Investigation of the effects of α-tocopherol on the levels of Fe,Cu, Zn,Mn, and carbonic anhydrase in rats with bleomycin-induced pulmonary fibrosis

This study was designed to examine the effects of vitamin E on the levels of Zn, Mn, Cu, Fe, and carbonic anhydrase in rats with bleomycin-induced pulmonary fibrosis. Twenty-one male Wistar albino rats were randomly divided into three groups: bleomycin alone, bleomycin+vitamin E, and saline alone (control group). The bleomycin group was given 7.5 mg/kg body weight (single dose) bleomycin hydrochloride intratracheally. The bleomycin+vitamin E group was also instilled with bleomycin hydrochloride but received injections of α-tocopherol twice a week. The control group was treated with saline alone. Animals were sacrified 14 d after intratracheal instillation of bleomycin. Tissue Zn, Mn, Cu, Fe, and carbonic anhydrase activities were measured in the lung and liver. Lung Cu, Fe, and carbonic anhydrase activity increase in both experimental groups. Zn and Mn levels decreased, except for the Mn level in the bleomycin group. Liver Zn, Mn, and Cu levels decreased in both experimental groups compared to the control group, whereas Fe and carbonic anhydrase activity increased in comparison to the control group. However, the liver tissue Fe level decreased compared to the control group. In the histopathologic assesment of lung sections in the bleomycin+vitamin E group, partial fibrotic lesions were observed, but the histopathologic changes were much less severe compared to the bleomycin-treated group.     

New mechanisms of pulmonary arterial hypertension: role of Ca²⁺ signaling

Pulmonary arterial hypertension (PAH) is a severe and progressive disease that usually culminates in right heart failure and death if left untreated. Although there have been substantial improvements in our understanding and significant advances in the management of this disease, there is a grim prognosis for patients in the advanced stages of PAH. A major cause of PAH is increased pulmonary vascular resistance, which results from sustained vasoconstriction, excessive pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular stiffness. In addition to other signal transduction pathways, Ca(2+) signaling in pulmonary artery smooth muscle cells (PASMCs) plays a central role in the development and progression of PAH because of its involvement in both vasoconstriction, through its pivotal effect of PASMC contraction, and vascular remodeling, through its stimulatory effect on PASMC proliferation. Altered expression, function, and regulation of ion channels and transporters in PASMCs contribute to an increased cytosolic Ca(2+) concentration and enhanced Ca(2+) signaling in patients with PAH. This review will focus on the potential pathogenic role of Ca(2+) mobilization, regulation, and signaling in the development and progression of PAH.     

Therapeutic effect of a peptide inhibitor of TGF-β on pulmonary fibrosis

Pulmonary fibrosis encompasses several respiratory diseases characterized by epithelial cell injury, inflammation and fibrosis. Transforming growth factor (TGF)-β1 is one of the main profibrogenic cytokines involved in the pathogenesis of lung fibrosis. It induces fibroblast differentiation into myofibroblasts, which produce high levels of collagen and concomitantly loss of lung elasticity and reduction of the respiratory function. In the present study, we have investigated the effects of P17 (a TGF-β inhibitor peptide) on IMR-90 lung fibroblast differentiation in vitro, as well as on the inhibition of the development of bleomycin-induced pulmonary fibrosis in mice. It was found that in IMR-90 cells, P17 inhibited TGF-β1-induced expression of connective tissue growth factor and α-smooth muscle actin. In vivo, treatment of mice with P17 2days after bleomycin administration decreased lung fibrosis, areas of myofibroblast-like cells and lymphocyte infiltrate. P17 also reduced mRNA expression of collagen type I, fibronectin and the fibronectin splice isoform EDA in the lung, and increased the expression of IFN-γ mRNA. Finally, therapeutic treatment with P17 in mice with already established fibrosis was able to significantly attenuate the progression of lung fibrosis. These results suggest that P17 may be useful in the treatment of pulmonary fibrosis.     

