Designing Hydrogels that Transform their Shape in Response to Molecular Cues

There has been considerable interest in developing shape-changing soft materials for potential applications in drug delivery, microfluidics and biosensing. These shape- changing materials are inspired by the morphological changes exhibited by plants in nature, such as the Venus flytrap. One specific class of shape-change is that from a flat sheet to a folded structure (e.g., a tube). Such “self-folding” materials are usually composed of polymer hydrogels, and these typically fold in response to external stimuli such as pH and temperature. In order to develop these hydrogels for the previously described applications, it is necessary to expand the range of triggers. The focus of this dissertation is the advancement of shape-changing polymer hydrogels that are sensitive to uncommon cues such as specific biomolecules (enzymes), the substrates for such enzymes, or specific multivalent cations. First, we describe a hybrid gel that responds to the presence of low concentrations of a class of enzymes known as matrix metalloproteinases (MMPs). The hybrid gel was created by utilizing photolithographic techniques to combine two or more gels with distinct chemical composition into the same material. Certain portions of the hybrid gel are composed of a biopolymer derivative with crosslinkable groups. The hybrid gel is flat in water; however, in the presence of MMPs, the regions containing the biopolymer are degraded and the flat sheet folds to form a 3D structure. We demonstrate that hydrogels with different patterns can transform into different 3D structures such as tubes, helices and pancakes. Furthermore, this shape change can be made to occur at physiological concentrations of enzymes. Next, we report a gel with two layers that undergoes a shape change in the presence of glucose. The enzyme glucose oxidase (GOx) is immobilized in one of the layers. GOx catalyzes the conversion of glucose to gluconic acid. The production of gluconic acid decreases the local pH. The decrease in local pH causes one of the layers to swell. As a result, the flat sheet folds to form a tube. The tube unfolds to form a flat sheet when it is transferred to a solution with no glucose present. Therefore, this biomolecule- triggered shape transformation is reversible, meaning the glucose sensing gel is reusable. Furthermore, this shape change only occurs in the presence of glucose and it does not occur in the presence of other small sugars such as fructose. In our final study, we report the shape change of a gel with two layers in the presence of multivalent ions such as Ca2+ and Sr2+. The gel consists of a passive layer and an active layer. The passive layer is composed of dimethylyacrylamide (DMAA), which does not interact with multivalent ions. The active layer consists of DMAA and the biopolymer alginate. In the presence of Ca2+ ions, the alginate chains crosslink and the active layer shrinks. As a result, the gel converts from a flat sheet to a folded tube. What is particularly unusual is the direction of folding. In most cases, when flat rectangular gels fold, they do so about their short-side. However, our gels typically fold about their long-side. We hypothesize that non-homogeneous swelling determines the folding axis.

Synthesis, Modification and Characterization of Polymeric Membranes for CO2 Separation

This doctorate focused on the development of dense polymeric membranes for carbon capture, mostly in post combustion applications, and for natural gas sweetening. The work was supported by the European Project NANOMEMC2 funded under H2020 program. Different materials have been investigated, that rely on two main transport mechanisms: the solution-diffusion and the facilitated transport. In both cases, proper nano-fillers have been added to the matrix, in order to boost the mechanical and permselective properties of the membranes. Facilitated transport membranes were based on the use of was polyvinylamine (PVAm), as main matrix with fixed-site carriers, and L-Arginine as mobile carrier; the filler, used mostly as reinforcer, was carboxymethylated nanocellulose (cNFC). Humid test showed interesting results, and especially the blend made of PVAm/cNFC/Arg in weight ratio 27,5/27,5/45 crossed the Robeson CO2/N2 upper bound, representing current state of the art membranes, with a CO2 permeability of 271 Barrer and CO2/N2 selectivity of 70. Solution diffusion membranes were based on Pebax®2533 matrix which was added with three different graphene oxide (GO)-based materials, namely pristine GO, Porous Graphene Oxide (PGO) and a GO functionalized with polyetheramine (PEAGO). All of them provided a modest but clear increment of permeability of the Pebax matrix, from plus 2% (GO) to plus 8% (PGO), with no change in selectivity. The gas tested with this type of composites were CO2 and N2, for Post combustion capture applications. Pebax®2533 was also chemically modified, obtaining the product called “Benzoyl-P2533”, that was fully characterized, and tested in term of permeation using five gas: CO2, N2, CH4, O2, and He. Modified material showed an increment of the overall permeability of the material of a fair 10% for all gases tested, apart from helium, that increased of almost 50%.

