Role of stromelysin 1 and gelatinase B in experimental acute lung injury

Matrix metalloproteinases (MMPs) are upregulated locally in sites of inflammation, including the lung. Several MMP activities are upregulated in acute lung injury models but the exact role that these MMPs play in the development of the lung injury is unclear due to the absence of specific inhibitors. To determine the involvement of individual MMPs in the development of lung injury, mice genetically deficient in gelatinase B (MMP-9) and stromelysin 1 (MMP-3) were acutely injured with immunoglobulin G immune complexes and the intensity of the lung injury was compared with genetically identical wild-type (WT) mice with normal MMP activities. In the WT mice there was upregulation of gelatinase B and stromelysin 1 in the injured lungs which, as expected, was absent in the genetically deficient gelatinase B- and stromelysin 1-deficient mice, respectively. In the deficient mice there was little in the way of compensatory upregulation of other MMPs. The gelatinase B- and the stromelysin 1-deficient mice had less severe lung injury than did the WT controls, suggesting that both MMPs are involved in the pathogenesis of the lung injury. Further, the mechanism of their involvement in the lung injury appears to be different, with the stromelysin 1-deficient mice having a reduction in the numbers of neutrophils recruited into the lung whereas the gelatinase B-deficient mice had the same numbers of lung neutrophils as did the injured WT controls. These studies indicate, first, that both gelatinase B and stromelysin 1 are involved in the development of experimental acute lung injury, and second, that the mechanisms by which these individual MMPs function appear to differ.     

Redox imbalance and ventilator-induced lung injury

Mechanical ventilation (MV) is an indispensable therapy in the care of critically ill patients with acute lung injury and the acute respiratory distress syndrome; however, it is also known to further lung injury in certain conditions of mechanical stress, leading to ventilator-induced lung injury (VILI). The mechanisms by which conventional MV exacerbates lung injury and inflammation are of considerable clinical significance. Redox imbalance has been postulated, among other mechanisms, to enhance/perpetuate susceptibility to VILI. A better understanding of these pathologic mechanisms will help not only in alleviating the side effects of mechanical forces but also in the development of new therapeutic strategies. Here, we review the relevance of oxidative stress in VILI from human studies as well as cellular and mouse models of mechanical stress. Potential therapeutic avenues for the treatment of VILI with exogenous administration of antioxidants also are discussed.     

Kinetic effects of carbon monoxide inhalation on tissue protection in ventilator-induced lung injury

Mechanical ventilation causes ventilator-induced lung injury (VILI), and contributes to acute lung injury/acute respiratory distress syndrome (ALI/ARDS), a disease with high morbidity and mortality among critically ill patients. Carbon monoxide (CO) can confer lung protective effects during mechanical ventilation. This study investigates the time dependency of CO therapy with respect to lung protection in animals subjected to mechanical ventilation. For this purpose, mice were ventilated with a tidal volume of 12 ml/kg body weight for 6 h with air in the absence or presence of CO (250 parts per million). Histological analysis of lung tissue sections was used to determine alveolar wall thickening and the degree of lung damage by VILI score. Bronchoalveolar lavage fluid was analyzed for total cellular influx, neutrophil accumulation, and interleukin-1β release. As the main results, mechanical ventilation induced pulmonary edema, cytokine release, and neutrophil recruitment. In contrast, application of CO for 6 h prevented VILI. Although CO application for 3 h followed by 3-h air ventilation failed to prevent lung injury, a further reduction of CO application time to 1 h in this setting provided sufficient protection. Pre-treatment of animals with inhaled CO for 1 h before ventilation showed no beneficial effect. Delayed application of CO beginning at 3 or 5 h after initiation of ventilation, reduced lung damage, total cell influx, and neutrophil accumulation. In conclusion, administration of CO for 6 h protected against VILI. Identical protective effects were achieved by limiting the administration of CO to the first hour of ventilation. Pre-treatment with CO had no impact on VILI. In contrast, delayed application of CO led to anti-inflammatory effects with time-dependent reduction in tissue protection.     

