Role of Matrix Metalloproteinases-1 and -2 in Interleukin-13-Suppressed Elastin in Airway Fibroblasts in Asthma.

Elastin synthesis and degradation in the airway and lung parenchyma contribute to airway mechanics, including airway patency and elastic recoil. IL-13 mediates many features of asthma pathobiology, including airway remodeling, but the effects of IL-13 on elastin architecture in the airway wall are not known. We hypothesized that IL-13 modulates elastin expression in airway fibroblasts from subjects with allergic asthma. Twenty-five subjects with mild asthma (FEV1, 89 ± 3% predicted) and 30 normal control subjects (FEV1, 102 ± 2% predicted) underwent bronchoscopy with endobronchial biopsy. Elastic fibers were visualized in airway biopsy specimens using Weigert's resorcin-fuchsin elastic stain. Airway fibroblasts were exposed to IL-13; a pan-matrix metalloproteinase (MMP) inhibitor (GM6001); specific inhibitors to MMP-1, -2, -3, and -8; and combinations of IL-13 with MMP inhibitors in separate conditions in serum-free media for 48 hours. Elastin (ELN) expression as well as MMP secretion and activity were quantified. Results of this study show that elastic fiber staining of airway biopsy tissue was significantly associated with methacholine PC20 (i.e., the provocative concentration of methacholine resulting in a 20% fall in FEV1 levels) in patients with asthma. IL-13 significantly suppressed ELN expression in asthmatic airway fibroblasts as compared with normal control fibroblasts. The effect of IL-13 on ELN expression was significantly correlated with postbronchodilator FEV1/FVC in patients with asthma. MMP inhibition significantly stimulated ELN expression in patients with asthma as compared with normal control subjects. Specific inhibition of MMP-1 and MMP-2, but not MMP-3 or MMP-8, reversed the IL-13-induced suppression of ELN expression. In asthma, MMP-1 and MMP-2 mediate IL-13-induced suppression of ELN expression in airway fibroblasts.

Emphysema in an adult with galactosialidosis linked to a defect in primary elastic fiber assembly.

Galactosialidosis is a lysosomal storage disorder caused by loss of function of protective protein cathepsin A, which leads to secondary deficiencies of ß-galactosidase and neuraminidase-1. Emphysema has not been previously reported as a possible complication of this disorder, but we now describe this condition in a 41-year-old, non-smoking male. Our patient did not display deficiency in α-1-antitrypsin, the most common cause of emphysema in non-smokers, which brings about disseminated elastolysis. We therefore hypothesized that loss of cathepsin A activity was responsible because of previously published evidence showing it is prerequisite for normal elastogenesis. We now present experimental evidence to support this theory by demonstrating impaired primary elastogenesis in cultures of dermal fibroblasts from our patient. The obtained data further endorse our previous finding that functional integrity of the cell surface-targeted molecular complex of cathepsin A, neuraminidase-1 and the elastin-binding protein (spliced variant of ß-galactosidase) is prerequisite for the normal assembly of elastic fibers. Importantly, we also found that elastic fiber production was increased after exposure either to losartan, spironolactone, or dexamethasone. Of immediate clinical relevance, our data suggest that surviving patients with galactosialidosis should have periodic assessment of their pulmonary function. We also encourage further experimental exploration of therapeutic potential of the afore-mentioned elastogenesis-stimulating drugs for the alleviation of pathological processes in galactosialidosis that could be mechanistically linked to impaired deposition of elastic fibers.

LOXL1 deficiency in the lamina cribrosa as candidate susceptibility factor for a pseudoexfoliation-specific risk of glaucoma.