Assessment of MMP-9, TIMP-1, and COX-2 in normal tissue and in advanced symptomatic and asymptomatic carotid plaques

Abstract Background Mature carotid plaques are complex structures, and their histological classification is challenging. The carotid plaques of asymptomatic and symptomatic patients could exhibit identical histological components. Objectives To investigate whether matrix metalloproteinase 9 (MMP-9), tissue inhibitor of MMP (TIMP), and cyclooxygenase-2 (COX-2) have different expression levels in advanced symptomatic carotid plaques, asymptomatic carotid plaques, and normal tissue. Methods Thirty patients admitted for carotid endarterectomy were selected. Each patient was assigned preoperatively to one of two groups: group I consisted of symptomatic patients (n = 16, 12 males, mean age 66.7 ± 6.8 years), and group II consisted of asymptomatic patients (n = 14, 8 males, mean age 67.6 ± 6.81 years). Nine normal carotid arteries were used as control. Tissue specimens were analyzed for fibromuscular, lipid and calcium contents. The expressions of MMP-9, TIMP-1 and COX-2 in each plaque were quantified. Results Fifty-eight percent of all carotid plaques were classified as Type VI according to the American Heart Association Committee on Vascular Lesions. The control carotid arteries all were classified as Type III. The median percentage of fibromuscular tissue was significantly greater in group II compared to group I (p < 0.05). The median percentage of lipid tissue had a tendency to be greater in group I than in group II (p = 0.057). The percentages of calcification were similar among the two groups. MMP-9 protein expression levels were significantly higher in group II and in the control group when compared with group I (p < 0.001). TIMP-1 expression levels were significantly higher in the control group and in group II when compared to group I, with statistical difference between control group and group I (p = 0.010). COX-2 expression levels did not differ among groups. There was no statistical correlation between MMP-9, COX-2, and TIMP-1 levels and fibrous tissue. Conclusions MMP-9 and TIMP-1 are present in all stages of atherosclerotic plaque progression, from normal tissue to advanced lesions. When sections of a plaque are analyzed without preselection, MMP-9 concentration is higher in normal tissues and asymptomatic surgical specimens than in symptomatic specimens, and TIMP-1 concentration is higher in normal tissue than in symptomatic specimens.

Activation of matrix metalloproteinase -9 (MMP-9) by heregulin in human breast cancer cells involves multiple signaling pathways

Matrix metalloproteinase-9 (MMP-9) plays an important role in tumor invasion and angiogenesis. Secretion of MMP-9 has been reported in various cancer types including lung cancer, brain cancer, colon cancer, and breast cancer. Heregulin is a growth factor that regulates growth and differentiation of normal breast cells as well as mammary tumor cells. To study the role of heregulin in breast cancer metastasis, we tested whether heregulin may regulate MMP-9 secretion. By screening a panel of breast cancer cell line for their ability to respond to heregulin and produce MMP-9, we have found that MMP-9 secretion can be induced by heregulin-β1 in two breast cancer cell lines, SKBr3 and MCF-7. In both cell lines, increase of MMP-9 activity as shown by zymography was accompanied by increased protein level as well as mRNA level of MMP-9. Using a reporter luciferase assay, we have identified that proximal −670bp promoter of MMP-9 had similar activity to a 2.2kb MMP-9 promoter in response to heregulin stimulation. Heregulin treatment of SKBr3 and MCF-7 activated multiple signaling pathways inside cells. These include the Erk pathway, the p38 kinase pathway, PKC pathway, and PI-3K pathway. To examine which pathways are involved in MMP-9 activation by heregulin, we have used a panel of chemical inhibitors to specifically inhibit each one of these pathways. Ro-31-8220 (PKC inhibitor) and SB203580 (p38 kinase inhibitor) completely blocked heregulin activation of MMP-9. On the other hand, PD098059 (MEK-1 inhibitor) partially blocked MMP-9 activation, whereas PI-3K inhibitor, wortmannin, had no effect. Therefore, at least three signaling pathways are involved in activation of MMP-9 by heregulin. Since MMP-9 is tightly associated with metastatic potential, our study also suggests that heregulin may enhance breast tumor metastasis through induction of MMP-9 expression. ^

Quantitative Proteomic Analysis Reveals Metabolic Alterations, Calcium Dysregulation, And Increased Expression Of Extracellular Matrix Proteins In Laminin α2 Chain-deficient Muscle.

Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain-deficient dy(3K)/dy(3K) mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain-deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978).

Effect of Laser Phototherapy on the Release of TNF-alpha and MMP-1 by Endodontic Sealer-Stimulated Macrophages

Objective: Our aim was to analyze the effect of laser phototherapy on the secretory activity of macrophages activated by interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS), and stimulated by substances leached from an epoxy resin-based sealer (AH-Plus) and a calcium hydroxide-based sealer (Sealapex). Background Data: Laser phototherapy can modulate the inflammatory process, improving wound healing. This type of therapy could be useful for modulating postoperative symptoms seen after endodontic treatment. Materials and Methods: Cytotoxicity was indirectly assessed by measuring mitochondrial activity. Macrophages were stimulated by the leached substances or not (controls), and the groups were then irradiated or not. The secretion of pro-inflammatory cytokines (TNF-alpha and MMP-1) was analyzed using ELISA. Two irradiations at 6-h intervals were done with an As-Ga-Al diode laser (780 nm, 70 mW, spot size 4.0 mm(2), 3 J/cm(2), for 1.5 sec) in contact mode. Results: The sealers were non-cytotoxic to macrophages. The production of TNF-alpha was significantly decreased by laser phototherapy, regardless of experimental group. The level of secretion of MMP-1 was similar in all groups. Conclusion: Based on the conditions of this study we concluded that in activated macrophages, laser phototherapy impairs the secretion of the pro-inflammatory cytokine TNF-alpha, but has no influence on MMP-1 secretion.

EXPRESSION OF MEMBRANE TYPE 1 MATRIX METALLOPROTEINASE IN MEDULLARY THYROID CARCINOMA: PROGNOSTIC IMPLICATIONS

Background. Clinical and pathologic examinations cannot always provide a prognosis for patients with medullary thyroid carcinoma. Membrane type 1 matrix metalloproteinase (MT1-MMP) can act directly on carcinogenesis and takes part in 1 of the processes of metalloproteinase 2 activation, an enzyme related to prognostic impairment of patients with such tumor. Methods. Thirty-five patients who were submitted to surgery were followed up for an average of 74 months, Postoperative and final medical conditions were characterized for comparison with MT1-MMP immunostainings, performed in surgical paraffin blocks. A value of p < .05 was considered statistically significant. Results. Proposed index (association of proportion and intensity of immunostaining) and proportion of immunostained cells in primary specimens were correlated with cure or persistence after initial operations (p = .0216 and p = .0098, respectively). Conclusion. MT1-MMP immunostaining in primary tumor specimens is a new and complementary prognostic predictor in patients with medullary thyroid carcinomas. (C) 2009 Wiley Periodicals, Inc. Head Neck 32: 58-67, 2010

Prognostic relevance of TTF-1 and MMP-9 expression in advanced lung adenocarcinoma

Background: The thyroid transcription factor-1 (TTF-1) is a tissue-specific transcription factor that Could playan important role in cell differentiation and morphogenesis of lung tumors. Matrix metalloproteinase-9 (MMP-9) is a protease commonly expressed in non-small cell lung cancer, conferring angiogenic and metastatic potential. Methods: We assessed TTF-1 and MMP-9 tumor expression by immunohistochemistry in 51 patients with lung adenocarcinoma, stage 11113 or IV, treated with platinum regimens. A bicategorical prognostic model was obtained using the Kaplan-Meier method, COX regression, and conjunctive consolidation. Results: The median expression of TTF-1 was 30.0% (range: 0-85.9%). All tumors expressed MMP-9 (median: 78.7%: range: 15.2-96.1%). Median survival was 41.6 weeks, with estimated 1- and 2-year survival rates of 45.0% and 22.0%, respectively. Poor performance status (Karnofsky scale) - hazards ratio(HR): 1.03. 95% confidence interval (CI): 1.01-1.06: low TTF-1 expression (<40%) - FIR: 4.00, 95% CI: 1.75-9.09: and high MMP-9 expression (>= 80%) - HR: 2.82, 95% CI: 1.30-6.08 were independent prognostic factors. Patients could be stratified in three death risk groups according to markers expression: low risk (high TTF-1 and low MMP-9; median survival: 127.6 weeks), intermediate risk (low TTF-1 OF high MMP-9; median survival: 39.0 weeks): and high risk (low TTF-1 and high MMP-9: median survival: 16.4 weeks). Conclusion: TTF-1 and MMP-9 tumor expression as detected by immunohistochemistry may allow identification of different, clinically meaningful, prognostic groups of advanced lung adenocarcinoma patients treated with platinum regimens. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Plasma Level of Mmp-9 as Predictive Factor for Response and Progression Free Survival in Patients Treated with Bevacizumab (Bev) and Pegylated Liposomal Doxorubicin (Pld): Sakk 24/06

