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Silica dust mainly attacks alveolar macrophages (AMs) and increases the apoptosis of AMs in silicosis patients. However, it is still unclear whether autophagy is affected. Autophagy mainly has defensive functions in response to stress, contributing to cell survival in adverse conditions, and conversely it has also been implicated in cell death. Lipopolysaccharide (LPS) induces autophagy and apoptosis in macrophages. The role of LPS in autophagy and apoptosis in AMs of silicosis patients is unknown. In this study, we collected AMs from 53 male workers exposed to silica and divided them into an observer (control) group, and stage I, II and III patient groups. We found increased levels of LC3B, SQSTM1/p62 and BECN1，whereas the phosphorylation of MTOR，and levels of LAMP2, TLR4, MYD88, TICAM1, as well as the number of lysosomes decreased with the development of silicosis. LPS stimulation triggered autophagy and increased levels of SQSTM1 in AMs. The autophagy inhibitor, 3-methyladenine (3MA), inhibited LPS-induced apoptosis in the AMs of silicosis patients. Moreover, 3MA reversed the LPS-induced decrease in BCL2 and the increase in BAX and CASP3 levels in AMs. These results suggest that autophagosomes accumulate in AMs during silicosis progression. LPS can induce the formation of autophagosomes through a TLR4-dependent pathway, and LPS may exacerbate the apoptosis in AMs. Blockade of the formation of autophagosomes may inhibit LPS-induced apoptosis via the intrinsic apoptotic pathway in AMs. These findings describe novel mechanisms that may lead to new preventive and therapeutic strategies for pulmonary fibrosis.

ABSTRACTThe migration of vascular smooth muscle cells (VSMCs) from the media to the intima is proposed to be a key event in the development of atherosclerosis. Recently, we reported that Chlamydia pneumoniaeinfection is involved in VSMC migration. However, the exact mechanisms for C. pneumoniaeinfection-induced VSMC migration are not yet well elucidated. In this study, we examined the role of the Toll-like receptor 2 (TLR2) activation-related signaling pathway in VSMC migration induced by C. pneumoniaeinfection. An Affymetrix-based gene expression array was conducted to identify the changes of gene expression in rat primary VSMCs (rVSMCs) infected with C. pneumoniae. Both the microarray analysis and quantitative real-time reverse transcription (RT)-PCR revealed that TLR2 mRNA expression was strongly upregulated 12 h after C. pneumoniaeinfection. RT-PCR and Western blot analysis further showed that the expression levels of TLR2 mRNA and protein significantly increased at the different time points after infection. Immunocytochemical analysis suggested a TLR2 recruitment to the vicinity of C. pneumoniaeinclusions. Cell migration assays showed that the TLR2-neutralizing antibody could significantly inhibit C. pneumoniaeinfection-induced rVSMC migration. In addition, C. pneumoniaeinfection stimulated Akt phosphorylation at Ser 473, which was obviously suppressed by the PI3K inhibitor LY294002, thereby inhibiting rVSMC migration caused by C. pneumoniaeinfection. Furthermore, both the infection-induced Akt phosphorylation and rVSMC migration were suppressed by the TLR2-neutralizing antibody. Taken together, these data suggest that C. pneumoniaeinfection can promote VSMC migration possibly through the TLR2-related signaling pathway.

Recent studies have shown that stromal cell derived factor‐1 (SDF‐1), first known as a cytokine involved in recruiting stem cells into injured organs, confers myocardial protection in myocardial infarction, which is not dependent on stem cell recruitment but related with modulation of ischemia‐reperfusion (I/R) injury. However, the effect of SDF has been studied only in a preischemic exposure model, which is not clinically relevant if SDF is to be used as a therapeutic agent. Our study was aimed at evaluating whether or not SDF‐1 confers cardioprotection during the reperfusion period. Hearts from SD rats were isolated and perfused with the Langendorff system. Proximal left coronary artery ligation, reperfusion, and SDF perfusion in KH buffer was done according to study protocol. Area of necrosis (AN) relative to area at risk (AR) was the primary endpoint of the study. Significant reduction of AN/AR by SDF in an almost dose‐dependent manner was noted during both the preischemic exposure and reperfusion periods. In particular, infusion of a high concentration of SDF (25 nM/L) resulted in a dramatic reduction of infarct size, which was greater than that achieved with ischemic pre‐ or postconditioning. SDF perfusion during reperfusion was associated with a similar significant reduction of infarct size as preischemic SDF exposure. Further studies are warranted to assess the potential of SDF as a therapeutic agent for reducing I/R injury in clinical practice.