PURPOSE: To test the hypothesis that a primary disturbance in lysyl oxidase-like 1 (LOXL1) and elastin metabolism in the lamina cribrosa of eyes with pseudoexfoliation syndrome constitutes an independent risk factor for glaucoma development and progression. DESIGN: Observational, consecutive case series. PARTICIPANTS: Posterior segment tissues obtained from 37 donors with early and late stages of pseudoexfoliation syndrome without glaucoma, 37 normal age-matched control subjects, 5 eyes with pseudoexfoliation-associated open-angle glaucoma, and 5 eyes with primary open-angle glaucoma (POAG). METHODS: Protein and mRNA expression of major elastic fiber components (elastin, fibrillin-1, fibulin-4), collagens (types I, III, and IV), and lysyl oxidase crosslinking enzymes (LOX, LOXL1, LOXL2) were assessed in situ by quantitative real-time polymerase chain reaction, (immuno)histochemistry, and light and electron microscopy. Lysyl oxidase-dependent elastin fiber assembly was assessed by primary optic nerve head astrocytes in vitro. MAIN OUTCOME MEASURES: Expression levels of elastic proteins, collagens, and lysyl oxidases in the lamina cribrosa. RESULTS: Lysyl oxidase-like 1 proved to be the major lysyl oxidase isoform in the normal lamina cribrosa in association with a complex elastic fiber network. Compared with normal and POAG specimens, lamina cribrosa tissues obtained from early and late stages of pseudoexfoliation syndrome without and with glaucoma consistently revealed a significant coordinated downregulation of LOXL1 and elastic fiber constituents on mRNA and protein level. In contrast, expression levels of collagens and other lysyl oxidase isoforms were not affected. Dysregulated expression of LOXL1 and elastic proteins was associated with pronounced (ultra)structural alterations of the elastic fiber network in the laminar beams of pseudoexfoliation syndrome eyes. Inhibition of LOXL1 interfered with elastic fiber assembly by optic nerve head astrocytes in vitro. CONCLUSIONS: The findings provide evidence for a pseudoexfoliation-specific elastinopathy of the lamina cribrosa resulting from a primary disturbance in LOXL1 regulation and elastic fiber homeostasis, possibly rendering pseudoexfoliation syndrome eyes more vulnerable to pressure-induced optic nerve damage and glaucoma development and progression.

Neuraminidase-1, a subunit of the cell surface elastin receptor, desialylates and functionally inactivates adjacent receptors interacting with the mitogenic growth factors PDGF-BB and IGF-2.

We recently established that the elastin-binding protein, which is identical to the spliced variant of beta-galactosidase, forms a cell surface-targeted complex with two proteins considered "classic lysosomal enzymes": protective protein/cathepsin A and neuraminidase-1 (Neu1). We also found that cell surface-residing Neu1 can desialylate neighboring microfibrillar glycoproteins and facilitate the deposition of insoluble elastin, which contributes to the maintenance of cellular quiescence. Here we provide evidence that cell surface-residing Neu1 contributes to a novel mechanism that limits cellular proliferation by desialylating cell membrane-residing sialoglycoproteins that directly propagate mitogenic signals. We demonstrated that treatment of cultured human aortic smooth muscle cells (SMCs) with either a sialidase inhibitor or an antibody that blocks Neu1 activity induced significant up-regulation in SMC proliferation in response to fetal bovine serum. Conversely, treatment with Clostridium perfringens neuraminidase (which is highly homologous to Neu1) decreased SMC proliferation, even in cultures that did not deposit elastin. Further, we found that pretreatment of aortic SMCs with exogenous neuraminidase abolished their mitogenic responses to recombinant platelet-derived growth factor (PDGF)-BB and insulin-like growth factor (IGF)-2 and that sialidosis fibroblasts (which are exclusively deficient in Neu1) were more responsive to PDGF-BB and IGF-2 compared with normal fibroblasts. Furthermore, we provide direct evidence that neuraminidase caused the desialylation of both PDGF and IGF-1 receptors and diminished the intracellular signals induced by the mitogenic ligands PDGF-BB and IGF-2.

Ultrastructural immunolocalization of lysyl oxidase in vascular connective tissue.

The localization of lysyl oxidase was examined in calf and rat aortic connective tissue at the ultrastructural level using polyclonal chicken anti-lysyl oxidase and gold conjugated rabbit anti-chicken immunoglobulin G to identify immunoreactive sites. Electron microscopy of calf aortic specimens revealed discrete gold deposits at the interface between extracellular bundles of amorphous elastin and the microfibrils circumferentially surrounding these bundles. The antibody did not react with microfibrils which were distant from the interface with elastin. There was negligible deposition of gold within the bundles of amorphous elastin and those few deposits seen at these sites appeared to be associated with strands of microfibrils. Lysyl oxidase was similarly localized in newborn rat aorta at the interface between microfibrils and nascent elastin fibers. Gold deposits were not seen in association with extracellular collagen fibers even after collagen-associated proteoglycans had been degraded by chondroitinase ABC. However, the antibody did recognize collagen-bound lysyl oxidase in collagen fibers prepared from purified collagen to which the enzyme had been added in vitro. No reaction product was seen if the anti-lysyl oxidase was preadsorbed with purified lysyl oxidase illustrating the specificity of the antibody probe. The present results are consistent with a model of elastogenesis predicting the radial growth of the elastin fiber by the deposition and crosslinking of tropoelastin units at the fiber-microfibril interface.