Background: There is currently no identified marker predicting benefit from Bev in patients with breast cancer (pts). We monitored prospectively 6 angiogenesis-related factors in the blood of advanced stage pts treated with a combination of Bev and PLD in a phase II trial of the Swiss Group for Clinical Cancer Research, SAKK.Methods: Pts received PLD (20 mg/m2) and Bev (10 mg/kg) every 2 weeks for a maximum of 12 administrations, followed by Bev monotherapy until progression or severe toxicity. Blood samples were collected at baseline, during treatment and at treatment discontinuation. Enzyme-linked immunosorbent assays (Quantikine, R&DSystems and Reliatech) were used to measure vascular endothelial growth factor (VEGF), placental growth factor (PlGF), matrix metalloproteinase 9 (MMP-9) and soluble VEGF receptors -1, -2 and -3. The natural log-transformed (ln) data for each factor was analyzed by analysis of variance (ANOVA) model to investigate differences between the mean values of the subgroups of interest (where a = 0.05), based on the best tumor response by RECIST.Results: 132 samples were collected in 41 pts. The mean of baseline ln MMP-9 levels was significantly lower in pts with tumor progression than those with tumor response (p=0.0202, log fold change=0.8786) or disease control (p=0.0035, log fold change=0.8427). Higher MMP-9 level was a significant predictor of superior progression free survival (PFS): p=0.0417, hazard ratio=0.574, 95% CI=0.336-0.979. In a multivariate cox proportional hazards model, containing performance status, disease free interval, number of tumor sites, visceral involvement and prior adjuvant chemotherapy, using stepwise regression baseline MMP-9 was still a statistically 117P Table 1. SOLTI-0701* AC01B07* NU07B1* SOR+CAP N=20 PL+CAP N=33 SOR+ GEM/CAP N=23 PL+ GEM/CAP N=27 SOR+PAC N=48 PL+PAC N=46 Baseline characteristics Age, median (range), y 49 (32-72) 53 (30-78 54 (32-69) 57 (31-82) 50 (27-80) 52 (23-74) AJCC stage, n (%) IIIB/IIIC 3 (15) 6 (18) 0 (0) 3 (11) 8 (17) 9 (20) IV 17 (85) 27 (82) 23 (100) 24 (89) 40 (83) 37 (80) Metastatic site, n (%) Non-visceral 3 (15) 6 (18) 7 (30) 6 (22) 9 (19) 17 (37) Visceral 17 (85) 27 (82) 16 (70) 21 (78) 39 (81) 29 (63) Prior metastatic chemo, n (%) 8 (40) 15 (45) 21 (91) 25 (93) - - Efficacy PFS, median, mo 4.3 2.5 3.1 2.6 5.6 5.5 HR (95% CI)_ 0.60 (0.31, 1.14) 0.57 (0.30, 1.09) 0.86 (0.50, 1.45) 1-sided P value_ 0.055 0.044 0.281 Overall survival, median, mo 17.5 16.1 Pending 14.7 18.2 HR (95% CI)_ 0.98 (0.50, 1.89) 1.11 (0.64, 1.94) 1-sided P value_ 0.476 0.352 Safety N=20 N=33 N=22 N=27 N=46 N=46 Tx-emergent Grade 3/4, n (%) 15 (75) 16 (48) 20 (91) 17 (63) 36 (78) 16 (35) Grade 3§ hand-foot skin reaction/ syndrome 8 (40) 5 (15) 8 (36) 0 (0) 14 (30) 2 (4) *Efficacy results based on intent-to-treat population and safety results based on safety population (pts who received study drug[s]); _Cox regression within each subgroup; _log-rank test within each subgroup; §maximum toxicity grade for hand-foot skin reaction/syndrome; AJCC, American Joint Committee on Cancer mittedabstractsª The Author 2011. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com Downloaded from annonc.oxfordjournals.org at Bibliotheque Cantonale et Universitaire on June 6, 2011 significant factor (p=0.0266). The results of the other measured factors were presented elsewhere.Conclusions: Higher levels of MMP-9 could predict tumor response and superior PFSin pts treated with a combination of Bev and PLD. These exploratory results justify further investigations of MMP-9 in pts treated with Bev combinations in order to assess its role as a prognostic and predictive factor.Disclosure: K. Zaman: Participation in advisory board of Roche; partial sponsoring ofthe study by Roche (the main sponsor was the Swiss Federation against Cancer (Oncosuisse)). B. Thu¨rlimann: stock of Roche; Research grants from Roche. R. vonMoos: Participant of Advisory Board and Speaker honoraria

