Expression Of Elastase Research Articles

MMP-12, An Old Enzyme Plays a New Role in the Pathogenesis of Rheumatoid Arthritis?

MMP-12, An Old Enzyme Plays a New Role in the Pathogenesis of Rheumatoid Arthritis?

Alterations in the renal elastin-elastase system in type 1 diabetic nephropathy identified by proteomic analysis.

Diabetes now accounts for >40% of patients with ESRD. Despite significant progress in understanding diabetic nephropathy, the cellular mechanisms that lead to diabetes-induced renal damage are incompletely defined. For defining changes in protein expression that accompany diabetic nephropathy, the renal proteome of 120-d-old OVE26 transgenic mice with hypoinsulinemia, hyperglycemia, hyperlipidemia, and proteinuria were compared with those of background FVB nondiabetic mice (n = 5). Proteins derived from whole-kidney lysate were separated by two-dimensional PAGE and identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Forty-one proteins from 300 visualized protein spots were differentially expressed in diabetic kidneys. Among these altered proteins, expression of monocyte/neutrophil elastase inhibitor was increased, whereas elastase IIIB was decreased, leading to the hypothesis that elastin expression would be increased in diabetic kidneys. Renal immunohistochemistry for elastin of 325-d-old FVB and OVE26 mice demonstrated marked accumulation of elastin in the macula densa, collecting ducts, and pelvicalyceal epithelia of diabetic kidneys. Elastin immunohistochemistry of human renal biopsies from patients with type 1 diabetes (n = 3) showed increased elastin expression in renal tubular cells and the interstitium but not glomeruli. These results suggest that coordinated changes in elastase inhibitor and elastase expression result in increased tubulointerstitial deposition of elastin in diabetic nephropathy. The identification of these coordinated changes in protein expression in diabetic nephropathy indicates the potential value of proteomic analysis in defining pathophysiology.

Cytokines in Sickle Cell Disease

Sickle red cells express adhesion molecules including integrin 4β1, CD36, band 3 protein, sulfated glycolipid, Lutheran protein, phosphatidylserine and integrin-associated protein. The proadhesive sickle cells may bind to endothelial cell P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), CD36 and integrins leading to its activation. Monocytes also activate endothelium by releasing proinflammatory cytokines like tumor necrosis factor alpha (TNF-) and interleukin 1β (IL-1β). Sickle monocytes also express increased surface CD11b and cytoplasmic cytokines TNF and IL-1β indicating activated state. Polymorphonuclear leukocytes (PMNs) are also activated with reduced L-selectin expression, enhanced CD64 expression and elevated levels of sL-selectin, sCD16 and elastase resulting in increased adhesiveness to the endothelium. Platelets are also activated and secrete thrombospondin (TSP) and cytokine IL-1. They also form platelet- monocytes aggregates causing endothelial cell P-selectin expression. Endothelial cell activation by these multiple mechanisms leads to a loss of vascular integrity, expression of leukocyte adhesion molecules, change in the surface phenotype from antithrombotic to prothrombotic, excessive cytokine production and upregulation of HLA molecules. Furthermore, contraction of these activated endothelial cells leads to exposure of extracellular matrix proteins, such as TSP, laminin, and fibronectin and their participation in adhesive interactions with bridging molecules from the plasma such as von Willebrand factor (vWf) released from endothelial cells, ultimately culminating in vasoocclusion and local tissue ischemia, the pathognomonic basis of vasoocclusive crisis.

Pseudomonas aeruginosa alginate is refractory to Th1 immune response and impedes host immune clearance in a mouse model of acute lung infection.

