The Role of Heparanase in Pulmonary Cell Recruitment in Response to an Allergic but Not Non-Allergic Stimulus

Heparanase is an endo-β-glucuronidase that specifically cleaves heparan sulfate proteoglycans in the extracellular matrix. Expression of this enzyme is increased in several pathological conditions including inflammation. We have investigated the role of heparanase in pulmonary inflammation in the context of allergic and non-allergic pulmonary cell recruitment using heparanase knockout (Hpa^-/-) mice as a model. Following local delivery of LPS or zymosan, no significant difference was found in the recruitment of neutrophils to the lung between Hpa^-/- and wild type (WT) control. Similarly neutrophil recruitment was not inhibited in WT mice treated with a heparanase inhibitor. However, in allergic inflammatory models, Hpa^-/- mice displayed a significantly reduced eosinophil (but not neutrophil) recruitment to the airways and this was also associated with a reduction in allergen-induced bronchial hyperresponsiveness, indicating that heparanase expression is associated with allergic reactions. This was further demonstrated by pharmacological treatment with a heparanase inhibitor in the WT allergic mice. Examination of lung specimens from patients with different severity of chronic obstructive pulmonary disease (COPD) found increased heparanase expression. Thus, it is established that heparanase contributes to allergen-induced eosinophil recruitment to the lung and could provide a novel therapeutic target for the development of anti-inflammatory drugs for the treatment of asthma and other allergic diseases.     

肺部微生物组通过炎症反应介导慢性阻塞性肺疾病转化为肺癌的研究进展

肺部微生物组存在于呼吸道和实质组织中,通过菌群紊乱、代谢产物、炎症反应、免疫反应、基因毒性等方面介导肺部损伤。随着肺部微生物组的深入研究,发现肺部微生物组的相关活动与慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD)和肺癌息息相关,能够促使COPD向肺癌的转变。本文主要介绍了肺部微生物组稳态及其通过炎症反应导致COPD和肺癌,重点探讨了肺部微生物组如何通过炎症反应介导COPD转化为肺癌,以期为COPD和肺癌的临床预防、优化治疗以及新型药物设计提供新的理论依据。     

Heparanase expression and localization in different types of human lung cancer

Background

Heparanase is the only known mammalian glycosidase capable of cleaving heparan sulfate chains. The expression of this enzyme has been associated with tumor development because of its ability to degrade extracellular matrix and promote cell invasion.

Methods

We analyzed heparanase expression in lung cancer samples to understand lung tumor progression and malignancy. Of the samples from 37 patients, there were 14 adenocarcinomas, 13 squamous cell carcinomas, 5 large cell carcinomas, and 5 small cell carcinomas. Immunohistochemistry was performed to ascertain the expression and localization of heparanase.

Results

All of the tumor types expressed heparanase, which was predominantly localized within the cytoplasm and nucleus. Significant enzyme expression was also observed in cells within the tumor microenvironment, such as fibroblasts, epithelial cells, and inflammatory cells. Adenocarcinomas exhibited the strongest heparanase staining intensity and the most widespread heparanase distribution. Squamous cell carcinomas, large cell carcinomas, and small cell carcinomas had a similar subcellular distribution of heparanase to adenocarcinomas but the distribution was less widespread. Heparanase expression tended to correlate with tumor node metastasis (TNM) staging in non-small cell lung carcinoma.

Conclusion

In this study, we showed that heparanase was localized to the cytoplasm and nucleus of tumor cells and to cells within the microenvironment in different types of lung cancer. This enzyme exhibited a differential distribution based on the type of lung tumor.General significanceElucidating the heparanase expression patterns in different types of lung cancer increased our understanding of the crucial role of heparanase in lung cancer biology. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Interleukin 1 bioactivity in the lungs of rats with monocrotaline-induced pulmonary hypertension