Macrophage elastase (MMP-12): a pro-inflammatory mediator?

As many metalloproteinases (MMPs), macrophage elastase (MMP-12) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes. Studies using animal models of acute and chronic pulmonary inflammatory diseases, such as pulmonary fibrosis and chronic obstrutive pulmonary disease (COPD), have given evidences that MMP-12 is an important mediator of the pathogenesis of these diseases. However, as very few data regarding the direct involvement of MMP-12 in inflammatory process in the airways were available, we have instilled a recombinant form of human MMP-12 (rhMMP-12) in mouse airways. Hence, we have demonstrated that this instillation induced a severe inflammatory cell recruitment characterized by an early accumulation of neutrophils correlated with an increase in proinflammatory cytokines and in gelatinases and then by a relatively stable recruitment of macrophages in the lungs over a period of ten days. Another recent study suggests that resident alveolar macrophages and recruited neutrophils are not involved in the delayed macrophage recruitment. However, epithelial cells could be one of the main targets of rhMMP-12 in our model. We have also reported that a corticoid, dexamethasone, phosphodiesterase 4 inhibitor, rolipram and a non-selective MMP inhibitor, marimastat could reverse some of these inflammatory events. These data indicate that our rhMMP-12 model could mimic some of the inflammatory features observed in COPD patients and could be used for the pharmacological evaluation of new anti-inflammatory treatment. In this review, data demonstrating the involvement of MMP-12 in the pathogenesis of pulmonary fibrosis and COPD as well as our data showing a pro-inflammatory role for MMP-12 in mouse airways will be summarized.

Clinical and radiographic findings in two brothers affected with a novel mutation in matrix metalloproteinase 2 gene.

The two well-described osteolysis syndromes associated with matrix metalloproteinase-2 deficiency and mutations in the metalloproteinase-2 gene are Torg-Winchester syndrome and nodulosis-arthropathy-osteolysis variant. They are characterized by carpal-tarsal destruction, subcutaneous nodules, and generalized osteoporosis and show autosomal recessive inheritance. Herein, we report two siblings affected with a novel mutation in matrix metalloproteinase 2 gene and discuss their clinical and radiographic findings.

Monitoring multiple angiogenesis-related molecules in the blood of cancer patients shows a correlation between VEGF-A and MMP-9 levels before treatment and divergent changes after surgical vs. conservative therapy.

Anti-angiogenic therapies are currently in cancer clinical trials, but to date there are no established tests for evaluating the angiogenic status of a patient. We measured 11 circulating angiogenesis-associated molecules in cancer patients before and after local treatment. The purpose of our study was to screen for possible relationships among the different molecules and between individual molecules and tumor burden. We measured VEGF-A, PlGF, SCF, MMP-9, EDB+ -fibronectin, sVEGFR-2, sVEGFR-1, salphaVbeta3, sTie-2, IL-8 and CRP in the blood of 22 healthy volunteers, 17 early breast, 17 early colorectal, and 8 advanced sarcoma/melanoma cancer patients. Breast cancer patients had elevated levels of VEGF-A and sTie-2, colorectal cancer patients of VEGF-A, MMP-9, sTie-2, IL-8 and CRP, and melanoma/sarcoma patients of sVEGFR-1. salphaVbeta3 was decreased in colorectal cancer patients. A correlation between VEGF-A and MMP-9 was found. After tumor removal, MMP-9 and salphaVbeta3 significantly decreased in breast and CRP in colorectal cancer, whereas sVEGFR-1 increased in colorectal cancer patients. In sarcoma/melanoma patients treated regionally with TNF and chemotherapy we observed a rise in VEGF-A, SCF, VEGFR-2, MMP-9, Tie-2 and CRP, a correlation between CRP and IL-8, and a decreased in sVEGFR-1 levels. In conclusion, among all factors measured, only VEGF-A and MMP-9 consistently correlated to each other, elevated CRP levels were associated with tumor burden, whereas sVEGF-R1 increased after tumor removal in colorectal cancer. Treatment with chemotherapy and TNF induced changes consistent with an angiogenic switch. These results warrant a prospective study to compare the effect of surgical tumor removal vs. chemotherapy on some of these markers and to evaluate their prognostic/predictive value.