Low molecular weight hyaluronan from stretched lung enhances interleukin-8 expression

Mechanical ventilation has been shown to cause ventilator-induced lung injury (VILI), probably by overdistending or stretching the lung. Hyaluronan (HA), a component of the extracellular matrix, in low molecular weight (LMW) forms has been shown to induce cytokine production. LMW HA is produced by hyaluronan synthase 3 (HAS 3). We found that HAS 3 mRNA expression was upregulated and that LMW HA accumulated in an animal model of VILI. We hypothesized that stretch-induced LMW HA production that causes cytokine release in VILI was dependent on HAS 3 mRNA expression. We explored this hypothesis with in vitro lung cell stretch. Cell stretch induced HAS 3 mRNA expression and LMW HA in fibroblasts. Nonspecific inhibitors of HAS 3 (cyclohexamide and dexamethasone), a nonspecific inhibitor of protein tyrosine kinases (genistein), and a janus kinase 2 inhibitor (AG490) blocked stretch-induced HAS 3 expression and synthesis of LMW HA. Stretch-induced LMW HA from fibroblasts caused a significant dose-dependent increase in interleukin-8 production both in static and stretched epithelial cells. These results indicated that de novo synthesis of LMW HA was induced in lung fibroblasts by stretch via tyrosine kinase signaling pathways, and may play a role in augmenting induction of proinflammatory cytokines in VILI.     

Quantifying the roles of tidal volume and PEEP in the pathogenesis of ventilator-induced lung injury

Management of patients with acute lung injury (ALI) rests on achieving a balance between the gas exchanging benefits of mechanical ventilation and the exacerbation of tissue damage in the form of ventilator-induced lung injury (VILI). Optimizing this balance requires an injury cost function relating injury progression to the measurable pressures, flows, and volumes delivered during mechanical ventilation. With this in mind, we mechanically ventilated naive, anesthetized, paralyzed mice for 4 h using either a low or high tidal volume (Vt) with either moderate or zero positive end-expiratory pressure (PEEP). The derecruitability of the lung was assessed every 15 min in terms of the degree of increase in lung elastance occurring over 3 min following a recruitment maneuver. Mice could be safely ventilated for 4 h with either a high Vt or zero PEEP, but when both conditions were applied simultaneously the lung became increasingly unstable, demonstrating worsening injury. We were able to mimic these data using a computational model of dynamic recruitment and derecruitment that simulates the effects of progressively increasing surface tension at the air–liquid interface, suggesting that the VILI in our animal model progressed via a vicious cycle of alveolar leak, degradation of surfactant function, and increasing tissue stress. We thus propose that the task of ventilating the injured lung is usefully understood in terms of the Vt–PEEP plane. Within this plane, non-injurious combinations of Vt and PEEP lie within a “safe region”, the boundaries of which shrink as VILI develops.     

Role of matrix metalloproteinases in models of macrophage-dependent acute lung injury. Evidence for alveolar macrophage as source of proteinases.

Matrix metalloproteinases (MMPs) have been implicated in the tissue injury seen in neutrophil-dependent models of acute lung injury. However, the role of MMPs in macrophage-dependent models of lung injury is unknown. To address this issue, the macrophage-dependent immunoglobulin A immune complex-induced lung injury model and the macrophage-dependent portion of the lipopolysaccharide-induced acute lung injury model in the rat were assessed for MMP involvement and for the source of these activities. In both models, injury was inhibited by the recombinant human tissue inhibitor of metalloproteinases-2. Bronchoalveolar lavage fluids (BALFs) from injured animals in both models showed increased levels of MMPs. Characterization of MMP production by isolated lung fibroblasts, endothelial cells, type II epithelial cells, and alveolar macrophages revealed that only the macrophage had the same spectrum of MMP activity as seen in the BALF. Further, isolated alveolar macrophages from injured lungs showed evidence of in vivo activation with the release of the same spectrum of MMP activities. Together these studies show that MMPs are produced during macrophage-dependent lung injury, that these MMPs play a role in the development of the lung injury, and that the alveolar macrophage is the likely source of these MMPs.     

