Malgorzata Litwiniuk

Airway Remodeling in Chronic Obstructive Pulmonary Disease and Asthma: the Role of Matrix Metalloproteinase-9

Chronic obstructive pulmonary disease (COPD) and asthma are both associated with airflow restriction and progressive remodeling, which affect the respiratory tract. Among various biological factors involved in the pathomechanisms of both diseases, proteolytic enzymes—matrix metalloproteinases (MMPs)—play an important role, especially MMP-9. In this review, the authors discuss the current topics of research concerning the possible role of MMP-9 in both mentioned diseases. They include the analysis of protein levels, nucleotide polymorphisms of MMP-9 gene and their possible correlation with asthma and COPD. Finally, the authors refer to the studies on MMP-9 inhibition as a new perspective for increasing the effectiveness of treatment in asthma and COPD.

Amniotic membrane: New concepts for an old dressing

The amniotic membrane is the innermost layer of fetal membranes that surrounds and protects the embryo. The amniotic epithelial cells are a rich source for biologically active factors known to promote cell proliferation and differentiation. Therefore, the amniotic membrane is considered to be an attractive wound dressing material. Despite a large number of publications reporting anti‐inflammatory, bacteriostatic, reepithelializing, and scar‐preventing properties of amniotic membrane, not all the molecular mechanisms underlying the beneficial actions of the amniotic membrane dressing have been fully elucidated. This review summarizes current knowledge on the properties of the amniotic membrane and its various clinical applications. It includes an overview of the main biologically active factors that may be responsible for the observed clinical effects of amnion dressings. This issue is briefly discussed in the context of the role of amnion processing and inter‐ and intradonor variations between amniotic membrane specimens. Finally, future directions for the use of amnion derivatives in wound care are indicated.

Potential role of metalloproteinase inhibitors from radiation‑sterilized amnion dressings in the healing of venous leg ulcers

Chronic wounds are a significant socio-economic problem, thus, the improvement of the effectiveness of their treatment is an important objective for public health strategies. The predominant stage of the chronic wound is the inflammatory reaction which is associated with the damage of tissues, possibly due to the excessive secretion and activation of matrix metalloproteinases (MMPs). Several reports have suggested that amnion dressing inhibits tissue destruction and accelerates wound healing. Our recent study revealed that sterilized amnion stimulates keratinocyte proliferation in vitro, while the present study focused on the clinical application of radiation-sterilized amnion in chronic venous leg ulcers and aimed to explain the possible mechanism of its in vivo action. The study involved 25 individuals suffering from venous leg ulceration with a surface area of 10-100 cm2 and a healing rate below 10% per week, as verified during a 2-week screening period. The effectiveness of the amnion dressing was estimated following 4 weeks of treatment. The wound assessment, based on a modified Bates-Jensen Questionnaire, revealed a good and satisfactory response to the treatment in 23 of the 25 patients. The measurement of MMP-2 and MMP-9 activities in wound exudates revealed a decrease in activity in response to amnion application. This effect resulted from the presence of the potent MMP inhibitors, tissue inhibitor of metalloproteinases-1 (TIMP-1), type-1 plasminogen activator inhibitor (PAI-1) and thrombospondin-1 (TSP-1) in the amnion dressings, as shown by real-time fluorescence zymography and protein microarrays. Thus, unlike modern synthetic dressing materials, radiation-sterilized amnion dressings may have a multidirectional beneficial effect on chronic wounds.

Prolonged Treatment with Inhaled Corticosteroids does not Normalize High Activity of Matrix Metalloproteinase-9 in Exhaled Breath Condensates of Children with Asthma.

The airway remodeling in asthma is associated with increased amount of matrix metalloproteinase (MMP)-9. High levels of MMP-9 were found in mucosal biopsies, sputum and in exhaled breath condensates (EBC) of asthma patients. However, there are no data concerning real in vivo activity. Inhaled corticosteroids are effective in asthma control, but it is unclear, whether they only attenuate inflammation, or also protect against progressive remodeling of respiratory tract. Therefore, the aim of the study was to assess the amount and activity of MMP-9 in context of pro-inflammatory cytokines (IL-6, IL-8 and tumor necrosis factor, TNF), measured in EBC of asthma-suffering children, treated with inhaled steroids. The study involved 27 children with asthma, continuously treated with inhaled fluticasone propionate, and 22 healthy controls. In addition to routine clinical screening, the selected cytokines in EBC were analyzed using Ultrasensitive ELISA, whereas activity of MMP-9 was assessed using a novel immunozymography method. Despite chronic treatment with inhaled steroids mean MMP-9/EBC activity in asthma group was significantly higher than in healthy controls. Moreover, high MMP-9/EBC in asthma-suffering children significantly correlated with IgE serum levels. The IL-6 and IL-8 concentration was below the detection limit in all EBC samples. TNF/EBC levels were similar in both, asthma and healthy children. We hypothesize that MMP-9 hyperactivity in asthma may be closely related to high IgE serum levels. Our results suggest that inhaled steroids may be ineffective to prevent asthma-associated airway remodeling. Finally, we emphasize the necessity of further research focused on MMP-9 inhibition in asthma treatment.

