activity in the lung. Fasudil reduced lung inflammation and lung collagen content, and attenuated the increased RVSP, RV hypertrophy, and pulmonary vascular remodeling in bleomycin-intoxicated mice. Fasudil inhibited the increased activity of RhoA/ROCK pathway, and partly altered bleomycin-associated activation of TGF-β1/Smad pathway, via inhibition of Smad2/3 phosphorylation. The efficacy of long-term treatment with fasudil suggests that the blockade of RhoA/ROCK pathway may be a promising therapy for patients with ILD-associated PH.

Amelioration of Systemic Fibrosis in Mice by Angiotensin II Receptor Blockade

OBJECTIVE Systemic sclerosis (SSc) is characterized by microvascular damage, fibrosis of skin and visceral organs, and autoimmunity. Previous studies have shown that angiotensin II is involved in the synthesis of type I collagen. We investigated whether the blockade of angiotensin II receptor type I (AT1 ) by irbesartan reduces skin and lung fibrosis in 2 murine models of SSc. METHODS SSc was induced by daily intradermal injection of HOCl into the backs of BALB/c mice (HOCl-induced SSc). Mice were treated daily with irbesartan by oral gavage. RESULTS Irbesartan reduced dermal thickness, collagen concentration, Smad2/3, and α-smooth muscle actin expression, as well as fibroblast proliferation and H-Ras expression in the skin of mice with HOCl-induced SSc. Mice treated with irbesartan also displayed less lung fibrosis, less inflammation, and a lower concentration of collagen in the lungs than untreated mice. Exhaled nitric oxide, inducible nitric oxide synthase, and 3-nitrotyrosine expression in the lungs were decreased following irbesartan treatment. Moreover, irbesartan reduced the number and the proliferation of splenic B and T cells and the serum levels of anti-DNA topoisomerase I autoantibodies. CONCLUSION Irbesartan, an AT1 antagonist, prevents fibrosis and inflammation and inhibits nitric oxide production in HOCl-induced models of systemic fibrosis. Our findings extend the indication of an AT1 antagonist to SSc patients with diffuse fibrosis, especially those with lung involvement.

Increased Rho-kinase expression and activity and pulmonary endothelial dysfunction in smokers with normal lung function

Endothelial dysfunction is one of the main consequences of the toxic effects of cigarette smoke on the vascular system. Increasing evidence suggests that the small G-protein RhoA and its downstream effectors, the Rho-kinases (ROCKs), are involved in systemic endothelial dysfunction induced by cigarette smoke. This study aimed to evaluate the role of the RhoA/ROCKs pathway in pulmonary artery endothelial function in current smokers with normal lung function. Lung tissues were obtained from nonsmokers and smokers who underwent lobectomy for lung carcinoma. Arterial relaxation in response to acetylcholine (ACh) was assessed in isolated pulmonary arterial rings. Protein expressions and activities of endothelial nitric oxide synthase (eNOS), ROCKs and the myosin phosphatase subunit 1 (MYPT-1) were sought. Relaxation in response to ACh was significantly lower in smokers as compared with nonsmokers (n = 8 in each group), consistent with reduced eNOS activity in the former compared with the latter. eNOS protein expression remained, however, the same in both groups. Expression of ROCKs, guanosine triphosphate-RhoA and phosphorylated MYPT-1 were significantly increased in smokers compared with controls. Pulmonary endothelial dysfunction is present in smokers whose lung function has not yet been impaired. Reduced activity of eNOS accounts at least in part for this endothelial dysfunction. Increased expression and activity of ROCKs accounts for another part through direct or indirect inhibition of the Rho-A/ROCKs pathway on nitric oxide synthesis and sustained pulmonary vasoconstriction through inhibition of myosin phosphatase.

Activation of RhoA/Rho‐kinase pathway accounts for pulmonary endothelial dysfunction in patients with chronic obstructive pulmonary disease

Recent evidence suggests that activation of RhoA/Rho‐kinase accounts for systemic and pulmonary endothelial dysfunction in smokers with normal lung function. However, its role in patients with chronic obstructive pulmonary disease (COPD) has not yet been investigated. The aim of this study was to evaluate the regulation of RhoA/Rho‐kinase pathway and pulmonary endothelial dysfunction in patients with COPD. Pulmonary arteries were obtained from nonsmokers (control subjects) and patients with nonhypoxemic and hypoxemic COPD (n = 6–7/group). Endothelium‐dependent and ‐independent relaxations were evaluated by acetylcholine and sodium nitroprusside, respectively. Gene and protein expressions of endothelial nitric oxide synthase (eNOS) were measured by RT‐PCR, Western blot, and immunohistochemistry. Nitrate, cGMP, and endothelin‐1 (ET‐1) concentrations, as well as Rho‐kinase activity were measured by ELISA. Protein expressions of total RhoA and GTP‐RhoA were measured by Western blot and pull‐down assay, respectively. Endothelium‐dependent relaxation, and nitrate and cGMP levels were significantly reduced in pulmonary arteries of COPD patients as compared with control subjects. Conversely, activity of RhoA/Rho‐kinase was increased in pulmonary arteries of COPD patients as compared with control subjects. In patients with COPD, pulmonary endothelial dysfunction was related to the downregulation of eNOS activity and upregulation of RhoA/Rho‐kinase activity.

Increased alveolar concentration of nitric oxide is related to serum-induced lung fibroblast proliferation in patients with systemic sclerosis.