Enhanced store-operated Ca²+ entry and TRPC channel expression in pulmonary arteries of monocrotaline-induced pulmonary hypertensive rats

Pulmonary hypertension (PH) is associated with profound vascular remodeling and alterations in Ca(2+) homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Previous studies show that canonical transient receptor potential (TRPC) genes are upregulated and store-operated Ca(2+) entry (SOCE) is augmented in PASMCs of chronic hypoxic rats and patients of pulmonary arterial hypertension (PAH). Here we further examine the involvement of TRPC and SOCE in PH with a widely used rat model of monocrotaline (MCT)-induced PAH. Rats developed severe PAH, right ventricular hypertrophy, and significant increase in store-operated TRPC1 and TRPC4 mRNA and protein in endothelium-denuded pulmonary arteries (PAs) 3 wk after MCT injection. Contraction of PA and Ca(2+) influx in PASMC evoked by store depletion using cyclopiazonic acid (CPA) were enhanced dramatically, consistent with augmented SOCE in the MCT-treated group. The time course of increase in CPA-induced contraction corresponded to that of TRPC1 expression. Endothelin-1 (ET-1)-induced vasoconstriction was also potentiated in PAs of MCT-treated rats. The response was partially inhibited by SOCE blockers, including Gd(3+), La(3+), and SKF-96365, as well as the general TRPC inhibitor BTP-2, suggesting that TRPC-dependent SOCE was involved. Moreover, the ET-1-induced contraction and Ca(2+) response in the MCT group were more susceptible to the inhibition caused by the various SOCE blockers. Hence, our study shows that MCT-induced PAH is associated with increased TRPC expression and SOCE, which are involved in the enhanced vascular reactivity to ET-1, and support the hypothesis that TRPC-dependent SOCE is an important pathway for the development of PH.     

Over-expression of PKGIα inhibits hypoxia-induced proliferation, Akt activation, and phenotype modulation of human PASMCs: the role of phenotype modulation of PASMCs in pulmonary vascular remodeling

The proliferation of pulmonary artery smooth muscle cells (PASMCs) plays a role in pulmonary vascular remodeling (PVR). Recently, it was shown that vascular smooth muscular cell phenotype modulation is important for their proliferation in other diseases. However, little is known about the role of human PASMC phenotype modulation in the proliferation induced by hypoxia and its molecular mechanism during PVR. In this study, we found using primary cultured human PASMCs that hypoxia suppressed the expression of endogenous PKGIα, which was reversed by transfection with a recombinant adenovirus containing the full-length cDNA of PKGIα (Ad-PKGIα). Ad-PKGIα transfection significantly attenuated the hypoxia-induced downregulation of the expression of smooth muscle α-actin (SM-α-actin), myosin heavy chain (MHC) and calponin in PASMCs, indicating that hypoxia-induced phenotype modulation was blocked. Furthermore, flow cytometry and ³H-TdR incorporation demonstrated that hypoxia-induced PASMC proliferation was suppressed by upregulation of PKGIα. These results suggest that enhanced PKGIα expression inhibited hypoxia-induced PASMC phenotype modulation and that it could reverse the proliferation of PASMCs significantly. Moreover, our previous work has demonstrated that Akt protein is activated in the process of hypoxia-induced proliferation of human PASMCs. Interestingly, we found that Akt was not activated by hypoxia when PASMC phenotype modulation was blocked by Ad-PKGIα. This result suggests that blocking phenotype modulation might be a key up-stream regulatory target.     

19,20-EpDPE, a bioactive CYP450 metabolite of DHA monoacyglyceride, decreases Ca²⁺ sensitivity in human pulmonary arteries

The aim of this study was to investigate the effect of docosahexaenoic acid monoacylglyceride (MAG-DHA) on human pulmonary arterial tone. Tension measurements on pulmonary arterial tissues demonstrated that MAG-DHA reduced U-46619-induced tone, which is highly sensitive to the H-1152 inhibitor. Results also showed that MAG-DHA treatments decreased RhoA activity levels, which in turn inactivated the Rho-kinase pathway, leading to a reduction in U-46619-induced Ca(2+) sensitivity of permeabilized pulmonary artery smooth muscle cells. According to the mechanical responses assessing U-46619-induced Ca(2+) sensitivity in the absence or presence of 3 μM MAG-DHA, MAG-DHA plus 1 μM N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH, a cytochrome P-450 epoxygenase inhibitor) and 300 nM 19,20-epoxydocosapentaenoic acid (a cytochrome P-450 epoxygenase-dependent DHA metabolite), our data suggest that the MAG-DHA is metabolized in a bioactive epoxymetabolite. This epoxyeicosanoid in turn decreases active tone and Ca(2+) sensitivity of smooth muscles cells through an inhibition of the Rho-kinase pathway. Together, these data provide primary evidence regarding the mode of action of MAG-DHA in human pulmonary arteries and suggest that this compound may be of pharmacological interest in patients with pulmonary hypertension to generate intracellular bioactive metabolites.     