The pathogenesis of photoaging: the role of neutrophils and neutrophil-derived enzymes.

The hallmark of photoaged skin is solar elastosis, which is probably an end product of elastic fiber degradation. Exposure of human skin to a certain threshold of UV, infrared radiation (IR), and heat leads to an influx of neutrophils. These neutrophils are packed with potent proteolytic enzymes capable of degrading collagen and, particularly, elastic fibers. Neutrophil-derived proteolytic enzymes are held responsible for the extracellular matrix (ECM) damage observed in several non-dermatological conditions. Furthermore, neutrophil elastase, a major product of neutrophils, is strongly associated with solar elastosis in mice. Taken together with our data that show in vivo proteolytic activity of neutrophil-derived elastase and matrix metalloproteinases (MMPs) in UV-exposed skin, we have hypothesized earlier that neutrophils are major contributors to the photoaging process. Although several groups have shown that MMPs are also induced in skin exposed to relatively low doses of UV, IR, and heat, clinical data indicate that high(er) doses of UV, IR, and heat are necessary to induce photoaging or photoaging-like pathology in the skin. Therefore, we propose that MMPs generated by suberythemogenic doses of UV and low doses of IR/heat are involved in cellular processes other than ECM degradation.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 67-72; doi:10.1038/jidsymp.2009.15.

The defensive role of lysozyme in human gingiva in inflammatory periodontal disease.

BACKGROUND AND OBJECTIVE: The presence of lysozyme in human gingiva has not previously been demonstrated. In this study, we looked for evidence for the potential role of lysozyme as a protector of gingival elastic fibres. The objective of this study was also to determine the ex vivo susceptibility to hydrolysis of gingival elastic fibres from patients with or without periodontal disease by human leukocyte elastase and by human cathepsin G. MATERIALS AND METHODS: Using gingival tissue sections from eight control, 10 gingivitis and 10 periodontitis patients, we evaluated the area fraction occupied by gingival elastic fibres (after selective staining) by the use of automated image analysis. In the ex vivo experiments, serial tissue sections from four control, four gingivitis, four young periodontitis and four aged periodontitis patients were submitted to the action of human leukocyte elastase and cathepsin G, after which enzymatic activities were determined by image analysis. Indirect immunodetection of lysozyme was also done on tissue sections for all patients included in this study. RESULTS: Large variations of the area fraction occupied by elastic fibres were observed in human gingiva from young and aged patients with and without periodontal disease. In control and gingivitis patients, leukocyte elastase and cathepsin G had high comparable elastin solubilizing activities. With young and aged periodontitis patients, the two serine proteinases had weak elastin solubilizing activities. Lysozyme appeared to be present at the periphery of gingival elastic fibres in periodontitis patients. CONCLUSION: Lysozyme can be considered an important natural protector of elastic fibres in pathological gingiva.

Increased choroidal neovascularization following laser induction in mice lacking lysyl oxidase-like 1.

PURPOSE: Age-related degradation of the elastic lamina in Bruch's membrane may have a permissive effect on the growth of choroidal neovascularization (CNV). This study investigated the influence of defective elastic fiber maintenance in the development of laser-induced CNV. METHODS: A mouse lacking lysyl oxidase-like (LOXL)-1, an enzyme essential for elastin polymerization, was studied. The morphologic characteristics of the elastic lamina within Bruch's membrane were examined in mutant and wild-type (WT) eyes. Laser-induced CNV was evaluated by fluorescein angiography and choroidal flat mounts. Immunohistochemistry for elastin was performed on the CNV lesions, and vascular endothelial growth factor (VEGF) levels were determined by ELISA. Soluble elastin and matrix metalloproteinase (MMPs) levels were also analyzed by immunoblotting. RESULTS: The elastic lamina of Bruch's membrane in the LOXL1-deficient mice was fragmented and less continuous than in the WT controls. The mutant mice showed increased levels of soluble elastin peptides and reduced elastin polymer deposition in neovascular membranes. Significantly larger CNV with greater leakage on fluorescein angiography developed in mutant mice. VEGF levels in the RPE/choroid were higher in the knockout mice on days 7 and 14 after laser (P < 0.05). MT1-MMP (MMP14) was also elevated after laser in the LOXL1 mutant eyes compared to the WT controls. CONCLUSIONS: These results show that a systemic defect in elastic fiber deposition affects Bruch's membrane integrity and leads to more aggressive CNV growth. The latter may be partially mediated by abnormal signaling from the accumulation of soluble elastin peptides.

