Mojtaba Abdul Roda

A Self-Propagating Matrix Metalloprotease-9 (MMP-9) Dependent Cycle of Chronic Neutrophilic Inflammation

Background Chronic neutrophilic inflammation is a poorly understood feature in a variety of diseases with notable worldwide morbidity and mortality. We have recently characterized N-acetyl Pro-Gly-Pro (Ac-PGP) as an important neutrophil (PMN) chemoattractant in chronic inflammation generated from the breakdown of collagen by the actions of MMP-9. MMP-9 is present in the granules of PMNs and is differentially released during inflammation but whether Ac-PGP contributes to this ongoing proteolytic activity in chronic neutrophilic inflammation is currently unknown. Methodology/Principal Findings Utilizing isolated primary blood PMNs from human donors, we found that Ac-PGP induces significant release of MMP-9 and concurrently activates the ERK1/2 MAPK pathway. This MMP-9 release is attenuated by an inhibitor of ERK1/2 MAPK and upstream blockade of CXCR1 and CXCR2 receptors with repertaxin leads to decreased MMP-9 release and ERK 1/2 MAPK activation. Supernatants obtained from PMNs stimulated by Ac-PGP generate more Ac-PGP when incubated with intact collagen ex vivo; this effect is inhibited by an ERK1/2 pathway inhibitor. Finally, clinical samples from individuals with CF demonstrate a notable correlation between Ac-PGP (as measured by liquid chromatography-tandem mass spectrometry) and MMP-9 levels even when accounting for total PMN burden. Conclusions/Significance These data indicate that ECM-derived Ac-PGP could result in a feed-forward cycle by releasing MMP-9 from activated PMNs through the ligation of CXCR1 and CXCR2 and subsequent activation of the ERK1/2 MAPK, highlighting for the first time a matrix-derived chemokine (matrikine) augmenting its generation through a discrete receptor/intracellular signaling pathway. These findings have notable implications to the development unrelenting chronic PMN inflammation in human disease.

Collagen degradation and neutrophilic infiltration: a vicious circle in inflammatory bowel disease

Objective Proline–glycine–proline (PGP) has been shown to have chemotactic effects on neutrophils via CXCR2 in several lung diseases. PGP is derived from collagen by the combined action of matrix metalloproteinase (MMP) 8 and/or MMP9 and prolyl endopeptidase (PE). We investigated the role of PGP in inflammatory bowel disease (IBD). Design In intestinal tissue from patients with IBD and mice with dextran sodium sulfate (DSS)-induced colitis, MMP8, MMP9 and PE were evaluated by ELISA, immunoblot and immunohistochemistry. Peripheral blood polymorphonuclear cell (PMN) supernatants were also analysed accordingly and incubated with collagen to assess PGP generation ex vivo. PGP levels were measured by mass spectrometry, and PGP neutralisation was achieved with a PGP antagonist and PGP antibodies. Results In the intestine of patients with IBD, MMP8 and MMP9 levels were elevated, while PE was expressed at similar levels to control tissue. PGP levels were increased in intestinal tissue of patients with IBD. Similar results were obtained in intestine from DSS-treated mice. PMN supernatants from patients with IBD were far more capable of generating PGP from collagen ex vivo than healthy controls. Furthermore, PGP neutralisation during DSS-induced colitis led to a significant reduction in neutrophil infiltration in the intestine. Conclusions The proteolytic cascade that generates PGP from collagen, as well as the tripeptide itself, is present in the intestine of patients with IBD and mice with DSS-induced colitis. PGP neutralisation in DSS-treated mice showed the importance of PGP-guided neutrophilic infiltration in the intestine and indicates a vicious circle in neutrophilic inflammation in IBD.

An Aberrant Leukotriene A4 Hydrolase–Proline-Glycine-Proline Pathway in the Pathogenesis of Chronic Obstructive Pulmonary Disease