Pseudomonas aeruginosa is an opportunistic respiratory pathogen that accounts for most of the morbidity and mortality in cystic fibrosis (CF) patients. In CF-affected lungs, the bacteria undergo conversion from a non-mucoid to a non-tractable mucoid phenotype, due to overproduction of alginate. The effect of alginate production on pathogenicity was investigated by using an acute lung infection mouse model that compared a non-mucoid P. aeruginosa strain, PAO1, to its constitutive alginate-overproducing derivative, Alg(+) PAOmucA22, and an alginate-defective strain, Alg(-) PAOalgD. Bacterial suspensions were instilled into the left bronchus and examined 24 and 48 h post-infection. The highest bacterial loads and the most severe lung pathology were observed with strain Alg(-) PAOalgD at 24 h post-infection, which may have been due to an increase in expression of bacterial elastase by the mutant. Significantly lower lung and spleen bacterial loads were found in the two non-mucoid (PAO1 and Alg(-) PAOalgD) groups, compared to the mucoid Alg(+) PAOmucA22 group, between 24 and 48 h post-infection. The positive correlation between lung bacteriology and lung macroscopic pathology in the Alg(+) PAOmucA22 group suggests that alginate production not only impedes pulmonary clearing, but also results in severe lung damage. Positive correlations between IL12 levels and lung macroscopic pathology, and between IL12 and IFN-gamma levels in the Alg(+) PAOmucA22 group, suggested a possible contribution of these pro-inflammatory cytokines to tissue damage. No significant differences were found between the three groups in lung cytokine responses at 24 or 48 h post-infection. However, on comparison within each group at 24 and 48 h post-infection, a significant increase in the pro-inflammatory cytokine IFN-gamma was observed. Higher ratios of IFN-gamma/IL4 and IFN-gamma/IL10, but lower IL10 levels, were also found in all three groups. These results indicate a Th1-predominated immune response in these animals. Such cytokine responses could have aided the clearance of non-mucoid P. aeruginosa, but were not sufficient to alleviate infection by the mucoid variants. Alginate production may promote survival and persistence of this pathogenic micro-organism in the lung.

Elevated expression of polymorphonuclear leukocyte elastase in breast cancer tissue is associated with tamoxifen failure in patients with advanced disease.

Besides a variety of other proteases, polymorphonuclear leukocyte elastase (PMN-E) is also suggested to play a role in the processes of tumour cell invasion and metastasis. Yet, there is only limited data available on the relation between the tumour level of PMN-E and prognosis in patients with primary breast cancer, and no published information exists on its relation with the efficacy of response to systemic therapy in patients with advanced breast cancer. In the present study, we have measured with enzyme-linked immunosorbent assay the levels of total PMN-E in cytosolic extracts of 463 primary breast tumours, and have correlated their levels with the rate and duration of response on first-line tamoxifen therapy (387 patients) or chemotherapy (76 patients) in patients with locally advanced and/or distant metastatic breast cancer. Furthermore, the probabilities of progression-free survival and postrelapse survival were studied in relation to the tumour levels of PMN-E. Our results show that in logistic regression analysis for response to tamoxifen treatment in patients with advanced disease, high PMN-E tumour levels were associated with a poor rate of response compared with those with low PMN-E levels (odds ratio: OR, 0.40; 95% CI, 0.22–0.73; P=0.003). After correction for the contribution of the traditional predictive factors in multivariate analysis, the tumour PMN-E status was an independent predictor of response (P=0.01). Furthermore, a high tumour PMN-E level was related with a poor progression-free survival (P<0.001) and postrelapse survival (P=0.002) in a time-dependent analysis. In contrast, the tumour level of PMN-E was not significantly related with the efficacy of response to first-line chemotherapy in patients with advanced breast cancer. Our present results suggest that PMN-E is an independent predictive marker for the efficacy of tamoxifen treatment in patients with advanced breast cancer.

Possibility of somatic mosaicism of ELA2 mutation overlooked in an asymptomatic father transmitting severe congenital neutropenia to two offspring

