Molecular mechanisms of pathogenesis of chronic obstructive pulmonary disease

Abstract

Kronična opstrukcijska plućna bolest (KOPB) najučestalija je kronična respiracijska bolest s progresivnim tijekom. Temeljni uzroci KOPB-a uključuju pušenje cigareta, čimbenike okoline te gensku predispoziciju. Dosadašnje spoznaje o razvoju upale, oksidacijskom stresu i neravnoteži proteaza i antiproteaza nisu dostatne za objašnjenje nastanka i progresije bolesti. Ovo istraživanje pokazalo je da ekstrakt dima cigareta u A549 alveolarnim epitelnim stanicama uzrokuje oksidacijska oštećenja staničnih lipida i proteina, što bi moglo voditi do gubitka važnih funkcija stanice, rezultirajući smrću stanice. Nakupljanje oštećenih proteina u stanicama vodi do aktivacije ER stresnog odgovora u vidu povećanja razine fosforiliranog eIF2α i smanjenja de novo sinteze proteina. Smanjena sinteza proteina vjerojatno pridonosi povećanju koncentracije slobodnih aminokiselina unutar stanica zajedno s razgradnjom proteina oštećenih dimom cigareta putem ubikvitin-proteasomalnog sustava. Iako se ER stresni odgovor obično povezuje s povećanom aktivnosti proteasoma, dim cigareta uzrokuje značajno smanjenje kaspazne, tripsinske i kimotripsinske aktivnosti proteasoma, što ih čini nedovoljno učinkovitima u procesu uklanjanja oštećenih proteina. Kao dio staničnog odgovora na stres dolazi i do porasta ekspresije šaperonskih proteina HSP32 i HSP27, što nismo pokazali i za HSP70. Također dolazi do promjena u MAPK signalnim putovima: značajno raste razina aktivirane p38, ne dolazi do aktivacije ERK, a aktivaciju JNK nismo uspjeli detektirati. Nadalje, rezultati su pokazali da A549 stanice ne sadrže MMP-9, ali pod utjecajem ekstrakta dima cigareta s vremenom u medij pojačano otpuštaju MMP-2, a TIMP-1 otpuštaju u manjoj količini, što bi moglo pridonijeti ukupnoj neravnoteži proteaza i antiproteaza. U bazalnim uvjetima polimorfonuklearni leukociti (PMN) bolesnika s KOPB-om otpuštaju značajno više MMP-9 u odnosu na zdrave kontrole. Stimulacijom PMN zdravih kontrola ekstraktom dima cigareta povećava se otpuštanje MMP-9, dok ista stimulacija u bolesnika s KOPB-om ne izaziva dodatno povećanje visoke razine otpuštanja MMP-9, što dokazuje kroničnu aktivaciju PMN bolesnika i njihovu moguću ulogu u razgradnji izvanstaničnog matriksa u plućima. Sistemska neravnoteža proteaza i antiproteaza u bolesnika s KOPB-om očituje se povećanom koncentracijom MMP-9 u plazmi. Analiza ROC krivulja pokazala je vrlo dobru dijagnostičku točnost za koncentraciju MMP-9, što je čini dobrim biljegom za razlikovanje bolesnika i zdravih osoba. Ispitivanje učestalosti polimorfizama MMP9 gena (-1562 C/T i (CA)n dinukleotidna ponavljanja u promotorskoj regiji te Q279R u egzonu 6) pokazalo je da nema značajnih razlika između bolesnika s KOPB-om i zdravih ispitanika, kao niti povezanosti koncentracije MMP-9 s genotipovima ispitanika. U serumu bolesnika s KOPB-om, kod kojih je dokazana sistemska upala (povećani ukupni leukociti, neutrofili i CRP), nalazimo smanjenu aktivnost DPPIV, proteaze koja bi mogla pridonositi razvoju upale. S obzirom na dokazanu vrlo dobru dijagnostičku točnost, ovaj enzim bi se također mogao koristiti kao učinkovit biokemijski pokazatelj KOPB-a.Chronic obstructive pulmonary disease (COPD) is the most common chronic respiratory disease with a progressive course. The principal causes of COPD include cigarette smoking, environmental factors and genetic predisposition. Current knowledge about the development of inflammation, oxidative stress and protease-antiprotease imbalance is not sufficient to explain the origin and progression of the disease. In this study we found that cigarette smoke extract causes oxidative damage to cellular lipids and proteins in A549 alveolar epithelial cells, which could lead to the loss of important cell functions, eventually resulting in cell death. The accumulation of damaged proteins in cells leads to the activation of ER stress response in the form of increased levels of phosphorylated eIF2α and reduced de novo protein synthesis. It is likely that reduced protein synthesis contributes to the increase in the level of free amino acids in the cells, together with the degradation of proteins damaged by cigarette smoke inside the ubiquitin-proteasome system. ER stress response is usually associated with increased proteasomal activity; however, cigarette smoke causes a significant reduction in caspase-, trypsin- and chymotrypsin-proteasomal activities, making the proteasome inefficient in the process of removing damaged proteins. As a part of the cellular stress response, we observed an increase in the expression of chaperone proteins HSP32 and HSP27, which was not shown for HSP70. Likewise, we noticed the changes in MAPK signaling pathways: a significant increase in the level of activated p38, no activation of ERK and undetectable activation of JNK. Furthermore, the results showed that A549 cells do not produce MMP-9, but after a while treatment with the cigarette smoke extract increases the release of MMP-2 in the medium and decreases the release of TIMP-1, which probably contributes to the overall protease-antiprotease imbalance. In basal conditions polymorphonuclear leukocytes (PMN) of patients with COPD release significantly much more MMP-9 compared to healthy controls. Stimulation of PMN of healthy controls with cigarette smoke extract increases the release of MMP-9, while in patients with COPD no further increase over the high secretion rate was found, suggesting the chronic activation of PMN of patients with COPD and their possible role in the degradation of extracellular matrix in the lungs. Systemic protease-antiprotease imbalance in plasma of patients with COPD is manifested by increased concentration of MMP-9. ROC curve analysis showed a very good diagnostic accuracy for concentration of MMP-9, making it a good biomarker for distinguishing patients from healthy individuals. Testing the frequency of MMP9 gene polymorphisms (-1562 C/T and (CA)n dinucleotide repeat in the promoter region, and Q279R in exon 6) showed no significant difference between patients with COPD and healthy subjects, and no correlation of MMP-9 concentration with subjects’ genotypes. In sera of patients with COPD, with the observed systemic inflammation (increased total leukocytes, neutrophils and CRP), we found a decrease in activity of DPPIV, a protease that could be contributing to the development of inflammation. As showing a very good diagnostic accuracy, this enzyme could also be used as an effective biochemical marker of COPD