Sulodexide inhibits angiogenesis via decreasing Dll4 and Notch1 expression in mouse proepicardial explant cultures.

Sulodexide (SDX) is a mixed drug containing low-molecular-weight heparin sulfate and dermatan sulfate. It exerts mild anticoagulant action but can also affect leukocytes, macrophages, and cell-cell adhesion and may interact with growth factors although its direct influence on endothelial cells is not well described. Clinically, SDX is used for the treatment of cardiovascular diseases, where it exerts anti-inflammatory and endothelial protective effects. The aim of this study was to determine the influence of SDX on tubule formation and angiogenesis-related proteins' mRNA expression in endothelial cell line C166 and mouse proepicardial explants. C166 cells and explants were stimulated with a proangiogenic cocktail containing bFGF/VEGF-A120 /VEGF-A164 enriched with SDX. After stimulation, the number and morphology of tubules stained with anti-CD31 antibody were examined under confocal microscope and expression of mRNA for VEGF-A, VEGF-B, VEGF-C, bFGF, IGF-1, Dll4, and Notch1 was measured with real-time PCR. In C166 cell line, there was no difference in tubule formation and mRNA expression, but in proepicardial explants, we observed reduction in tubule number and in mRNA level for DLL4 and Notch1 after SDX administration. In conclusion, SDX indirectly inhibits angiogenesis in mouse proepicardial explant cultures but has no direct effect on the C166 endothelial cell line.

Decreased expression of G-protein-coupled receptors GPR43 and GPR109a in psoriatic skin can be restored by topical application of sodium butyrate.

The G-protein-coupled receptors GPR43 and GPR109a are known to play an important role in mediating anti-inflammatory and anti-cancer functions in the gut. Short-chain fatty acids, such as sodium butyrate (SB), are activators of GPR43 and GPR109a and thereby promote anti-inflammatory effects. The present study aimed to examine the expression of these receptors and their reaction to SB in psoriasis. Lesional and non-lesional biopsies of 6 psoriasis patients and of 4 controls were obtained and stained for GPR109a and GPR43. Ex vivo stimulation with SB was performed on fresh biopsy material. Lesional and non-lesional psoriatic skin showed a decreased expression of GPR109a and GPR43 on keratinocytes in comparison with control skin. Topical application of SB was able to increase the low-level expression of both receptors. The data suggest that SB by restoring the impaired expression of GPR109a and GPR43 might exert anti-inflammatory effects and may be utilized as a topical tool for the treatment of psoriasis, which has to be proven in future clinical trials.

Matrix metalloproteinase 9 in exhaled breath condensate in patients with stable chronic obstructive pulmonary disease: an observational study.

Introduction Data on the measurement of matrix metalloproteinase 9 (MMP­9) in exhaled breath condensate (EBC) from patients with chronic obstructive pulmonary disease (COPD) are scarce and inconsistent. Objectives We aimed to assess the usefulness of enzyme­linked immunosorbent assay (ELISA) and immunoenzymatic assay (IEA) for the measurement of MMP­9 in EBC, the agreement between the results of both methods, and the relationships between total and active MMP­9 in EBC and clinical and functional COPD characteristics. Patients and methods Total (ELISA and IEA) and active (IEA) MMP­9 levels were assessed in EBC from 70 patients with stable COPD and 21 controls and correlated with pulmonary function and COPD symptom severity. Results MMP­9 levels did not reach the sensitivity threshold of the ELISA kit in any of the COPD patients and in 11 controls. Total and active MMP­9 (IEA) levels did not differ between COPD patients and controls. In COPD patients, total MMP­9 levels correlated positively with forced expiratory volume in 1 second (FEV1) and FEV1 to forced vital capacity ratio and inversely with residual volume to total lung capacity ratio. A weak positive correlation between active MMP­9 concentrations and COPD Assessment Test (CAT) score was found (r = 0.31, P = 0.01). Conclusions The utility of ELISA in MMP­9 assessment in EBC is limited in COPD patients, while MMP­9 measurement in EBC by IEA is feasible. The positive correlation between active MMP­9 and CAT score in our patients and the inverse relationship between total MMP­9 concentration and the degree of airway obstruction reflect the complex role of MMP­9 in COPD.

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.

Pentoxifylline inhibits angiogenesis via decreasing Dll4 and Notch1 expression in mouse proepicardial explant cultures.