The article by Germeshausen et al (2001) describes a pedigree in which severe congenital neutropenia (CN) has been transmitted by a father to two children. Sequence analysis of the neutrophil elastase gene (ELA2) identified heterozygous changes at amino acid 122 in both the affected children as well as the father. The authors interpret the lack of neutropenia in the father to be evidence that the mutation in ELA2 is not sufficient to cause CN. CN is known to be primarily a sporadic disease and as such the authors have overlooked the most likely interpretation of their results which is that the father is mosaic for the C122Y mutation. Somatic and germline mosaicism is a common occurrence in severe genetic diseases in which reduced genetic fitness mandates that de novo mutations account for a substantial proportion of new cases. These include Duchenne's muscular dystrophy, achondroplasia, tuberous sclerosis, Hunter's disease, osteogenesis imperfecta, retinoblastoma, neurofibromatosis type 1 and haemophilia A (Leuer et al, 2001 and references within). Most recently, Ancliff et al (2001a) have demonstrated somatic mosaicism of a C42R ELA2 mutation in the father of a girl presenting with CN. Further analysis determined that, while about half the father's lymphocytes and other cells contained the C42R ELA2 mutation found in his daughter, < 5% of his neutrophils contained a mutant allele, unequivocally demonstrating the pathogenic nature of ELA2 mutation on neutrophil production and CN genesis. The authors go on to suggest that their one case proves that mutations in the ELA2 gene are not the single cause of CN; however, it has never been stated that there is a single cause of CN. An elegant study by Ancliff et al (2001b) has shown that mutations in ELA2 account for approximately 75% of sporadic cases of CN, a figure similar to that obtained by Dale et al (2000). ELA2 mutations are less common in familial cases of the disease and linkage analysis and the exclusion of the 19p13.3 region in one pedigree with autosomal dominant CN suggests that at least one other gene is involved (Ancliff et al, 2001b). The possibility that this other gene or genes may function in a common pathway or actually directly regulate neutrophil elastase expression is supported by the authors themselves who have pointed out that CN patients with undetectable mutations in the ELA2 gene nonetheless demonstrate reduced elastase activity (Germeshausen et al, 2000). In closing, the demonstration of somatic mosaicism in the case of CN presented by Germeshausen et al (2001), which we believe to be a likely scenario that has not been addressed, would lead to the opposite conclusion as that proffered by the authors: mutations in ELA2 are causative of CN and sufficient.

Functional dissociation between proforms and mature forms of proteinase 3, azurocidin, and granzyme B in regulation of granulopoiesis.

We previously demonstrated that secreted proform(s) of the neutrophil serine protease PR3 (proteinase 3) can down-modulate the fraction of normal human colony-forming unit granulocyte-macrophage (CFU-GM) in S-phase, whereas PR3 extracted from mature neutrophils lacks this ability. The objective of this study was to characterize the structural and functional dissociation between secreted proforms and granule-stored mature forms and to extend the investigation to other related hematopoietic serine proteases. Conditioned media containing secreted proteases from transfectant cell lines with stable expression of human PR3, neutrophil elastase, cathepsin G, azurocidin, and granzymes A, B, H, K, and M were tested for their ability to reduce the fraction of normal human CFU-GM in S phase. Furthermore, recombinant PR3, azurocidin, and granzyme B with defined N-terminal propeptides, and the respective mature forms without propeptide, were functionally characterized. In addition to PR3, secreted proforms of azurocidin and granzymes A, B, H, K, and M, but not cathepsin G or neutrophil elastase, have S-phase reducing activity. This activity is restricted to the dipeptide proforms, whereas mature forms without propeptide have no S-phase reducing activity. On the other hand, only the mature forms of PR3 and granzyme B could bind the serine protease inhibitor diisopropylfluorophosphate (DFP), or aprotinin in the case of azurocidin. We also demonstrate that granulocyte colony-stimulating factor-stimulated CD34+ cells and interleukin-2-stimulated lymphocytes secrete active proforms of PR3 and granzyme B, respectively. These results demonstrate distinctive functional and conformational differences between proforms and mature forms of these hematopoietic serine proteases and suggest novel growth regulatory mechanisms in granulopoiesis.

Antibodies induced in mice by a DNA-construct coding for the elastase of Schistosoma mansoni recognize the enzyme in secretions and preacetabular glands of cercariae.