Short-term exposure to high-pressure ventilation leads to pulmonary biotrauma and systemic inflammation in the rat

Though often lifesaving, mechanical ventilation itself bears the risk of lung damage [ventilator-induced lung injury (VILI)]. The underlying molecular mechanisms have not been fully elucidated, but stress-induced mediators seem to play an important role in biotrauma related to VILI. Our purpose was to evaluate an animal model of VILI that allows the observation of pathophysiologic changes along with parameters of biotrauma. For VILI induction, rats (n=16) were ventilated with a peak airway pressure (pmax) of 45 cm H2O and end-expiratory pressure (PEEP) of 0 for 20 min, followed by an observation time of 4 h. In the control group (n=8) the animals were ventilated with a pmax of 20 cm H2O and PEEP of 4. High-pressure ventilation resulted in an increase in paCO2 and a decrease in paO2 and mean arterial pressure. Only 4 animals out of 16 survived 4 h and VILI lungs showed severe macroscopic and microscopic damage, oedema and neutrophil influx. High-pressure ventilation increased the cytokine levels of macrophage inflammatory protein-2 and IL-1beta in bronchoalveolar lavage and plasma. VILI also induced pulmonary heat shock protein-70 expression and the activity of matrix metalloproteinases. The animal model used enabled us to observe the effect of high-pressure ventilation on mortality, lung damage/function and biotrauma. Thus, by combining barotrauma with biotrauma, this animal model may be suitable for studying therapeutical approaches to VILI.     

中性粒细胞明胶酶相关脂质运载蛋白在小鼠呼吸机所致肺损伤模型中的表达

 目的：中性粒细胞明胶酶相关脂质运载蛋白（NGAL）是25 kD大小的脂质运载蛋白超家族一员。我们通过观察NGAL在小鼠呼吸机所致肺损伤(VILI)模型中不同机械通气策略对其表达的影响,探讨NGAL是否为VILI新的生物标志物。方法：采用不同的机械通气策略作用于小鼠,构建不同的急性肺损伤模型。利用实时定量RT-PCR观察小鼠肺组织NGAL mRNA的变化;利用Western blotting检测NGAL蛋白在肺组织、血清和支气管肺泡灌洗液（BALF）中的变化;采用免疫组化检测NGAL蛋白在损伤性通气肺组织中的空间定位。结果：不同的通气策略都能引起小鼠NGAL的表达升高,在损伤性通气策略组升高显著,高吸气末峰压组和大潮气量通气组NGAL mRNA和蛋白的表达明显升高,并且在肺组织的上皮细胞、血管内皮细胞和浸润的中性粒细胞中均有表达,提示NGAL是对机械刺激敏感的传感蛋白,可能参与了VILI的发病机制。结论：NGAL可能是一种新的、能早期判断VALI的生物标志物。监测血清和BALF中的NGAL水平有可能成为诊断VILI高危病人的有效工具。     

Expression of gelatinase B and the extracellular matrix metalloproteinase inducer EMMPRIN in benign and malignant pigment cell lesions of the skin.

By the degradative effect on basement membrane collagen type IV, matrix metalloproteinases (MMPs) or gelatinases are important in the early invasion of malignant tumors. These enzymes may be released by the tumor cells themselves or may be derived from nearby fibroblasts that have been stimulated by the extracellular MMP inducer EMMPRIN. We studied the distribution of 92-kd gelatinase B (MMP-9) and of EMMPRIN in 33 benign and 41 malignant, paraffin-embedded pigment cell lesions using immunohistochemistry and monoclonal antibodies. In benign pigment cell lesions, EMMPRIN but not gelatinase B was expressed in cellular blue nevi whereas all other benign lesions, including common blue nevi, were negative. In malignant melanomas (MMs), both gelatinase B and EMMPRIN were variably expressed in the pure and invasive radial growth phase but not in the vertical growth phase. All lentigo maligna cases and all metastatic lesions were negative. Of MMs with thickness < 1.6 mm, 63% expressed gelatinase B and 70% expressed EMMPRIN, whereas in MMs with > 1.6 mm thickness, only 10% expressed gelatinase B and only 25% expressed EMMPRIN. We conclude that early invasion of MM is associated with de novo expression of gelatinase B and EMMPRIN by neoplastic melanocytes. Expression of EMMPRIN and MMP-9 may be partly responsible for the stromal changes observed in thin MM. Their absence in the vertical growth phase and in metastatic lesions suggests that other factors are involved in tissue degradation during later stages of tumor progression in MM. The lack of both gelatinase B and EMMPRIN in lentigo maligna may contribute to the indolent behavior of this type of pigment cell lesion.     