Pentoxifylline (PTX), a non-specific inhibitor of cAMP phosphodiesterases, is commonly used for treatment of peripheral vascular disorders although its direct action on endothelial cells is not well described. The aim of this study was to determine the influence of PTX on tubule formation and mRNA expression for angiogenesis-related proteins in endothelial cell line C166 and mouse proepicardial explants cultured on collagen. C166 cells and explants were stimulated with proangiogenic cocktail containing bFGF/VEGF-A120/VEGF-A164 and with proangiogenic cocktail enriched with PTX. After stimulation the number and morphology of tubules stained with anti-CD31 antibody was examined under a confocal microscope and expression of mRNA for VEGF-A, VEGF-B, VEGF-C, bFGF, IGF-1, Dll4 and Notch1 was measured with RealTime PCR. In C166 cell line there was no significant difference in tubule formation and mRNA expression, but in proepicardial explants we observed a considerable reduction in tubule number and in mRNA levels for Dll4 and Notch1 after PTX administration. In conclusion, PTX indirectly inhibits angiogenesis in mouse proepicardial explant cultures but has no significant effect on C166 endothelial cell line.

The MMP-9/TIMP-1 imbalance and the reduced level of TGF-ß 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-ß 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-ß 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-ß 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-ß may be possible risk factors of pre-term labor and PROM.

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.

LL-37 but Not 25-Hydroxy-Vitamin D Serum Level Correlates with Healing of Venous Leg Ulcers.

Human cathelicidin, LL-37, is small antimicrobial peptide, which reveals also some immunomodulatory and proangiogenic properties and, therefore, may promote wound healing. The expression of LL-37 is controlled by various factors, including vitamin D. Thus, any disturbances in vitamin D level may influence LL-37 production and, possibly, affect wound healing. Since deficiency of vitamin D was identified as a common problem in the population, this proof of concept study aimed to verify the relationship between serum levels of LL-37, vitamin D, and healing rate of venous leg ulcers. The study involved small group (n = 19) of patients with venous leg ulcers. Apart from non-venous ulcer aethiology, compression intolerance, active vein thrombosis, and wound infection, the exclusion criteria concerned also kidney insufficiency. The results of the analysis of wound healing rates were correlated with patients' serum levels of 25(OH) vitamin D and LL-37. In addition, serum levels of pro-inflammatory cytokines (IL-6, IL-8, and TNF) were analyzed. We have found strong association between serum concentrations of LL-37 and the healing rates in patients with leg ulcers. Despite the fact that 25(OH) vitamin D levels in all patients were below the normal range, they did not show any correlation with healing rates. Furthermore, no association was observed between serum levels of 25(OH) vitamin D and LL-37. No significant correlation between tested pro-inflammatory cytokines and healing rate, LL-37, or 25(OH) vitamin D levels was also observed. Regardless of small study group, our results suggest that the assessment of serum concentration of LL-37, but not 25-hydroxy vitamin D, may help in predicting the wound healing efficacy. Moreover, this assessment may be useful in pre-selection of patients, which could benefit from local treatment with exogenous LL-37.

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.

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 ß-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.

Hyaluronic Acid in Inflammation and Tissue Regeneration.

Hyaluronic acid (HA), the main component of extracellular matrix, is considered one of the key players in the tissue regeneration process. It has been proven to modulate via specific HA receptors, inflammation, cellular migration, and angiogenesis, which are the main phases of wound healing. Studies have revealed that most HA properties depend on its molecular size. High molecular weight HA displays anti-inflammatory and immunosuppressive properties, whereas low molecular weight HA is a potent proinflammatory molecule. In this review, the authors summarize the role of HA polymers of different molecular weight in tissue regeneration and provide a short overview of main cellular receptors involved in HA signaling. In addition, the role of HA in 2 major steps of wound healing is examined: inflammation and the angiogenesis process. Finally, the antioxidative properties of HA are discussed and its possible clinical implication presented.

Modulation of matrix metalloproteinases MMP-2 and MMP-9 activity by hydrofiber-foam hybrid dressing - relevant support in the treatment of chronic wounds.

Success in chronic wound therapy requires careful selection of appropriate dressing, which enables effective management of wound exudate. According to current knowledge, exudate may contain large quantities of proteases, including matrix metalloproteinases, MMP-2 and MMP-9, which are responsible for delay in wound healing. Therefore, neutralization of MMPs may be beneficial for treatment efficacy. The aim of the study was to test whether AQUACEL Foam, a novel, technologically advanced hydrofiber-foam hybrid dressing (HFHD), may interfere with proteolytic activity of MMPs in vitro. The assessment included in vitro tests of liquid retention and measurement of human recombinant MMP-2 and MMP-9 activity. The MMPs activity was measured before and after their interaction with HFHD, using a fluorescent gelatinase assay kit and Real-Time PCR device. The in vitro tests have shown that the hydrofiber layer of HFHD revealed significant potential to decrease the activity of MMPs in the experimental system. The mentioned modulatory properties of AQUACEL Foam may contribute to a composed mechanism of its beneficial action. Furthermore, our finding may explain clinical effectiveness of HFHD observed in clinical settings.

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.