A single subcutaneous injection of monocrotaline in rats provokes lung injury, inflammation, and progressive pulmonary hypertension. The specific mediators of the lung injury and inflammation and the relation of these events to the ensuing hypertensive pulmonary vascular disease are not understood. Since the monokine interleukin 1 (IL-1) has been implicated in acute inflammatory reactions, the present study tested the hypotheses that monocrotaline promotes the appearance of IL-1 in the bronchoalveolar spaces of treated rats and that accumulation of the monokine coincides temporally with development of lung injury, inflammation, and/or pulmonary hypertension. As expected, monocrotaline administration was associated with an early phase of pulmonary edema, manifest at Day 7 post-treatment as an increase in the lung wet-to-dry weight ratio, followed at Day 14 post-treatment by development of pulmonary hypertension as evidenced by progressive right ventricular hypertrophy. Lung inflammation also was present at Days 14 and 21 after monocrotaline as indicated by the accumulation of leukocytes in the bronchoalveolar lavage fluid and by an increase in the lung tissue activity of the granulocyte-specific enzyme myeloperoxidase. Interleukin 1, bioassayed in bronchoalveolar lavage fluid using the standard D10 T-cell assay system, was increased slightly at Day 4 postmonocrotaline, returned to baseline at Day 7, and was markedly elevated at Days 14 and 21 after monocrotaline treatment. These observations indicate that increases in the bronchoalveolar lavage fluid content of IL-1 bioactivity are temporally related to the evolution of monocrotaline-induced lung injury, inflammation, and pulmonary hypertension and suggest that the monokine may play a pathogenetic role in these events.     

Unscheduled apoptosis during acute inflammatory lung injury

Apoptosis is a mode of cell death currently thought to occur in the absence of inflammation. In contrast, inflammation follows unscheduled events such as acute tissue injury which results in necrosis, not apoptosis. We examined the relevance of this paradigm in three distinct models of acute lung injury; hyperoxia, oleic acid, and bacterial pneumonia. In every case, it was found that apoptosis is actually a prominent component of the acute and inflammatory phase of injury. Moreover, using strains of mice that are differentially sensitive to hyperoxic lung injury we observed that the percent of apoptotic cells was well correlated with the severity of lung injury. These observations suggest that apoptosis may be one of the biological consequences during acute injury and the failure to remove these apoptotic cells may also contribute to the inflammatory response.     

Deciphering Mode of Action of Heparanase Using Structurally Defined Oligosaccharides

Heparan sulfate (HS) is a highly sulfated polysaccharide that serves many biological functions, including regulating cell growth and inflammatory responses as well as the blood coagulation process. Heparanase is an enzyme that cleaves HS and is known to display a variety of pathophysiological effects in cancer, diabetes, and Alzheimer disease. The link between heparanase and diseases is a result of its selective cleavage of HS, which releases smaller HS fragments to enhance cell proliferation, migration, and invasion. Despite its importance in pathological diseases, the structural cues in HS that direct heparanase cleavage and the steps of HS depolymerization remain unknown. Here, we sought to probe the substrate specificity of heparanase using a series of structurally defined oligosaccharide substrates. The sites of heparanase cleavage on the oligosaccharide substrates were determined by mass spectrometry and gel permeation chromatography. We discovered that heparanase cleaves the linkage of glucuronic acid linked to glucosamine carrying 6-O-sulfo groups. Furthermore, our findings suggest that heparanase displays different cleavage modes by recognizing the structures of the nonreducing ends of the substrates. Our results deepen the understanding of the action mode of heparanase.     

Heparanase expression in invasive trophoblasts and acute vascular damage

Heparan sulfate proteoglycans play a pivotal role in tissue function, development, inflammation, and immunity. We have identified a novel cDNA encoding human heparanase, an enzyme thought to cleave heparan sulfate in physiology and disease, and have located the HEP gene on human chromosome 4q21. Monoclonal antibodies against human heparanase located the enzyme along invasive extravillous trophoblasts of human placenta and along endothelial cells in organ xenografts targeted by hyperacute rejection, both sites of heparan sulfate digestion. Heparanase deposition was evident in arterial walls in normal tissues; however, vascular heparan sulfate cleavage was coincident with heparanase enzyme during inflammatory episodes. These findings suggest that heparanase elaboration and control of catalytic activity may contribute to the development and pathogenesis of vascular disease and suggest that heparanase intervention might be a useful therapeutic target.     