Tumorigenic potential of extracellular matrix metalloproteinase inducer

Extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein present on the cancer cell plasma membrane, enhances fibroblast synthesis of matrix metalloproteinases (MMPs). The demonstration that peritumoral fibroblasts synthesize most of the MMPs in human tumors rather than the cancer cells themselves has ignited interest in the role of EMMPRIN in tumor dissemination. In this report we have demonstrated a role for EMMPRIN in cancer progression. Human MDA-MB-436 breast cancer cells, which are tumorigenic but slow growing in vivo, were transfected with EMMPRIN cDNA and injected orthotopically into mammary tissue of female NCr nu/nu mice. Green fluorescent protein was used to visualize metastases. In three experiments, breast cancer cell clones transfected with EMMPRIN cDNA were considerably more tumorigenic and invasive than plasmid-transfected cancer cells. Increased gelatinase A and gelatinase B expression (demonstrated by in situ hybridization and gelatin substrate zymography) was demonstrated in EMMPRIN-enhanced tumors. In contrast to de novo breast cancers in humans, human tumors transplanted into mice elicited minimal stromal or inflammatory cell reactions. Based on these experimental studies and our previous demonstration that EMMPRIN is prominently displayed in human cancer tissue, we propose that EMMPRIN plays an important role in cancer progression by increasing synthesis of MMPs.     

Structural characterization of the mesangial cell type IV collagenase and enhanced expression in a model of immune complex-mediated glomerulonephritis.

Secretion of glomerular cell-derived matrix metalloproteinases (MMPs) and their specific inhibitors, TIMP-1,2, may play an important role in the turnover of the glomerular extracellular matrix under basal and pathologic conditions. A 66-68 kd MMP secreted by cultured mesangial cells (MC) with activity against Type IV collagen and gelatin was purified and shown by amino-acid sequence analysis to be identical with a Type IV collagenase/gelatinase secreted by certain transformed tumor cell lines. The expression of the mesangial MMP in vivo was limited within the kidney to a small subset of the intrinsic glomerular mesangial cell population. After induction of acute anti-Thy 1.1 glomerulonephritis, there was a large increment in the number of Type IV collagenase-secreting MC, temporally coincident with the development of mesangial hypercellularity. The expression of the MMP inhibitor protein, TIMP-1, was not changed over this period. Ultrastructural studies localized the mesangial MMP to areas of evolving mesangiolysis and at sites of glomerular basement membrane disruption. Enhanced expression of the mesangial cell-derived Type IV collagenase may contribute to the evolution of glomerular injury in this model of immune complex-mediated glomerulonephritis or may be involved in the extensive matrix remodeling process that accompanies this form of glomerular injury.     

Effect and Mechanism Study of Sodium Houttuyfonate on Ventilator-Induced Lung Injury by Inhibiting ROS and Inflammation

PurposeVentilator-induced lung injury (VILI) is a serious complication of mechanical ventilation (MV) that increases morbidity and mortality of patients receiving ventilator treatment. This study aimed to reveal the molecular mechanism of sodium houttuyfonate (SH) on VILI.Materials and MethodsThe male mice VILI model was established by high tidal volume ventilation. The cell model was established by performing cell stretch (CS) experiments on murine respiratory epithelial cells MLE-15. In addition, the JNK activator Anisomycin and JNK inhibitor SP600125 were used on VILI mice and CS-treated cells.ResultsVILI modeling damaged the structural integrity, increased apoptosis and wet-to-dry (W/D) ratio, enhanced the levels of inflammatory factors, reactive oxygen species (ROS) and malonaldehyde (MDA), and activated JNK pathway in lung tissues. SH gavage alleviated lung injury, decreased apoptosis and W/D ratio, and reduced levels of inflammatory factors, ROS and MDA, and p-JNK/JNK expression of lung tissues in VILI mice. However, activation of JNK wiped the protective effect of SH on VILI. Contrary results were found in experiments with JNK inhibitor SP600125.ConclusionSH relieved VILI by inhibiting the ROS-mediated JNK pathway.     