Granulocyte macrophage colony-stimulating factor deficiency affects vascular elastin production and integrity of elastic lamellae.

BACKGROUND: Granulocyte macrophage colony-stimulating factor (GM-CSF) deficiency affects the production and fiber assembly/organization of the vascular collagenous matrix; structural alterations to the elastic system were observed. The present study elaborates the effect of GM-CSF deficiency on the vascular elastin system. METHODS AND RESULTS: Histological examination of the aorta of GM-CSF-deficient mice revealed structurally altered elastic fibers. The elastic fiber area was significantly enhanced, whereas the remaining medial area was not affected. Aortic size was significantly increased. Reverse transcription polymerase chain reaction demonstrated decreased expression levels of tropoelastin, lysyl oxidase and bone morphogenetic protein 1 (BMP-1). Cell culture studies on vascular smooth muscle cells showed that after clearance of GM-CSF with GM-CSF antibodies, the tropoelastin mRNA expression was markedly reduced. Concomitantly, lysyl oxidase and BMP-1 mRNA levels were decreased. Treatment with GM-CSF stimulated the expression of these mRNAs. CONCLUSIONS: Our studies demonstrate that disorganization of elastic lamellae as induced by GM-CSF deficiency is associated with adaptive vascular remodeling. The decreased tropoelastin expression observed is associated with elastic fiber hypertrophy. This paradox effect may be explained by decreased expression levels of lysyl oxidase and BMP-1, both mediating cross-linkage and thus assembly and organization of elastic fibers. From our data, we conclude that GM-CSF is a prerequisite for the maintenance of structural integrity of the vessel wall.

Matrix metalloproteinase promotes elastic fiber degradation in ligamentum flavum degeneration.

Ligamentum flavum (LF) hypertrophy in lumbar spinal canal stenosis (LSCS) is characterized by a loss of elastic fibers and fibrosis. Chronic inflammation is thought to be responsible for the histological change but the mechanism underlying elastic fiber degradation remains unclear. Given that matrix metalloproteinase (MMP)-2 and -9 have elastolytic activity and are partly regulated by inflammatory cytokines such as interleukin (IL)-6, in this study, we investigated whether MMPs mediate LF degeneration using 52 LF samples obtained during lumbar surgery, including 31 LSCS and 21 control specimens. We confirmed by histological analysis that the LSCS samples exhibited severe degenerative changes compared with the controls. We found that MMP-2 was upregulated in LF tissue from patients with LSCS at the mRNA and protein levels, whereas MMP-9 expression did not differ between the two groups. The MMP-2 level was positively correlated with LF thickness and negatively correlated with the area occupied by elastic fibers. IL-6 mRNA expression was also increased in LF tissue from patients with LSCS and positively correlated with that of MMP-2. Signal transducer and activator of transcription (STAT)3, a component of the IL-6 signaling pathway, was activated in hypertrophied LF tissues. Our in vitro experiments using fibroblasts from LF tissue revealed that IL-6 increased MMP-2 expression, secretion, and activation via induction of STAT3 signaling, and this effect was reversed by STAT3 inhibitor treatment. Moreover, elastin degradation was promoted by IL-6 stimulation in LF fibroblast culture medium. These results indicate that MMP-2 induction by IL-6/STAT3 signaling in LF fibroblasts can degrade elastic fibers, leading to LF degeneration in LSCS.

Fibrillin-2 degradation by matrix metalloproteinase-2 in periodontium.