A DNA-construct coding for the elastase of the parasite Schistosoma mansoni was prepared from adult S. mansoni worm RNA which was reverse transcribed into cDNA. The gene coding for the elastase was amplified using primers specific for the sequence of cercarial elastase and was cloned into a mammalian expression vector. Expression of the elastase gene at the transcriptional level was achieved for the first time in transfected mammalian cells (COS-7) and was also successful in muscle tissue of mice injected with the DNA-construct. These mice developed antibodies recognizing in Western blots the elastase from cercarial secretions. Also, these antibodies reacted in immunofluorescence tests with the preacetabular glands of cercariae, i.e. the site of origin for elastase. Thus, the DNA-construct induced the expression of elastase in mice and formation of antibodies that recognized the native antigen.

Differential proteinase expression by Pseudomonas aeruginosa derived from chronic leg ulcers.

Pseudomonas aeruginosa colonizes 20-30% of all venous leg ulcers. Hypothetically, P. aeraginosa could release proteases and cytotoxic substances in the environment of chronic ulcers, thus negatively affecting the wound-healing activity in this patient group. Here we show that P. aeruginosa isolates from leg ulcers exhibit a highly variable expression of the proteinases elastase and alkaline proteinase. We propose that bacterial phenotype should be taken into account in future studies on the clinical outcome of leg ulcers colonized by P. aeruginosa.

Macrophage cyclooxygenase expression, immunosuppression, and cardiopulmonary dysfunction after blunt chest trauma.

Two series of experiments were performed in swine who received severe blunt chest trauma. The goals were to determine the time course of constitutive and inducible cyclooxygenase (COX) isozyme expression in pulmonary macrophages (Mphis), and to determine whether COX expression and cardiopulmonary dysfunction were altered when neutrophils (PMNs) were pharmacologically depleted with cyclophosphamide (CYC). In series 1 (n = 17), anesthetized, mechanically ventilated swine were subjected to right chest trauma via captive bolt gun, hemorrhage, and a 60-minute shock period. In series 2 (n = 41), CYC (50 mg/kg intravenously) was administered 4 days before trauma, and the shock period was shortened to 30 minutes. In both series, hemodynamic support and supplemental oxygen were provided for an additional 60 to 90 minutes after shock. Mphis were isolated from serial bilateral bronchoalveolar lavages (BALs) and COX protein expression was measured with Western blots. In series 1, death occurred in 11 of 17. In survivors, Mphi COX-1 peaked at > 100 times baseline in both right BAL and left BAL by 60 minutes (before resuscitation). Changes in Mphi COX-2 were minimal. In series 2, before trauma, CYC (n = 16) reduced circulating and BAL PMNs by > 90% relative to control (n = 25, both p < 0.05) with no complicating side effects. After trauma, death occurred in 11 of 25 controls versus 9 of 16 with CYC. In survivors, PaO2/FIO2 was < 250 and PaCO2 was 25% higher on constant minute ventilation, indicating mismatched ventilation/perfusion; both changes were reduced with CYC (p < 0.05). In controls, bilateral histologic damage included edema, alveolar hemorrhage, and interstitial infiltrates. These changes were reduced by one third with CYC (p = 0.08). Trauma-induced changes in BAL protein, BAL elastase, or Mphi COX expression were not lessened by CYC. After unilateral chest trauma, Mphi COX-1, not COX-2, is induced bilaterally and before fluid resuscitation; CYC prevented PMN infiltration and attenuated structural and functional changes after resuscitation, which suggests that PMNs have a role in the pathogenic mechanism of secondary lung injury; Mphi COX expression and other injury markers were not altered by CYC; and since Mphis continued to express proinflammatory COX protein even after pretreatment with a powerful nonspecific immunosuppressant, and since there is residual alveolar capillary damage even in the absence of PMNs, it is logical to conclude that no single cell type or mediator is a practical therapeutic target and that novel resuscitation strategies must address multiple elements in the inflammatory cascade.