Differential expression of extracellular matrix metalloproteinase inducer (CD147) in normal and ulcerated corneas: role in epithelio-stromal interactions and matrix metalloproteinase induction

Extracellular matrix metalloproteinase inducer (EMMPRIN) was originally identified on the tumor cell surface as an inducer of matrix metalloproteinase (MMP) production in neighboring fibroblasts. Here we demonstrate a role for EMMPRIN in MMP induction during corneal wound healing. MMP and EMMPRIN expression was analyzed in normal and ulcerated human corneas, as well as in corneal epithelial and stromal cells in culture using confocal microscopy, zymography, immunoblots, and real-time polymerase chain reaction. In normal cornea EMMPRIN was predominantly expressed in the epithelium but was markedly induced in the anterior stroma of ulcerated corneas. This coincided with MMP-2 induction that co-localized with EMMPRIN at the epithelio-stromal boundary. The role of epithelial-stromal interaction in MMP induction was investigated in an in vitro co-culture system and demonstrated an induction and co-localization of EMMPRIN and MMP-2 in the fibroblasts at the interface with epithelial cells. Direct contact of fibroblasts with EMMPRIN-containing purified epithelial cell membranes also induced MMP-1, MMP-2, and EMMPRIN and this was inhibited by a blocking anti-EMMPRIN antibody, suggesting that EMMPRIN was primarily responsible for this induction. These findings, and the up-regulation of EMMPRIN by epidermal growth factor and transforming growth factor-beta, demonstrate a role for EMMPRIN in wound healing and suggest that sustained local up-regulation of EMMPRIN and MMPs in chronic situations in which healing is delayed may lead to excessive matrix degradation and corneal melts.     

EGFR-p38 MAPK信号通路参与机械通气肺损伤大鼠肺组织HMGB1的表达

 目的：研究表皮生长因子受体(epidermal growth factor receptor, EGFR)-p38丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)信号通路在机械通气肺损伤(ventilator-induced lung injury, VILI)大鼠肺组织高迁移率族盒蛋白1 (high mobility group box 1 protein, HMGB1)表达中的作用。方法：健康SD大鼠32只随机分为4组：对照组（A组）不行机械通气,保留自主呼吸;小潮气量通气组（B组）潮气量(VT)为8 mL/kg;大潮气量通气组（C组）VT为40 mL/kg;大潮气量通气+EGFR拮抗剂AG-1478组为D组。机械通气4 h后处死动物,测定支气管肺泡灌洗液中总蛋白水平、白细胞计数以及肺湿干重比值(W/D)和髓过氧化物酶(MPO)活性,采用HE染色观察肺组织病理学改变,Western blotting方法检测肺组织磷酸化EGFR、磷酸化p38和HMGB1蛋白表达,RT-PCR方法检测EGFR mRNA的表达。结果：通气4 h后,与A组比较,C组肺组织病理学改变明显,总蛋白水平、白细胞计数、肺W/D、MPO活性、EGFR mRNA表达和磷酸化水平、p38磷酸化水平以及HMGB1蛋白表达均显著增加（P<0.05）;与C组比较,D组上述各项指标的变化均显著降低（P<0.05）。结论：大潮气量机械通气可引起大鼠急性肺损伤,其机制可能与通过EGFR-p38 MAPK信号通路介导HMGB1蛋白的表达有关。     

Local blockage of EMMPRIN impedes pressure ulcers healing in a rat model

Excessive extracellular matrix degradation caused by the hyperfunction of matrix metalloproteinases (MMPs) has been implicated in the failure of pressure ulcers healing. EMMPRIN, as a widely expressed protein, has emerged as an important regulator of MMP activity. We hypothesize that EMMPRIN affects the process of pressure ulcer healing by modulating MMP activity. In the rat pressure ulcer model, the expression of EMMPRIN in ulcers detected by Western blot was elevated compared with that observed in normal tissue. To investigate the role of EMMPRIN in regulating ulcer healing, specific antibodies against EMMPRIN were used via direct administration on the pressure ulcer. Local blockage of EMMPRIN resulted in a poor ulcer healing process compared with control ulcers, which was the opposite of our expectation. Furthermore, inhibiting EMMPRIN minimally impacted MMP activity. However, the collagen content in the pressure ulcer was reduced in the EMMPRIN treated group. Angiogenesis and the expression of angiogenic factors in pressure ulcers were also reduced by EMMPRIN local blockage. The results in the present study indicate a novel effect of EMMPRIN in the regulation of pressure ulcer healing by controlling the collagen contents and angiogenesis rather than MMPs activity.     