Platelet-derived growth factor receptor-β and epidermal growth factor receptor in pulmonary vasculature of systemic sclerosis-associated pulmonary arterial hypertension versus idiopathic pulmonary arterial hypertension and pulmonary veno-occlusive disease: a case-control study

Introduction

Systemic sclerosis (SSc) complicated by pulmonary arterial hypertension (PAH) carries a poor prognosis, despite pulmonary vascular dilating therapy. Platelet-derived growth factor receptor-β (PDGFR-β) and epidermal growth factor receptor (EGFR) are potential therapeutic targets for PAH because of their proliferative effects on vessel remodelling. To explore their role in SScPAH, we compared PDGFR- and EGFR-mmunoreactivity in lung tissue specimens from SScPAH. We compared staining patterns with idiopathic PAH (IPAH) and pulmonary veno-occlusive disease (PVOD), as SScPAH vasculopathy differs from IPAH and sometimes displays features of PVOD. Immunoreactivity patterns of phosphorylated PDGFR-β (pPDGFR-β) and the ligand PDGF-B were evaluated to provide more insight into the patterns of PDGFR-b activation.

Methods

Lung tissue specimens from five SScPAH, nine IPAH, six PVOD patients and five controls were examined. Immunoreactivity was scored for presence, distribution and intensity.

Results

All SScPAH and three of nine IPAH cases (P = 0.03) showed PDGFR-β-immunoreactivity in small vessels (arterioles/venules); of five SScPAH vs. two of nine IPAH cases (P = 0.02) showed venous immunoreactivity. In small vessels, intensity was stronger in SScPAH vs. IPAH. No differences were found between SScPAH and PVOD. One of five normal controls demonstrated focally mild immunoreactivity. There were no differences in PDGF-ligand and pPDGFR-b-immunoreactivity between patient groups; however, pPDGFR-b-immunoreactivity tended to be more prevalent in SScPAH small vasculature compared to IPAH. Vascular EGFR-immunoreactivity was limited to arterial and arteriolar walls, without differences between groups. No immunoreactivity was observed in vasculature of normals.

Conclusions

PDGFR-β-immunoreactivity in SScPAH is more common and intense in small- and post-capillary vessels than in IPAH and does not differ from PVOD, fitting in with histomorphological distribution of vasculopathy. PDGFR-β immunoreactivity pattern is not paralleled by pPDGFR-β or PDGF-B patterns. PDGFR-β- and EGFR-immunoreactivity of pulmonary vessels distinguishes PAH patients from controls.     

Hypoxic vasoconstriction of cyclostome systemic vessels: the antecedent of hypoxic pulmonary vasoconstriction?

Hypoxic vasoconstriction (HV) is an intrinsic response of mammalian pulmonary vascular smooth muscle (VSM). In the present study, HV was examined by myography of vessel rings from three primitive vertebrates: New Zealand hagfish (NZH), Pacific hagfish (PH), and sea lamprey (SL). Hypoxia dilated pre-gill arteries (ventral aorta, afferent branchial) from all species, whereas it contracted systemic arteries [dorsal aorta (DA), efferent branchial, celiacomesenteric]. DA HV was reproducible over several days, and it could be sustained in NZH for 8 h without adverse effects. Tension was proportional to PO(2), and half-maximal HV was obtained at PO(2) (mmHg) of 4.7 +/- 0. 2 (NZH), 0.8 +/- 0.1 (PH), and 10.7 +/- 1.9 (SL). HV did not require preconditioning (preexisting contractile stimulus) and was unaffected by elevated extracellular potassium (200 mM NZH; 80 mM SL); removal of the endothelium (NZH); or inhibitors of cyclooxygenase, lipoxygenase, cytochrome P-450 or antagonists of alpha-adrenergic, muscarinic, nicotinic, purinergic, or serotoninergic receptors. These results show that HV is an intrinsic feature of systemic VSM in cyclostomes and suggest that HV has been in the repertoire of VSM responses, since the origin of vertebrates. The exceptionally hardy HV in cyclostome DA may provide a useful model with which to examine both the phylogeny and mechanisms of this response.     