Elastase expression by infiltrating neutrophils in gliomas

Polymorphonuclear neutrophil elastase is a neutral protease released by activated polymorphonuclear neutrophils and plays a crucial role in maintaining host defense. However, under certain conditions, this enzyme damages normal tissue and facilitates infiltration of tumor cells. In this study, surgical specimens were obtained from the tumor core and infiltrating margin of the glioma of 72 patients with astrocytoma of varying degrees of malignancy, and the specimens were tested for the presence of elastase by immunohistochemical analysis. Polymorphonuclear neutrophil elastase was not present in the tumor core of any of the 12 cases. Elastase was expressed in areas of tumor infiltration of the brain in all four glioblastoma cases, three of the four anaplastic astrocytoma cases, and none of the four low-grade astrocytoma cases. There was a higher percentage of elastase-positive polymorphonuclear neutrophils in the infiltrating margin of tumors with greater degree of malignancy. Polymorphonuclear neutrophils are recruited to malignant gliomas, and the elastase released by these cells aids in the infiltration of gliomas [Neurol Res 2000; 22: 465-468]

C-C Chemokines in Allergen-Induced Late-Phase Cutaneous Responses in Atopic Subjects: Association of Eotaxin with Early 6-Hour Eosinophils, and of Eotaxin-2 and Monocyte Chemoattractant Protein-4 with the Later 24-Hour Tissue Eosinophilia, and Relationship to Basophils and Other C-C Chemokines (Monocyte Chemoattractant Protein-3 and RANTES)

The relationship of expression of the C-C chemokines eotaxin, eotaxin 2, RANTES, monocyte chemoattractant protein-3 (MCP-3), and MCP-4 to the kinetics of infiltrating eosinophils, basophils, and other inflammatory cells was examined in allergen-induced, late-phase allergic reactions in the skin of human atopic subjects. EG2+ eosinophils peaked at 6 h and correlated significantly with eotaxin mRNA and protein, whereas declining eosinophils at 24 h correlated significantly with eotaxin-2 and MCP-4 mRNA. In contrast, no significant correlations were observed between BB1+ basophil infiltrates, which peaked at 24 h, and expression of eotaxin, eotaxin-2, RANTES, MCP-3, and MCP-4 or elastase+ neutrophils (6-h peak), CD3+ and CD4+ T cells (24 h), and CD68+ macrophages (72 h). Furthermore, 83% of eosinophils, 40% of basophils, and 1% of CD3+ cells expressed the eotaxin receptor CCR3, while eotaxin protein was expressed by 43% of macrophages, 81% of endothelial cells, and 6% of T cells (6%). These data suggest that 1) eotaxin has a role in the early 6-h recruitment of eosinophils, while eotaxin-2 and MCP-4 appear to be involved in later 24-h infiltration of these CCR3+ cells; 2) different mechanisms may guide the early vs late eosinophilia; and 3) other chemokines and receptors may be involved in basophil accumulation of allergic tissue reactions in human skin.

Expression and localization of macrophage elastase (matrix metalloproteinase-12) in abdominal aortic aneurysms.

Elastolytic matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of abdominal aortic aneurysms (AAA), a disorder characterized by chronic aortic wall inflammation and destruction of medial elastin. The purpose of this study was to determine if human macrophage elastase (HME; MMP-12) might participate in this disease. By reverse transcription-polymerase chain reaction, HME mRNA was consistently demonstrated in AAA and atherosclerotic occlusive disease (AOD) tissues (six of six), but in only one of six normal aortas. Immunoreactive proteins corresponding to proHME and two products of extracellular processing were present in seven of seven AAA tissue extracts. Total HME recovered from AAA tissue was sevenfold greater than normal aorta (P < 0.001), and the extracted enzyme exhibited activity in vitro. Production of HME was demonstrated in the media of AAA tissues by in situ hybridization and immunohistochemistry, but HME was not detected within the media of normal or AOD specimens. Importantly, immunoreactive HME was specifically localized to residual elastin fragments within the media of AAA tissue, particularly areas adjacent to nondilated normal aorta. In vitro, the fraction of MMP-12 sequestered by insoluble elastin was two- to fivefold greater than other elastases found in AAA tissue. Therefore, HME is prominently expressed by aneurysm-infiltrating macrophages within the degenerating aortic media of AAA, where it is also bound to residual elastic fiber fragments. Because elastin represents a critical component of aortic wall structure and a matrix substrate for metalloelastases, HME may have a direct and singular role in the pathogenesis of aortic aneurysms.