Elastic system fibers, comprised of microfibrils and tropoelastin, are extracellular components of periodontal tissue. During development, the microfibrils act as a template on which tropoelastin is deposited. However, the process of elastic system fiber remodeling is not fully understood. Therefore, we examined whether matrix metalloproteinases (MMPs) are involved in the remodeling of fibrillins (major components of microfibrils) by human gingival fibroblasts and periodontal ligament (PDL) fibroblasts. Gingival and PDL fibroblasts were cultured for 6 weeks. In some cultures, MMP inhibitor or tissue inhibitor of matrix metalloproteinsase-2 (TIMP-2) was added to the medium for an additional 2 weeks. Active MMP-2 (62 kDa) appeared as cell-membrane-associated or in extracellular matrix only in PDL fibroblast cell layers. The addition of MMP inhibitor or TIMP-2 significantly increased fibrillin-2 accumulation in PDL fibroblast cell layers, and decreased the amount of fibrillin-2 fragments, suggesting that active MMP-2 may degrade fibrillin-2, and that MMPs may play a role in the remodeling of elastic system fibers in PDL.

Degradation of elastic fiber and elevated elastase expression in long head of biceps tendinopathy.

Tendinopathy of the long head of the biceps (TLHB) involves various types of extracellular matrix degeneration, but previous studies have not evaluated elastic fibers. The purpose of this study was to investigate elastic fiber distribution in long head of the biceps (LHB). The TLHB tendons of 16 consecutive patients (eight men and eight women; average age of 55.75 years; age range of 40-71 years) were transected and harvested. Three cadaveric LHB tendons were used as the control group. The expression of collagen type I was decreased, but type III was increased in TLHB. Disruption of elastic fibers was particularly observed in grade II specimens where the level of elastase-positive staining was significantly higher than in grade I specimens. Elastic fibers were not observed in the grade III area, implying a higher expression of elastase than in the grade I area. Results of Western blotting showed that the expression of elastin was higher in the control group and the levels of elastin significantly decreased in grades II and III of TLHB. Levels of osteopontin and elastase were increased in primary culture of human tenocytes after experiencing elastic derived peptide treatment. These results suggested that elastase may be caused by the disruption of elastic fibers in the development of chronic tendinopathy and that elastic derived peptide may enhance elastase and osteopontin expression. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1919-1926, 2017.

Metalloproteinase-2 and -9 in giant cell arteritis: involvement in vascular remodeling.

BACKGROUND: Both matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) have been postulated to play roles in the pathophysiology of giant cell arteritis (GCA) because of their ability to degrade elastin. Understanding the specific mediators of arterial damage in GCA could lead to new therapeutic targets in this disease. METHODS AND RESULTS: Temporal artery biopsy specimens were obtained from 147 consecutive patients suspected of GCA. Clinical and histopathological data were collected according to protocol. Using immunohistochemistry, we compared the expression of MMP-2 and MMP-9 in the temporal artery biopsies of both GCA cases (n=50) and controls (n=97). MMP-9 was found more frequently in positive than in negative temporal artery biopsies (adjusted odds ratio [OR], 3.20; P=0.01). In contrast, the frequency of MMP-2 was not significantly different between positive and negative biopsies (adjusted OR, 2.18; P=0.22). Both MMP-2 and MMP-9 were found in macrophages and giant cells near the internal elastic lamina and in smooth muscle cells and myofibroblasts of the media and intima. MMP-9 was also found in the vasa vasorum. MMP-9 but not MMP-2 was associated with internal elastic lamina degeneration, intimal hyperplasia, and luminal narrowing, even after adjustment for possible confounding variables. CONCLUSIONS: MMP-9 appears more likely than MMP-2 to be involved in the pathophysiology of GCA. MMP-9 not only participates in the degradation of elastic tissue but also is associated with intimal hyperplasia, subsequent luminal narrowing, and neoangiogenesis. The expression of MMP by smooth muscle cells implicates these cells as potential secretory cells in GCA.

The Pathogenesis of floppy eyelid syndrome: involvement of matrix metalloproteinases in elastic fiber degradation.