Amelioration of Duchenne muscular dystrophy in mdx mice by elimination of matrix-associated fibrin-driven inflammation coupled to the αMβ2 leukocyte integrin receptor

In Duchenne muscular dystrophy (DMD), a persistently altered and reorganizing extracellular matrix (ECM) within inflamed muscle promotes damage and dysfunction. However, the molecular determinants of the ECM that mediate inflammatory changes and faulty tissue reorganization remain poorly defined. Here, we show that fibrin deposition is a conspicuous consequence of muscle-vascular damage in dystrophic muscles of DMD patients and mdx mice and that elimination of fibrin(ogen) attenuated dystrophy progression in mdx mice. These benefits appear to be tied to: (i) a decrease in leukocyte integrin α(M)β(2)-mediated proinflammatory programs, thereby attenuating counterproductive inflammation and muscle degeneration; and (ii) a release of satellite cells from persistent inhibitory signals, thereby promoting regeneration. Remarkably, Fib-gamma(390-396A) (Fibγ(390-396A)) mice expressing a mutant form of fibrinogen with normal clotting function, but lacking the α(M)β(2) binding motif, ameliorated dystrophic pathology. Delivery of a fibrinogen/α(M)β(2) blocking peptide was similarly beneficial. Conversely, intramuscular fibrinogen delivery sufficed to induce inflammation and degeneration in fibrinogen-null mice. Thus, local fibrin(ogen) deposition drives dystrophic muscle inflammation and dysfunction, and disruption of fibrin(ogen)-α(M)β(2) interactions may provide a novel strategy for DMD treatment.

Pain-evoked alterations on gingival blood flow and gingival crevicular fluid (GCF) neuropeptide SP and collagenase-2 (MMP-8) levels