RATIONALE Chronic neutrophilic inflammation is a hallmark in the pathogenesis of chronic obstructive pulmonary disease (COPD) and persists after cigarette smoking has stopped. Mechanisms involved in this ongoing inflammatory response have not been delineated. OBJECTIVES We investigated changes to the leukotriene A4 hydrolase (LTA4H)-proline-glycine-proline (PGP) pathway and chronic inflammation in the development of COPD. METHODS A/J mice were exposed to air or cigarette smoke for 22 weeks followed by bronchoalveolar lavage and lung and cardiac tissue analysis. Two human cohorts were used to analyze changes to the LTA4H-PGP pathway in never smokers, control smokers, COPD smokers, and COPD former smokers. PGP/AcPGP and LTA4H aminopeptidase activity were detected by mass spectroscopy, LTA4H amounts were detected by ELISA, and acrolein was detected by Western blot. MEASUREMENTS AND MAIN RESULTS Mice exposed to cigarette smoke developed emphysema with increased PGP, neutrophilic inflammation, and selective inhibition of LTA4H aminopeptidase, which ordinarily degrades PGP. We recapitulated these findings in smokers with and without COPD. PGP and AcPGP are closely associated with cigarette smoke use. Once chronic inflammation is established, changes to LTA4H aminopeptidase remain, even in the absence of ongoing cigarette use. Acrolein modifies LTA4H and inhibits aminopeptidase activity to the same extent as cigarette smoke. CONCLUSIONS These results demonstrate a novel pathway of aberrant regulation of PGP/AcPGP, suggesting this inflammatory pathway may be intimately involved in disease progression in the absence of ongoing cigarette smoke exposure. We highlight a mechanism by which acrolein potentiates neutrophilic inflammation through selective inhibition of LTA4H aminopeptidase activity. Clinical trial registered with www.clinicaltrials.gov (NCT 00292552).

The matrikine N-α-PGP couples extracellular matrix fragmentation to endothelial permeability.

Organ tissue breakdown can induce vascular leak in lung injury. The compartmentalization and transport of proteins and solutes across the endothelium is a critical biologic function altered during inflammation and disease, leading to pathology in multiple disorders. The impact of tissue damage and subsequent extracellular matrix (ECM) fragmentation in regulating this process is unknown. We demonstrate that the collagen-derived matrikine acetylated proline-glycine-proline (N-α-PGP) serves as a critical regulator of endothelial permeability. N-α-PGP activates human endothelial cells via CXC-chemokine receptor 2 (CXCR2), triggering monolayer permeability through a discrete intracellular signaling pathway. In vivo, N-α-PGP induces local vascular leak after subcutaneous administration and pulmonary vascular permeability after systemic administration. Furthermore, neutralization of N-α-PGP attenuates lipopolysaccharide-induced lung leak. Finally, we demonstrate that plasma from patients with acute respiratory distress syndrome (ARDS) induces VE-cadherin phosphorylation in human endothelial cells, and this activation is attenuated by N-α-PGP blockade with a concomitant improvement in endothelial monolayer impedance. These results identify N-α-PGP as a novel ECM-derived matrikine regulating paracellular permeability during inflammatory disease and demonstrate the potential to target this ligand in various disorders characterized by excessive matrix turnover and vascular leak such as ARDS.

Targeting Prolyl Endopeptidase with Valproic Acid as a Potential Modulator of Neutrophilic Inflammation

A novel neutrophil chemoattractant derived from collagen, proline-glycine-proline (PGP), has been recently characterized in chronic obstructive pulmonary disease (COPD). This peptide is derived via the proteolytic activity of matrix metalloproteases (MMPs)-8/9 and PE, enzymes produced by neutrophils and present in COPD serum and sputum. Valproic acid (VPA) is an inhibitor of PE and could possibly have an effect on the severity of chronic inflammation. Here the interaction site of VPA to PE and the resulting effect on the secondary structure of PE is investigated. Also, the potential inhibition of PGP-generation by VPA was examined in vitro and in vivo to improve our understanding of the biological role of VPA. UV- visible, fluorescence spectroscopy, CD and NMR were used to determine kinetic information and structural interactions between VPA and PE. In vitro, PGP generation was significantly inhibited by VPA. In vivo, VPA significantly reduced cigarette-smoke induced neutrophil influx. Investigating the molecular interaction between VPA and PE showed that VPA modified the secondary structure of PE, making substrate binding at the catalytic side of PE impossible. Revealing the molecular interaction VPA to PE may lead to a better understanding of the involvement of PE and PGP in inflammatory conditions. In addition, the model of VPA interaction with PE suggests that PE inhibitors have a great potential to serve as therapeutics in inflammatory disorders.