OBJECTIVE: To investigate histopathologic alterations of eyelid biopsy specimens from patients with floppy eyelid syndrome (FES) with special regard to elastic fiber content and ultrastructure as well as to the expression of elastin-degrading enzymes to elucidate the pathogenesis of this disorder. DESIGN: Retrospective, interventional case series. PARTICIPANTS AND CONTROLS: Eleven consecutive patients with FES and 10 age-matched control patients with basal cell carcinoma of the eyelid. METHODS: Horizontal pentagonal eyelid resections of 16 upper lids were performed in 11 patients with FES. Full-thickness eyelid biopsy specimens from study and control patients were examined by light and transmission electron microscopy, semiquantitative morphometry, and immunohistochemistry using antibodies against matrix metalloproteinase (MMP)-2, MMP-7, MMP-9, and MMP-12 and neutrophil elastase. RESULTS: All patients treated with surgical horizontal eyelid shortening were asymptomatic at follow-up. Histopathologic analysis of the surgical specimens showed, apart from unspecific signs of chronic inflammation, a significant decrease in the amount of elastin within the tarsal plate and eyelid skin as compared with controls. Residual elastic fibers revealed an abnormal ultrastructure with a diminished elastin core. Immunohistochemistry demonstrated an increased immunoreactivity for elastolytic proteases, particularly MMP-7 and MMP-9, in areas of elastin depletion in FES specimens as compared with controls. CONCLUSIONS: The findings indicate that upregulation of elastolytic enzymes, most probably induced by repeated mechanical stress, participates in elastic fiber degradation and subsequent tarsal laxity and eyelash ptosis in FES.

Is neutrophil elastase associated with elastic tissue in emphysema?

To test the role of elastase in the pathogenesis of emphysema human neutrophil elastase (HNE) was localised by electron microscopy using an immunogold staining technique. Specific localisation of HNE to elastic tissue in emphysema did not occur, but non-specific binding of immunoglobulin G (IgG) to elastic tissue in emphysematous and normal lung tissue, which was completely blocked by the non-immune serum that was homologous to the gold labelled second antibody, was found. HNE was also present, however, in the granules of neutrophils in the same sections. Non-specific labelling associated with elastin was probably due to binding of IgG to the high numbers of hydrophobic and charged regions known to be present in this molecule, and it is concluded that our findings do not support the existence of high concentrations of elastase in association with elastin in emphysematous lung tissue.

Elastin degradation in the superficial temporal arteries of patients with intracranial aneurysms reflects changes in plasma elastase.

OBJECTIVE: alpha 1-Antitrypsin (AAT) and alpha 2-macroglobulin (AMG) are elastase inhibitors that bind the enzyme and reduce measured levels of free elastase. It was recently demonstrated that some patients with intracranial aneurysms have significantly elevated plasma elastase (PE) levels. Although this elevation is unrelated to plasma AAT, it is unknown whether abnormal AAT phenotypes or reduced AMG levels play a role. Moreover, the pathological significance of this elevation is not understood. METHODS: Plasma from 24 patients with aneurysms (ruptured, n = 15; unruptured, n = 9) and 10 age-matched patients who comprised a control group was analyzed for PE and AMG levels by enzyme-linked immunosorbent assay and for AAT phenotype by isoelectric focusing. Sections of superficial temporal temporal artery obtained from these patients at the time of surgery were examined for evidence of elastin degradation by using a van Gieson stain, with scoring on a nine-point quantitative scale. RESULTS: Patients with aneurysms showed significantly elevated PE levels (119 +/- 28 versus 17 +/- 7 micrograms/ml, P < 0.05), but AMG levels were not decreased. AAT phenotypic abnormalities were observed in 10% (2 of 20) of the patients with aneurysms, but this was not different from the expected population incidence (7%). Elastin degradation scores were significantly higher in patients with aneurysms than in patients control group (4.26 +/- 0.54 versus 1.21 +/- 0.43, P < 0.05). In addition, patients with higher elastase levels (> 80 micrograms/ml) demonstrated 55% higher degradation scores than did those with lower elastase levels (< 80 micrograms/ml). CONCLUSION: These data suggest that high PE levels may play a role in systemic arterial elastin degradation seen in patients with intracranial aneurysms. These data also support the contention that elevated elastase levels are not the result of decreased protease inhibitor levels. Although PE levels were significantly higher for the entire group of patients with aneurysms, this assay has relatively low sensitivity for predicting the presence of unruptured aneurysms. Additional study is necessary to determine whether serum elastase levels greater than 80 micrograms/ml, in the setting of other risk factors, are useful in identifying asymptomatic patients for additional screening.

Ultrastructural changes of the external elastic lamina in experimental hypercholesterolemic porcine coronary arteries.