Starvation selection restores elastase and rhamnolipid production in a Pseudomonas aeruginosa quorum-sensing mutant.

The las quorum-sensing system of Pseudomonas aeruginosa controls the expression of elastase and rhamnolipid. We report that starvation can select a mutant producing these virulence factors in spite of a lasR deletion. Expression of the autoinducer synthase gene rhlI was increased in this suppressor mutant, suggesting compensation by the rhl system. These data show that P. aeruginosa can restore elastase and rhamnolipid production in the absence of a functional las quorum-sensing system.

Mast cell collagenase correlates with regression of pulmonary vascular remodeling in the rat.

Pulmonary vascular remodeling, produced by cell hypertrophy and extracellular matrix protein synthesis in response to hemodynamic stress, regresses after reduction of blood pressure, possibly by proteolysis of structural proteins. To test this postulate, we assessed the breakdown of extracellular matrix proteins and expression of collagenase and elastase in pulmonary arteries of rats exposed to hypoxia (10% O2 for 10 d) followed by normoxia. During hypoxia, contents of collagen and elastin increased in pulmonary arteries and latent rat interstitial collagenase was expressed without increased collagenolytic activity or mRNA levels. At 3 days after normoxia, collagen and elastin contents decreased coincident with the new appearance of activated collagenase and transient increases in collagenolytic and elastolytic activities. The amount of immunoreactive collagenase, localized predominately in connective tissue-type mast cells, was increased in the adventitia and media of hypertensive vessels. We conclude that mast cells containing latent collagenase are recruited into the outer walls of pulmonary arteries during remodeling. It is possible that mast cell-derived collagenase contributes to collagen breakdown in pulmonary arteries during early recovery from hypoxia and plays a role in restoration of vascular architecture.

Involvement of STAT3 in the Granulocyte Colony-stimulating Factor-induced Differentiation of Myeloid Cells

Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation and differentiation of the progenitor cells of neutrophilic granulocytes. The binding of G-CSF to its receptor specifically activates JAK1 and JAK2 kinases, as well as STAT3, a signal transducer and activator of transcription (STAT). To examine the role of STAT3 in G-CSF receptor-mediated signal transduction, two different forms of the dominant negative STAT3 were introduced into a mouse myeloid cell line that exogenously expresses the mouse G-CSF receptor. In response to G-CSF, the parental myeloid cells grew for about 4 days, and then they stopped dividing and differentiated into cells with lobulated nuclei. During this period, the expression of the myeloperoxidase (MPO) gene was induced, while c-myc gene expression was down-regulated. In contrast, in the cells expressing the dominant negative STAT3, G-CSF could induce neither growth arrest nor morphological change. However, the induction of the MPO gene by G-CSF was not affected by the dominant negative STAT3. These results indicate that STAT3 activation is responsible for part of the G-CSF-induced differentiation of neutrophils but that another pathway, involving the expression of the MPO gene, that does not utilize the activated STAT3, is also required to fully differentiate the cells.

136 Wrinkle effacement in mouse skin by a sphingosine derivative with a potential to supress elastase expression

136 Wrinkle effacement in mouse skin by a sphingosine derivative with a potential to supress elastase expression

Metalloelastase expression in a mouse macrophage cell line--regulation by 4beta-phorbol 12-myristate 13-acetate, lipopolysaccharide and dexamethasone.