The matrikine PGP as a potential biomarker in COPD

The lack of a well-characterized biomarker for the diagnosis of chronic obstructive pulmonary disease (COPD) has increased interest toward finding one, because this would provide potential insight into disease pathogenesis and progression. Since persistent neutrophilia is an important hallmark in COPD Pro-Gly-Pro (PGP), an extracellular matrix-derived neutrophil chemoattractant, has been suggested to be a potential biomarker in COPD. The purpose of this review is to critically examine both biological and clinical data related to the role of PGP in COPD, with particular focus on its role as a clinical biomarker and potential therapeutic target in disease. The data provided in this review will offer insight into the potential use of PGP as end point for future clinical studies in COPD lung disease. Following PGP levels during disease might serve as a guide for the progression of lung disorders.

Angiotensin-converting enzyme defines matrikine-regulated inflammation and fibrosis

The neutrophil chemoattractant proline-glycine-proline (PGP) is generated from collagen by matrix metalloproteinase-8/9 (MMP-8/9) and prolyl endopeptidase (PE), and it is concomitantly degraded by extracellular leukotriene A4 hydrolase (LTA4H) to limit neutrophilia. Components of cigarette smoke can acetylate PGP, yielding a species (AcPGP) that is resistant to LTA4H-mediated degradation and can, thus, support a sustained neutrophilia. In this study, we sought to elucidate if an antiinflammatory system existed to degrade AcPGP that is analogous to the PGP-LTA4H axis. We demonstrate that AcPGP is degraded through a previously unidentified action of the enzyme angiotensin-converting enzyme (ACE). Pulmonary ACE is elevated during episodes of acute inflammation, as a consequence of enhanced vascular permeability, to ensure the efficient degradation of AcPGP. Conversely, we suggest that this pathway is aberrant in chronic obstructive pulmonary disease (COPD) enabling the accumulation of AcPGP. Consequently, we identify a potentially novel protective role for AcPGP in limiting pulmonary fibrosis and suggest the pathogenic function attributed to ACE in idiopathic pulmonary fibrosis (IPF) to be a consequence of overzealous AcPGP degradation. Thus, AcPGP seemingly has very divergent roles: it is pathogenic in its capacity to drive neutrophilic inflammation and matrix degradation in the context of COPD, but it is protective in its capacity to limit fibrosis in IPF.

Cigarette smoke-induced collagen destruction: Key to chronic neutrophilic airway inflammation

* Zahid Shakoor, Musibeeh, Najd S, Al-Anazi Sara R, Al-Hassan Ghaida A, Al-Mesned Rawan K, Al-Yahya and Maha Y. Prof. Department of Pathology (32), College of Medicine, King Saud University, Riyadh 11461, Kingdom of Saudi Arabia. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript HistoryC is defined as a disease state characterized by airflow limitation that is not fully reversible. In COPD, multiple classes of proteases are released from neutrophils in the airway compartment, including endopeptidases, serine proteases and matrix metalloproteinases (MMPs). We have characterized a novel neutrophil chemoattractant, proline-glycine-proline (PGP) which is derived from collagen. PGP is increased in the bronchoalveolar lavage fluid, sputum and serum of COPD patients. An acetylated form of this peptide (N-α-PGP) is also detected and demonstrates increased chemotactic properties compared to non-acetylated PGP. PGP acts as a neutrophil chemoattractant in vitro and induces neutrophilic inflammation when instilled into the airways of mice in vivo. PGP is known to act on CXC receptors 1 and 2 (CXCR1, CXCR2) on neutrophils due to a structural homology with ELR+ chemokines, such as interleukin-8 (IL-8). Chronic N-α-PGP administration into murine airways for 12 weeks at biweekly intervals leads to the development of neutrophilic airway inflammation, alveolar enlargement and right ventricular hypertrophy, all of which are features of COPD. The degree of alveolar enlargement is similar to that seen with mice exposed to cigarette smoke 6 times per week for 24 weeks. Generation of PGP occurs via initial cleavage of collagen by matrix metalloproteases (MMP-8, MMP9) and subsequently by prolylendopeptidase (PE). This occurs when there is some initial insult to the epithelial layer, which leads to an exposure of collagen. Collagen is then cleaved by MMP-8 and/or -9 into fragments. PE further degrades the fragments into the tripeptide PGP. It has been shown that all three enzymes, MMP-8, 9 and PE are found in neutrophils and are present in COPD serum and sputum. The experimental and clinical therapeutical possibilities for COPD in the cascade of PGP generation will be highlighted during the presentation.