The external elastic lamina (EEL) serves as a barrier for cells and macromolecules between the media and adventitia in the vascular wall. We evaluated the morphological changes and quantitative assessments of the EEL architecture in the coronary circulation of pigs fed with a high cholesterol diet. Confocal microscopy analysis of the EEL from hypercholesterolemic coronary arteries revealed an altered pattern characterized by fragmentation and disorganization of the EEL associated with an increase in the thickness. Computerized digital analysis of the images obtained by confocal scanning microscopy demonstrated that compared to normal coronary arteries, the EEL of hypercholesterolemic coronary arteries decreased in the percentage of their elastin content (30.80 +/- 1.64% vs. 47.85 +/- 1.82%, p = 0.001). The percentage of elastin content was negatively correlated with the vessel wall area (r = -0.82, p = 0.001). The immunoreactivity for matrix metalloproteinase-3 (MMP-3) increased in cholesterol-fed coronary arteries, predominantly in the neointima and adventitia. This study demonstrates that experimental hypercholesterolemia induced ultrastructural changes of the EEL in coronary circulation. The EEL may also be an atherosclerosis-prone area compared with the intima. The EEL may play an important role in the development of structural changes which characterizes the early phase of coronary atherosclerosis and vascular remodeling.

Damaging role of neutrophil elastase in the elastic fiber and basement membrane in human emphysematous lung.

The immunolocalization of human neutrophil elastase (HNE) in the alveolar interstitium of 6 patients with emphysema was investigated by immunochemical electron microscopy. The results showed that HNE is localized in the azurophil granules of neutrophils, and extracellularly on the elastic fibers of alveolar interstitium and basement membranes of epithelium and endothelium. The damage of elastic fibers and basement membranes could be observed. The HNE level of the alveolar interstitium was obviously elevated and closely related to the severity of emphysematous lesions as shown by measuring the mean linear intercept (MLI) in 4 emphysematous lungs with chronic bronchitis (r = 0.84). This suggests that HNE might play an important role in the pathogenesis of emphysema. Although enlarged airspace and increased MLI data were observed in 2 patients, one with asthma and the other, an elderly patient, without lung diseases, the HNE level of alveolar interstitium is much less than that of the other 4 chronic obstructive pulmonary disease patients. This implies that the mechanism of the the airspace enlargement occurring in the 2 patients may be different from that of others. These findings support the hypothesis of elastase-antielastase imbalance on the pathogenesis of pulmonary emphysema.

Altered vascular activation due to deficiency of the NADPH oxidase component p22phox.

BACKGROUND: Reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase play important roles in vascular activation. The p22(phox) subunit is necessary for the activity of NADPH oxidase complexes utilizing Nox1, Nox2, Nox3, and Nox4 catalytic subunits. METHODS: We assessed p22(phox)-deficient mice and human tissue for altered vascular activation. RESULTS: Mice deficient in p22(phox) were smaller than their wild-type littermates but showed no alteration in basal blood pressure. The wild-type littermates were relatively resistant to forming intimal hyperplasia following carotid ligation, and the intimal hyperplasia that developed was not altered by p22(phox) deficiency. However, at the site of carotid artery ligation, the p22(phox)-deficient mice showed significantly less vascular elastic fiber loss compared with their wild-type littermates. This preservation of elastic fibers was associated with a reduced matrix metallopeptidase (MMP) 12/tissue inhibitor of metalloproteinase (TIMP) 1 expression ratio. A similar decrease in the relative MMP12/TIMP1 expression ratio occurred in human coronary artery smooth muscle cells upon knockdown of the hydrogen peroxide responsive kinase CK1αLS. In the ligated carotid arteries, the p22(phox)-deficient mice showed reduced expression of heterogeneous nuclear ribonucleoprotein C (hnRNP-C), suggesting reduced activity of CK1αLS. In a lung biopsy from a human patient with p22(phox) deficiency, there was also reduced vascular hnRNP-C expression. CONCLUSIONS: These findings indicate that NADPH oxidase complexes modulate aspects of vascular activation including vascular elastic fiber loss, the MMP12/TIMP1 expression ratio, and the expression of hnRNP-C. Furthermore, these findings suggest that the effects of NADPH oxidase on vascular activation are mediated in part by protein kinase CK1αLS.