Although the adenosine A(3) receptor agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) has been reported to be cardioprotective at reperfusion, little is known about the mechanisms underlying the protection. We hypothesized that IB-MECA may protect the heart at reperfusion by preventing the opening of mitochondrial permeability transition pore (mPTP) through inactivation of glycogen synthase kinase (GSK) 3beta. IB-MECA (1 muM) applied during reperfusion reduced infarct size in isolated rat hearts, an effect that was abrogated by the selective A(3) receptor antagonist 1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylic acid 3-ethyl-5-[(3-nitrophenyl)-methyl]ester (MRS1334) (100 nM). The effect of IB-MECA was abrogated by the mPTP opener atractyloside (20 muM), implying that the action of IB-MECA may be mediated by inhibition of the mPTP opening. In cardiomyocytes, IB-MECA attenuated oxidant-induced loss of mitochondrial membrane potential (DeltaPsi(m)), which was reversed by MRS1334. IB-MECA also reduced Ca(2+)-induced mitochondrial swelling. IB-MECA enhanced phosphorylation of GSK-3beta (Ser(9)) upon reperfusion, and the GSK-3 inhibitor 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) (3 muM) mimicked the protective effect of IB-MECA by attenuating both infarction and the loss of DeltaPsi(m). In addition, the effect of IB-MECA on GSK-3beta was reversed by wortmannin (100 nM), and IB-MECA was shown to enhance Akt phosphorylation upon reperfusion. In contrast, rapamycin (2 nM) failed to affect GSK-3beta phosphorylation by IB-MECA, and IB-MECA did not alter phosphorylation of either mTOR (Ser(2448)) or 70s6K (Thr(389)). Taken together, these data suggest that IB-MECA prevents myocardial reperfusion injury by inhibiting the mPTP opening through the inactivation of GSK-3beta at reperfusion. IB-MECA-induced GSK-3beta inhibition is mediated by the PI3-kinase/Akt signal pathway but not by the mTOR/p70s6K pathway.

Pre-ischemic treatment is seldom possible in the clinical setting of acute myocardial infarction. Thus, to successfully save myocardium from infarction, it is required that protective interventions must be effective when applied after ischemia has begun or at the onset of reperfusion. Unfortunately, in spite of a large body of experimental data showing that various interventions are cardioprotective at reperfusion, no specific therapy has yet been established to be clinically applicable. However, recent data from several laboratories have shown that adenosine and its analogues given at reperfusion can markedly protect the heart from ischemia/reperfusion injury. While the experimental data suggest that factors such as adenosine A2receptor activation, anti-neutrophil effect, attenuation of free radical generation, increased nitric oxide (NO) availability, activation of the PI3-kinase/Akt pathway and ERK, prevention of mitochondrial damage, and anti-apoptotic effects may be involved in the protective effect of adenosine or its analogues, the exact receptor subtype(s), the detailed signaling mechanisms, and interaction between those individual factors are still unknown. A definite answer to these unsolved problems will offer insights into the mechanisms of cardioprotection at reperfusion, and will be critical for developing a successful therapeutic strategy to salvage ischemic myocardium in patients with acute myocardial infarction.