Objective. Lung inflammation is present in patients with systemic sclerosis (SSc) and interstitial lung disease (ILD), but the mechanisms linking inflammatory and fibrotic processes in ILD are unknown. Our aim was to investigate whether alveolar inflammation, reflected by increased alveolar concentration of exhaled nitric oxide (CANO), is related to the ability of serum from patients with SSc to induce pulmonary fibroblast proliferation (PFP) and myofibroblast conversion. Methods. CANO was measured in all subjects (37 patients with SSc and 10 healthy controls) whose sera were used to stimulate PFP (assessed by BrdU labeling index) and myofibroblast conversion (detected by α-smooth muscle actin expression). The PFP index in patients with SSc was compared to control values, and between patients with SSc who had elevated (> 4.3 ppb) and normal (≤ 4.3 ppb) CANO values. Results. Both CANO and the PFP index were significantly greater in patients with SSc compared to controls. In patients with SSc, the PFP index was directly related to CANO levels (r = 0.48; p = 0.002). The median PFP index was significantly higher in patients with SSc who had elevated CANO (> 4.3 ppb; n = 25, median 1.1, range 0.98–1.23) than in patients with SSc who had normal CANO (≤ 4.3 ppb; n = 12, median 0.93, range 0.82–1.08; p = 0.01). Similarly, myofibroblast conversion induced by SSc serum was significantly greater in patients with CANO > 4.3 ppb than in patients whose CANO was ≤ 4.3 ppb (p < 0.001) and controls (p < 0.001). Conclusion. Alveolar inflammation reflected by increased nitric oxide production was related to serum-induced PFP and myofibroblast conversion, linking the active alveolitis process to cell proliferation and lung fibrosis in patients with SSc.

Cellular and molecular mechanisms in the pathophysiology of systemic sclerosis.

Fibrosis is characterized by disproportionate accumulation of collagens and other extracellular matrix substances, resulting in organ dysfunction and failure. In systemic sclerosis, cellular and molecular mechanisms involved in the pathophysiology of fibrosis are highly complex and yet barely understood. Anatomopathological findings showed the coexistence of patchy inflammatory cell infiltration, microvascular injuries, and fibrotic foci. One of the most commonly accepted hypotheses considers endothelial activation as the triggering phenomenon inducing inflammatory and autoimmunity activation. The resulting cytokines and autoantibodies production accelerates the proliferating rate of normal fibroblasts and their transformation into myofibroblasts, leading to diffuse fibrosis. This review aims to focus on cellular and molecular mechanisms implicated in the fibrogenesis of systemic sclerosis.

Early inhaled nitric oxide at high dose enhances rat lung development after birth

RATIONAL Inhaled nitric oxide (NO) is frequently administered to full term and preterm newborns in various clinical settings in order to alleviate pulmonary hypertension whilst improving oxygenation. However, the physiological effect of NO on early postnatal lung development has not yet been clearly described. We therefore investigated whether NO administered by inhalation affects lung development at early postnatal life. METHODS Pregnant rats were placed in a chamber containing 5 ppm (iNO-5 ppm group) and 20 ppm NO (iNO-20 ppm group), started from the last day of their pregnancy in order to keep rat pups under ambient NO from birth to 7 days postnatal. Control animals were kept at room air and all rat pups were sacrificed at postnatal day 7 and day 14. RESULTS Lung-to-body weight and wet-to-dry lung weight ratios did not significantly differ among 3 groups at postnatal day 7 and day 14. Vascular volume densities (Vv) in both NO groups (5 and 20 ppm) were higher than controls (P<0.05; P<0.001). Pulmonary vessel number was significantly increased in iNO-20 ppm group. Radial alveolar counts (RAC) and mean linear intercepts (MLI) markedly increased (consistent with increased alveolarization) in iNO-20 ppm group. This was associated with upregulation of VEGF/VEGFR-2, MT1-MMP/MMP2 and HO-1 protein expression in iNO-20 ppm group. CONCLUSIONS We concluded that inhaled NO at 20 ppm enhanced lung development possibly through increased expression of HO-1, VEGF/VEGFR-2, and MMP2 at early stage of postnatal rat life.

Exhaled NO predicts cyclophosphamide response in scleroderma-related lung disease.

Cyclophosphamide (CYC) is not always effective in patients with scleroderma-related interstitial lung disease (SSc-ILD), hence the need for biomarkers able to predict beneficial responses to CYC therapy. We therefore assessed whether baseline alveolar concentration of nitric oxide (CANO) could predict the favourable response to CYC therapy in patients with SSc-ILD. Nineteen non-smoker patients with SSc-ILD, were enrolled and treated with 6 courses of CYC (0.75 g/m2/monthly) for lung function decline the year before inclusion, and followed-up for 2 years period. We assessed the proportion of favourable response to CYC, defined as improvement of forced vital capacity (FVC) or total pulmonary capacity (TLC) more than 10% between the inclusion and each following visit, according to the validated cut-off of CANO at 8.5 ppb identifying progressive SSc-ILD subset. At inclusion, 7 patients out of 19 had CANO >8.5 ppb. Clinical parameters were comparable between patients with high (>8.5 ppb) and low level of CANO (≤8.5 ppb). After CYC therapy, and during the follow-up, 9 out of 19 patients had favourable response to CYC therapy, 10 did not meet responders criteria, from whom 4 patients died from respiratory failure. Six out of 7 patients with CANO >8.5 ppb at inclusion had favourable response to CYC therapy, while only 3 out of 12 patients with CANO ≤8.5 ppb responded favourably to CYC therapy (p=0.001). High level of CANO >8.5 ppb reflecting alveolar inflammation identify SSc patients with a greater chance to benefit from CYC treatment with a significant lung function improvement.