Previous studies have demonstrated that clinical pulpal pain can induce the expression of pro-inflammatory neuropeptides in the adjacent gingival crevice fluid (GCF). Vasoactive agents such as substance P (SP) are known to contribute to the inflammatory type of pain and are associated with increased blood flow. More recent animal studies have shown that application of capsaicin on alveolar mucosa provokes pain and neurogenic vasodilatation in the adjacent gingiva. Pain-associated inflammatory reactions may initiate expression of several pro- and anti-inflammatory mediators. Collagenase-2 (MMP-8) has been considered to be the major destructive protease, especially in the periodontitis-affected gingival crevice fluid (GCF). MMP-8 originates mostly from neutrophil leukocytes, the first line of defence cells that exist abundantly in GCF, especially in inflammation. With this background, we wished to clarify the spatial extensions and differences between tooth-pain stimulation and capsaicin-induced neurogenic vasodilatation in human gingiva. Experiments were carried out to study whether tooth stimulation and capsaicin stimulation of alveolar mucosa would induce changes in GCF MMP-8 levels and whether tooth stimulation would release neuropeptide SP in GCF. The experiments were carried out on healthy human volunteers. During the experiments, moderate and high intensity painful tooth stimulation was performed by a constant current tooth stimulator. Moderate tooth stimulation activates A-delta fibres, while high stimulation also activates C-fibres. Painful stimulation of the gingiva was achieved by topical application of capsaicin-moistened filter paper on the mucosal surface. Capsaicin is known to activate selectively nociceptive C-fibres of stimulated tissue. Pain-evoked vasoactive changes in gingivomucosal tissues were mapped by laser Doppler imaging (LDI), which is a sophisticated and non-invasive method for studying e.g. spatial and temporal characteristics of pain- and inflammation-evoked blood flow changes in gingivomucosal tissues. Pain-evoked release of MMP-8 in GCF samples was studied by immunofluorometric assay (IFMA) and Western immunoblotting. The SP levels in GCF were analysed by Enzyme immunoassay (EIA). During the experiments, subjective stimulus-evoked pain responses were determined by a visual analogue pain scale. Unilateral stimulation of alveolar mucosa and attached gingiva by capsaicin evoked a distinct neurogenic vasodilatation in the ipsilateral gingiva, which attenuated rapidly at the midline. Capsaicin stimulation of alveolar mucosa provoked clear inflammatory reactions. In contrast to capsaicin stimuli, tooth stimulation produced symmetrical vasodilatations bilaterally in the gingiva. The ipsilateral responses were significantly smaller during tooth stimulation than during capsaicin stimuli. The current finding – that tooth stimulation evokes bilateral vasodilatation while capsaicin stimulation of the gingiva mainly produces unilateral vasodilatation – emphasises the usefulness of LDI in clarifying spatial features of neurogenic vasoactive changes in the intra-oral tissues. Capsaicin stimulation of the alveolar mucosa induced significant elevations in MMP-8 levels and activation in GCF of the adjacent teeth. During the experiments, no marked changes occurred in MMP-8 levels in the GCF of distantly located teeth. Painful stimulation of the upper incisor provoked elevations in GCF MMP-8 and SP levels of the stimulated tooth. The GCF MMP-8 and SP levels of the non-stimulated teeth were not changed. These results suggest that capsaicin-induced inflammatory reactions in gingivomucosal tissues do not cross the midline in the anterior maxilla. The enhanced reaction found during stimulation of alveolar mucosa indicates that alveolar mucosa is more sensitive to chemical irritants than the attached gingiva. Analysis of these data suggests that capsaicin-evoked neurogenic inflammation in the gingiva can trigger the expression and activation of MMP-8 in GCF of the adjacent teeth. In this study, it is concluded that experimental tooth pain at C-fibre intensity can induce local elevations in MMP-8 and SP levels in GCF. Depending on the role of MMP-8 in inflammation, in addition to surrogated tissue destruction, the elevated MMP-8 in GCF may also reflect accelerated local defensive and anti-inflammatory reactions.

MMP-1/PAR-1 signal transduction axis and its prognostic impact in esophageal squamous cell carcinoma

The matrix metalloprotease-1 (MMP-1)/protease-activated receptor-1 (PAR-1) signal transduction axis plays an important role in tumorigenesis. To explore the expression and prognostic value of MMP-1 and PAR-1 in esophageal squamous cell carcinoma (ESCC), we evaluated the expression of two proteins in resected specimens from 85 patients with ESCC by immunohistochemistry. Sixty-two (72.9%) and 58 (68.2%) tumors were MMP-1- and PAR-1-positive, respectively, while no significant staining was observed in normal esophageal squamous epithelium. MMP-1 and PAR-1 overexpression was significantly associated with tumor node metastasis (TNM) stage and regional lymph node involvement. Patients with MMP-1- and PAR-1-positive tumors, respectively, had poorer disease-free survival (DFS) than those with negative ESCC (P = 0.002 and 0.003, respectively). Univariate analysis showed a significant relationship between TNM stage [hazard ratio (HR) = 2.836, 95% confidence interval (CI) = 1.866-4.308], regional lymph node involvement (HR = 2.955, 95%CI = 1.713-5.068), MMP-1 expression (HR = 2.669, 95%CI = 1.229-6.127), and PAR-1 expression (HR = 1.762, 95%CI = 1.156-2.883) and DFS. Multivariate analysis including the above four parameters identified TNM stage (HR = 2.035, 95%CI = 1.167-3.681), MMP-1 expression (HR = 2.109, 95%CI = 1.293-3.279), and PAR-1 expression (HR = 1.967, 95%CI = 1.256-2.881) as independent and significant prognostic factors for DFS. Our data suggest for the first time that MMP-1 and PAR-1 were both overexpressed in ESCC and are novel predictors of poor patient prognosis after curative resection. The MMP-1/PAR-1 signal transduction axis might be a new therapeutic target for future therapies tailored against ESCC.