AMP 579, an adenosine A1/A2receptor agonist, is cardioprotective when administered at reperfusion. Pretreatment with the Na/Hexchanger inhibitor cariporide or ischemic preconditioning (PC) also limits infarct size. To gain insight into the mechanism of AMP 579 we investigated whether its protection could be added to that from either cariporide or PC. In siturabbit hearts were subjected to 45 min of regional ischemia followed by 3 h of reperfusion. Infarct size in the control group was 55.8 ± 3.9 of the risk zone. PC significantly reduced infarct size to 26.0 ± 6.7 (p<0.05). AMP 579 (30 g/kg) given just before reperfusion followed by 3 g/kg/min infusion for 70 min also limited infarct size (32.1 ± 1.8,) but the combination of AMP 579 and PC showed a significantly greater limitation of infarct size (5.5 ± 2.7, p < 0.05). Because cariporide pretreatment was so protective (8.5 ± 3.7 infarction), we had to increase the ischemic insult to 60 min to test for any additive effect of the combination of AMP 579 cariporide. Infarct size in the untreated group was 66.0 ± 4.9 of the risk zone. Cariporide (0.5 mg/kg) 5 min prior to ischemia significantly reduced infarct size to 41.5 ± 7.7. When cariporide pre-treatment was combined with AMP 579 at reperfusion, infarction was further limited (14.2 ± 4.5). Because AMP 579's protection can be added to that of either cariporide or PC, AMP 579's mechanism of protection probably differs from either of them. The combination of AMP 579 cariporide was particularly efficacious and could be useful in the surgical setting.

To determine the mechanism by which AMP 579, an adenosine A1A2agonist, administered at reperfusion protects ischemic myocardium, bufferperfused rabbit hearts were subjected to 30 min of global ischemia and 2 h of reperfusion. AMP 579 500 nMwas included in the reperfusate for the first 70 min. Average left ventricular diastolic pressure during reperfusion in hearts receiving AMP 579 was lower than that in control hearts 17.9 ± 2.4 vs. 39.0 ± 6.5 mm Hg, p < 0.05, indicating attenuation of contracture. Left ventricular developed pressure and coronary flow during reperfusion were also significantly improved with AMP 579 treatment. AMP 579's anticontracture effect was blocked by the adenosine A2receptor antagonist 83chlorostyrylcaffeine CSC, but not by the A1antagonist 8cyclopentyl1,3dipropylxanthine DPCPX. CSC, but not DPCPX, also blocked AMP 579's ability to preserve developed pressure and coronary flow in these hearts. AMP 579 significantly reduced infarction in isolated hearts subjected to regional ischemia. The antiinfarct effect again was abolished by CSC but not by DPCPX. Finally, we tested whether 5′Nethylcarboxamidoadenosine NECA, another A1A2agonist, also administered for the initial 70 min of reperfusion, could duplicate the antiinfarct effect of AMP 579. Onehundrednanomolar NECA duplicated the protection, but neither 50 nMCGS21680, a selective A2agonist, nor 100 Madenosine was protective. Therefore, AMP 579 given at reperfusion reduces contracture and infarction. Anticontracture and antiinfarct effects require the adenosine A2, but not the A1, receptor suggesting that prevention of contracture and tissue salvage are mechanistically related. Not all A2agonists were able to duplicate the antiinfarct effect, suggesting something unique about AMP579.

The novel A1/A2adenosine receptor agonist AMP 579 has been reported to reduce myocardial infarct size in pig and dog. The present study tested the effect of AMP 579 in two rabbit models. In open-chest rabbits undergoing 30 min of regional ischemia and 3 h of reperfusion AMP 579 (3 μg/min/kg) reduced infarct size when treatment was started either 10 min before ischemia or 10 min prior to reperfusion from 36.4±3.1% of the risk zone in untreated hearts to 11.8±4.4 and 12.3±1.0%, respectively. To determine whether protection observed when the drug was administered shortly before reperfusion represented a long-lasting effect rather than merely a transient delay of necrosis, the chest wound was closed in layers and the rabbits permitted to recover. After 3 days the hearts were removed to evaluate infarct size. Continued limitation of infarct size after 3 days of reperfusion (8.2±2.8% of the risk zone) confirmed that sustained tissue salvage had been conferred by the drug. In isolated, buffer-perfused rabbit hearts undergoing 30 min of regional ischemia and 2 h of reperfusion, AMP 579 again limited infarct size (8.6±2.9% of the risk zone) when treatment started 10 min prior to reperfusion, arguing against an anti-leukocyte mechanism of protection. AMP 579's protective effect in this in vitromodel was abrogated by 8-(p-sulfophenyl)theophylline, indicating that it was mediated through adenosine receptors. We conclude that AMP 579 given just prior to reperfusion may be an effective anti-infarct intervention.