Genetic polymorphisms of matrix metalloproteinases in lung, breast and colorectal cancer

The matrix metalloproteinases (MMPs) can degrade various components of the extracellular matrix and are implicated in the development and progression of cancer. There is evidence suggesting an association of MMP gene polymorphisms with cancer susceptibility and/or metastasis. This paper reviews the findings on several single nucleotide polymorphisms in the collagenase, stromelysin and gelatinase genes in lung cancer, breast cancer and colorectal cancer.     

Extracellular matrix metalloproteinase inducer in interstitial pneumonias

Extracellular matrix metalloproteinase inducer (EMMPRIN), a glycosylated transmembrane protein that induces matrix metalloproteinases (MMPs), is minimally expressed in the normal adult lung. We previously reported that it is up-regulated in murine bleomycin-induced lung injury. In this study, we determined the expression of EMMPRIN and its association with MMP-2, MMP-7, and MMP-9 in interstitial pneumonias (IPs). We performed immunohistochemistry for EMMPRIN and MMPs on lung tissue from 22 subjects with various IPs. We did bronchoalveolar lavage (BAL) on 9 of these subjects and 13 others with IPs to measure the soluble EMMPRIN in BAL fluid. For comparison, immunohistochemistry or BAL was done on 14 subjects without IPs. The staining intensity for each protein was scored from 0 to 3 in various epithelial cell types. Soluble EMMPRIN in BAL fluid was measured by an enzyme-linked immunosorbent assay. Extracellular matrix metalloproteinase inducer was prominent in abnormal epithelial cells. It was more prominent in hyperplastic type II cells, compared with epithelium in alveolar bronchiolization. It was also elevated in BAL fluid from the subjects with IPs. Matrix metalloproteinases were expressed in cells expressing EMMPRIN, although the profile of MMPs varied among the different abnormal epithelial cell types with MMP-2 and MMP-7 in hyperplastic type II cells and MMP-7 and MMP-9 in cells showing squamous metaplasia and cells comprising bronchiolization. These results suggest a role of EMMPRIN in reepithelialization in IPs.     

Neutrophil proteinases in hydrochloric acid- and endotoxin-induced acute lung injury: evaluation of interstitial protease activity by in situ zymography

We investigated the role of polymorphonuclear neutrophil (PMN) proteinases, elastase, and gelatinase B in rat models of acute lung injury. Three groups of rats were studied 6 hours after unilateral instillation of hydrochloric acid (HCl; 0.1 N), lipopolysaccharide (LPS) (4 microg), or saline. The results demonstrated that HCl-induced lung injury, as compared with LPS-induced lung injury, was associated with an increase in permeability (wet/dry weight ratio and proteins in bronchoalveolar lavage fluid). In contrast, there was similar PMN recruitment (in bronchoalveolar lavage fluid and myeloperoxidase activity in lung homogenates) and similar proteinase exocytosis (residual alveolar PMN content of elastase and gelatinase B) in both types of lung injury. In situ zymography, evaluating interstitial protease/inhibitor balance, demonstrated a decrease in gelatinolytic activity in both HCl- and LPS-injured lungs compared with normal lung. The increase in interleukin 6 concentration in lung homogenates, which is observed after both injuries compared with saline-instilled animals, could be involved in up-regulation of tissue inhibitor of matrix metalloproteinase-1, shown by immunocytochemistry to participate in antiproteinase excess. Neither inhibition of alveolar neutrophil influx using a leukocyte elastase inhibitor (EPI-hNE-4) nor inhibition of gelatinase activities by recombinant adenovirus for the human tissue inhibitor of matrix metalloproteinase 1 gene transfer decreased lung edema in HCl-induced injury. These data suggest that PMN proteinases do not contribute to HCl-induced acute lung injury in rats.