Inhaled corticosteroids do not reduce initial high activity of matrix metalloproteinase (MMP)-9 in exhaled breath condensates of children with asthma exacerbation: a proof of concept study

Inhaled corticosteroids (ICS) are the key component of asthma treatment. However, it is unclear whether they could control the activity and level of matrix metalloproteinase (MMP)-9, which is an important factor in asthma-associated inflammation and airway remodeling. Therefore, the aim of this proof of concept study was to analyze the influence of increased doses of ICS on MMP-9 in exhaled breath condensates (EBC) of patients with allergic asthma exacerbation. Apart from MMP-9, the assessment concerned selected inflammation markers – exhaled nitric oxide (eNO) and cytokines (IL-8 and TNF). The study involved a small group (n = 4) of individuals with asthma exacerbation. The intervention concerned increased doses of ICS with β-mimetics for 4 weeks. In addition to clinical evaluation, eNO measurements and EBC collections were done before and after 4 weeks of intense ICS treatment. The biochemical assessment of EBC concerned MMP-9, IL-8 and TNF. The data were compared to results of healthy controls (n = 6). The initial levels of eNO, MMP-9 and TNF in EBC were higher in the asthma group than in controls. In all subjects IL-8 levels were below the detection limit. After 4 weeks of ICS treatment in all patients we observed improvement of clinical and laboratory parameters. Interestingly, despite reduction of eNO and TNF, the activity of MMP-9/EBC remained on the initial level. Practical relevance of our results is limited by a small group. Nevertheless, our data suggest that ICS, although sufficient to control symptoms and inflammatory markers, may be ineffective to reduce MMP-9/EBC activity in asthma exacerbation and, possibly, airway remodeling.

Matrix metalloproteinases in aortic aneurysm - executors or executioners?

Despite numerous studies focusing on the aortic aneurysm pathogenesis, the mechanism of aneurysm formation, especially – initiation of this process, remains unclear. The research concerning both, structural and molecular studies, is based on two main data sources. The first source of information are patients with already formed aneurysm, and with well defined biochemical and morphological changes in aortic wall architecture. The other source of data are experimental studies based on laboratory animals with artificially induced aneurysms. This approach enables verification of various hypotheses concerning pathogenesis of aortic aneurysm. Regrettably, animal aneurysm models, although similar, are not exactly the same, as human pathology. Thus, since the link between both mentioned data sources is still lacking, the knowledge achieved to date, even being highly profound, is not sufficient to fully understand this disease. Besides well defined factors, predisposing to formation of aortic aneurysm (patient’s age, cigarette smoking, arterial hypertension, atherosclerosis, as well as the Marfan’s and the Ehlers-Danlos’s syndrome-associated mutations), increasing popularity is currently being gained by the hypothesis concerning the pivotal role of proteolytic enzymes matrix metalloproteinases (MMPs) in aortic wall destruction. The involvement of MMPs in extracellular matrix damage in aortic aneurysm is doubtless. However, it needs to be elucidated, what the sequence of events is and what the exact role of MMPs is in these events. MMPs could play a role of “executioners”, that are produced and activated in the aortic wall as constituents of inflammatory reaction, in response to some yet poorly defined triggers. On the other hand, it is plausible, that aortic wall destruction, followed by inflammatory response to tissue degradation products, results from primary local overproduction and/or activation of proteases. It could be due to some mutations or polymorphisms of MMP genes, or some impairment in their controlling mechanisms. In that circumstance MMPs could rather be considered as “executors”, with causative role in aortic aneurysm pathogenesis. Although the majority of studies suggest the first scenario as being more possible, there is some evidence, that could support the second alternative, too.