The regulation of the expression of mouse macrophage elastase (MME) was investigated using the murine tumor cell line P388D1. The effects of three factors were studied: a phorbol ester (4beta-phorbol 12-myristate 13-acetate, PMA), an endotoxin (lipopolysaccharide, LPS) and a corticosteroid (dexamethasone). Both in situ hybridization and northern blot analysis showed that P388D1 cells constitutively express the MME gene. Quantification of the MME mRNA by northern blot analysis showed that only PMA and dexamethasone significantly regulate MME gene expression in a time-dependent and dose-dependent manner. After PMA treatment, the MME mRNA level was maximal between 4 h and 9 h (medium-term response), and the mean amplitude of the response to a concentration of 100 nM was 2.5-fold (P<0.01). LPS did not induce any significant change in MME mRNA level even when 1% serum was added to the cultures. Following dexamethasone treatment, the MME mRNA level was minimal between 21 h and 33 h (long-term response), and the mean amplitude of the response to a concentration of 100 nM was 0.49-fold (P < 0.05). Using actinomycin D, it appeared that the inhibition of RNA synthesis reduces the ulterior stimulating effect of PMA from 184% to 121%, and that MME mRNA has a half-life longer than 8 h, which is not diminished by dexamethasone. These results strongly suggest that the two factors modify MME mRNA level by stimulating (PMA) or inhibiting (dexamethasone) the transcription of the gene, rather than by modifying the transcript stability. Analysis of the cell-conditioned media by elastin zymography showed the MME as a lysis band in the 22-kDa region, the intensity of which varied with the treatments. The MME secretion is stimulated by PMA, inhibited by dexamethasone and does not show any variation after LPS treatment.

Increased expression of promatrix metalloproteinase-9 and neutrophil elastase in canine dilated cardiomyopathy.

Canine dilated cardiomyopathy, commonly affecting Doberman pinschers, results in extracellular matrix remodelling within the myocardium. The aim of this study was to examine the proteolytic activity in myocardium from Doberman pinschers with dilated cardiomyopathy. Samples of myocardium, obtained rapidly post mortem from the left ventricular free wall of Dobermans with dilated cardiomyopathy, clinically normal Dobermans and control dogs (non-Dobermans), were examined for proteolytic activity using substrate gel zymography. Gels were analysed by scanning densitometry. Promatrix metalloproteinase-9 activity was significantly increased in all Doberman myocardium when compared to controls. A significant increase in an enzyme, identified to be neutrophil elastase by inhibition of its activity by Elastatinal and Western blotting, was also detected in all Dobermans when compared to controls. The results indicate that promatrix metalloproteinase-9 and neutrophil elastase, both of which are implicated in inflammatory responses, are present in significantly elevated levels in Doberman dilated cardiomyopathy and are raised in clinically normal Dobermans. Both proteolytic enzymes degrade a wide variety of connective tissue components and thus the increased levels found may play an important role in the structural remodelling seen in the myocardium and subsequent heart failure. Increased proteolytic enzyme levels in clinically normal Dobermans may be indicative of the predisposition of the breed to dilated cardiomyopathy.

Pancreatic reg gene expression is inhibited during cellular differentiation.

Factors that control pancreatic regenerating (reg I) gene expression are unknown, but it is believed that its expression may correspond with cellular differentiation. The authors recently demonstrated that reg I is expressed in AR42J, a rat acinar cell line whose state of differentiation can be modulated by dexamethasone. They used this line to study reg I expression during cellular proliferation and differentiation. After treatment of cells with 10 nmol/L dexamethasone, proliferation was assayed by thymidine incorporation; differentiation by expression of elastase I mRNA. Reg I mRNA levels were measured using a rat reg I cDNA probe, and reg I protein levels assayed by enzyme-linked immunosorbent assay of cellular lysates with a polyclonal antibody. The effect of gastrin, cholecystokinin and glucagon on reg I expression was also studied. When compared with controls, treatment with dexamethasone caused thymidine incorporation to decrease and elastase mRNA levels to increase. Reg I mRNA decreased from controls of 100 +/- 16% to 40 +/- 18% (p < 0.05), and reg I protein levels decreased as well. Gastrointestinal hormones had no significant effect on either elastase or reg I gene expression. Expression of reg I inversely correlates with the level of cellular differentiation, can be modulated via the glucocorticoid receptor, and is a potential marker of gastrointestinal epithelial differentiation. Despite its presence within a pancreatic acinar cell line, reg I gene expression is not modulated by gastrointestinal hormones.