Medical treatment of pulmonary hydatid disease: for which child?

There have been many encouraging studies on medical treatment of pulmonary hydatid disease due to Echinococcus granulosus infection. Our aims were to demonstrate the safety and efficacy of medical treatment in pulmonary hydatid disease and to describe a pediatric population who would benefit from medical treatment, especially in respect to the diameter of the hydatid cyst. All patients were treated with mebendazole or albendazole. Treatment outcome was defined as cure, improvement or failure. Among 82 patients, 34.1% were cured, 34.1% improved and 31.8% failed. When 102 cysts were individually evaluated, 36.31% were cured, 32.4% improved and 31.3% failed. The cure and the failure rates were statistically insignificant in cysts treated with mebendazole and albendazole; however statistically significantly more cysts were improved with albendazole. The results were statistically insignificant between continuous and cyclic albendazole treatment. The mean size of successfully treated cysts was 5.3+/-3.4 cm, but "failed" for cyst with a mean size of 7.3+/-4.3 cm. There was a positive, weak and statistically significant correlation between the cyst size and treatment results. The major complication was infection. We suggest that selected pediatric patients with uncomplicated pulmonary hydatid cysts sized less than 5 cm should have a trial of medical treatment with a very close follow up.     

Heterogeneous distribution of type I nitric oxide synthase in pulmonary vasculature of ovine fetus

The nitric oxide/guanosine 3',5'-cyclic monophosphate pathway plays an essential role in mediating pulmonary vasodilation at birth. Small resistance arteries in the fetal lung are vessels of major significance in the regulation of pulmonary vascular tone. The present study is to determine that type I nitric oxide synthase (NOS-I) is present in ovine fetal pulmonary vasculature and that NOS-I is distributed heterogeneously in ovine fetal pulmonary circulation. We used reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and NOS-I immunohistochemistry to localize NOS-I in fetal sheep lungs and showed a colocalization for NADPH-d activity with NOS-I immunoreactivity. Strong NOS-I immunoreactivity was observed exclusively in the endothelium of the terminal bronchiole and respiratory bronchiole-associated arteries. As a comparison, adult sheep lung did not show positive immunoreactivity in the pulmonary endothelium. NOS-I was absent in the umbilical or systemic arteries from the ovine fetus, whereas abundant NOS-III immunoreactivity was present in these arteries. We conclude that NOS-I is present uniquely in the ovine fetal pulmonary circulation as opposed to the adult pulmonary or the fetal systemic circulation. NOS-I is distributed heterogeneously in the ovine pulmonary vasculature. We speculate that NOS-I plays an active role in the regulation of perinatal pulmonary circulation.     

Effect of pulmonary TNF-α overexpression on mouse isolated skeletal muscle function

TNF-α is a proinflammatory cytokine that is involved in numerous pathological processes including chronic obstructive pulmonary disease (COPD). In the present study, we used a transgenic mouse model that overexpresses TNF-α in the lung (Tg(+)) to test the hypothesis that chronic exposure to TNF-α (as seen in COPD) reduces skeletal muscle force production and fatigue resistance, particularly under low Po(2) conditions. At 7-12 mo, body and muscle weight of both extensor digitorum longus (EDL) and soleus were significantly smaller in Tg(+) compared with littermate wild-type (WT) mice; however, the body-to-muscle weight ratio was not different between groups. EDL and soleus muscles were subjected to in vitro fatiguing contractile periods under high (～550 Torr) and low Po(2) (～40 Torr). Although all muscles were less fatigue-resistant during low Po(2) compared with high Po(2), only the soleus fatigued more rapidly in Tg(+) mice (～12%) compared with WT at high Po(2). The maximal tension of EDL was equally reduced in Tg(+) mice (28-34% decrease from WT under both Po(2) conditions); but for soleus this parameter was smaller only under low Po(2) in Tg(+) mice (～31% decrease from WT). The peak rate of relaxation and the peak rate of contraction were both significantly reduced in Tg(+) EDL muscles compared with WT EDL under low Po(2) conditions, but not in soleus. These results demonstrate that TNF-α upregulation in the lung impairs peripheral skeletal muscle function but affects fast- and slow-twitch muscles differentially at high and low Po(2).