The MMP-9/TIMP-1 imbalance and the reduced level of TGF-β in the cervical area of amniotic membrane is a possible risk factor of PROM and premature labor — proof-of-concept study

OBJECTIVES To assess the level MMP-9, TIMP-1 and TGF-β in placental and cervical region of amniotic membranes derived from at-term, pre-term and PROM deliveries. MATERIAL AND METHODS 14 amniotic membranes have been assessed; the quantitative analysis of MMP-9, TGF-β and TIMP-1 was assayed using respective Quantikine Immunoassay Kit. RESULTS The MMP-9 level in PROM samples was similar to the level of MMP-9 in at-term membranes and comparable between the cervical and placental region of these membranes. The concentration of TGF-β and TIMP-1 was decreased in the cervical area of AM derived from deliveries complicated with PROM. CONCLUSION The MMP9/TIMP-1 imbalance, as well as the reduced level of TGF-β may be possible risk factors of pre-term labor and PROM.

Amount and distribution of selected biologically active factors in amniotic membrane depends on the part of amnion and mode of childbirth. Can we predict properties of amnion dressing? A proof-of-concept study

Aim of the study The amniotic membrane is used as a dressing material, e.g. in ocular surgery or treatment of non-healing wounds. Of note, results of previous studies differ significantly, presumably due to the biological properties of amnion. Some authors suggest that these properties may depend on inter-donor variations, as well as the method of delivery. The aim of our study was to analyse the content of selected factors important for tissue regeneration in various areas of amnion samples originating from elective caesarean sections and on-term natural deliveries. Material and methods Cervical and placental samples of amniotic membranes originating from physiological deliveries and caesarean sections have been collected with subsequent preparation of amniotic membrane extracts. The screening of amnion samples was performed using a proteome microarray system. Results In all of the amnion samples high amounts of angiogenin, IGF-binding proteins-1, -2, and -3, serine protease inhibitor E1, and TIMP-1 were detected. Important variations in the content of these factors were observed between physiological delivery and caesarean section-derived membranes, as well as between placental and cervical portions of the same membrane. Conclusions Our study has shown that the content of selected growth factors and regulators of ECM turnover in amniotic membrane samples vary between various donations, and that they depend on the region of the membrane or delivery method. This may determine its potential applications in wound treatment and ophthalmologic surgery. However, our observations require further verification in clinical settings.

Polymorphic Variants 279R and 668Q Augment Activity of Matrix Metalloproteinase-9 in Breath Condensates of Children with Asthma

Matrix metalloproteinase (MMP)-9 is involved in pathophysiology of asthma, mainly asthma-associated airway remodeling. Exhaled breath condensates (EBC) of asthmatics contain increased amounts of MMP-9 with activity higher, than in healthy controls. The increased activity of MMP-9 may originate from its excessive production and activation, but may also result from variations in MMP-9 structure, which are determined by single nucleotide polymorphisms (SNPs). In this pilot study we aimed to assess the possible influence of two functional MMP-9 polymorphisms, Q279R and R668Q, on enzymatic activity of MMP-9, measured in EBC of asthmatic children. The concentration and activity of MMP-9 were analyzed in EBC of 20 children with allergic asthma using specific standard ELISA and novel immunoenzymatic activity assay. The SNPs of MMP-9 were assessed using real-time PCR-based genotyping test. We have found that MMP-9 concentration in breath condensates of children with stable asthma was slightly higher in ELISA, than in the activity assay. Moreover, these results and activity-to-amount ratio have revealed some relationship with a presence of specific 279R and/or 668Q MMP-9 gene variants. Our observation suggests that at least in some patients MMP-9 hyperactivity may result from genetic predisposition, determined by polymorphic variants of MMP-9 gene. Moreover, it supports previous reports postulating significance of MMP-9 in pathogenesis of asthma. However, this issue still requires further studies.

Increased angiogenic factors in exhaled breath condensate of children with severe asthma - New markers of disease progression?

Asthma progression is associated with airway remodeling and neo-vascularization. However, role of angiogenesis in these changes remains unclear and available data still incomplete. In this pilot study we verify usefulness of proteome profiler assay in screening of angiogenesis-related factors in exhaled breath condensates (EBC) collected from children with asthma. EBC samples from patients with mild or severe asthma and healthy controls were tested using protein array. In EBC samples from patients with severe asthma we have found large quantities of several angiogenesis regulators, including thrombospondin (TSP)-1, angiogenin, dipeptidyl peptidase (DPP) IV, matrix metalloproteinase (MMP)-9 and its inhibitor TIMP-1. Small amounts of angiopoietin (Ang)-2 and vascular endothelial growth factor (VEGF) were also present. In contrast to them, in EBC from mild asthma group we have detected TSP-1 and small quantities of Ang-2. EBC samples from healthy controls contained only TSP-1. Our preliminary report suggests that, since increased amounts of angiogenesis-related factors in EBC seem to correlate with asthma severity, they may be considered as convenient non-invasive markers of disease progression. However, further research is necessary.