Involvement of heparanase in atherosclerosis and other vessel wall pathologies

Heparanase, the sole mammalian endoglycosidase degrading heparan sulfate, is causally involved in cancer metastasis, angiogenesis, inflammation and kidney dysfunction. Despite the wide occurrence and impact of heparan sulfate proteoglycans in vascular biology, the significance of heparanase in vessel wall disorders is underestimated. Blood vessels are highly active structures whose morphology rapidly adapts to maintain vascular function under altered systemic and local conditions. In some pathologies (restenosis, thrombosis, atherosclerosis) this normally beneficial adaptation may be detrimental to overall function. Enzymatic dependent and independent effects of heparanase on arterial structure mechanics and repair closely regulate arterial compliance and neointimal proliferation following endovascular stenting. Additionally, heparanase promotes thrombosis after vascular injury and contributes to a pro-coagulant state in human carotid atherosclerosis. Importantly, heparanase is closely associated with development and progression of atherosclerotic plaques, including stable to unstable plaque transition. Consequently, heparanase levels are markedly increased in the plasma of patients with acute myocardial infarction. Noteworthy, heparanase activates macrophages, resulting in marked induction of cytokine expression associated with plaque progression towards vulnerability. Together, heparanase emerges as a regulator of vulnerable lesion development and potential target for therapeutic intervention in atherosclerosis and related vessel wall complications.     

Inter-alpha-trypsin inhibitor attenuates complement activation and complement-induced lung injury

Complement activation is a central component of inflammation and sepsis and can lead to significant tissue injury. Complement factors are serum proteins that work through a cascade of proteolytic reactions to amplify proinflammatory signals. Inter-alpha-trypsin inhibitor (IaI) is an abundant serum protease inhibitor that contains potential complement-binding domains, and has been shown to improve survival in animal sepsis models. We hypothesized that IaI can bind complement and inhibit complement activation, thus ameliorating complement-dependent inflammation. We evaluated this hypothesis with in vitro complement activation assays and in vivo in a murine model of complement-dependent lung injury. We found that IaI inhibited complement activation through the classical and alternative pathways, inhibited complement-dependent phagocytosis in vitro, and reduced complement-dependent lung injury in vivo. This novel function of IaI provides a mechanistic explanation for its observed salutary effects in sepsis and opens new possibilities for its use as a treatment agent in inflammatory diseases.     

Susceptibility to neoplastic and non-neoplastic pulmonary diseases in mice: genetic similarities

Chronic inflammation predisposes toward many types of cancer. Chronic bronchitis and asthma, for example, heighten the risk of lung cancer. Exactly which inflammatory mediators (e.g., oxidant species and growth factors) and lung wound repair processes (e.g., proangiogenic factors) enhance pulmonary neoplastic development is not clear. One approach to uncover the most relevant biochemical and physiological pathways is to identify genes underlying susceptibilities to inflammation and to cancer development at the same anatomic site. Mice develop lung adenocarcinomas similar in histology, molecular characteristics, and histogenesis to this most common human lung cancer subtype. Over two dozen loci, called Pas or pulmonary adenoma susceptibility, Par or pulmonary adenoma resistance, and Sluc or susceptibility to lung cancer genes, regulate differential lung tumor susceptibility among inbred mouse strains as assigned by QTL (quantitative trait locus) mapping. Chromosomal sites that determine responsiveness to proinflammatory pneumotoxicants such as ozone (O3), particulates, and hyperoxia have also been mapped in mice. For example, susceptibility QTLs have been identified on chromosomes 17 and 11 for O3-induced inflammation (Inf1, Inf2), O3-induced acute lung injury (Aliq3, Aliq1), and sulfate-associated particulates. Sites within the human and mouse genomes for asthma and COPD phenotypes have also been delineated. It is of great interest that several susceptibility loci for mouse lung neoplasia also contain susceptibility genes for toxicant-induced lung injury and inflammation and are homologous to several human asthma loci. These QTLs are described herein, candidate genes are suggested within these sites, and experimental evidence that inflammation enhances lung tumor development is provided.     

Erythromycin and clarithromycin modulation of growth factor-induced expression of heparanase mRNA on human lung cancer cells in vitro.

Heparanase activity is correlated with the metastatic potential of several cancer cells and is a key enzyme in the breakdown of tissue barriers. It is also involved in the regulation of growth factor and cytokine activity. However, little is known about the factors that induce heparanase in cancer cells. We investigated the effect of three growth factors, platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF), on heparanase mRNA induction in lung cancer cells in vitro. In addition, we examined the effect of erythromycin (EM) and clarithromycin (CAM), which are 14-membered ring macrolide antibiotics that act as biological response modifiers, on the expression of heparanase mRNA induced by growth factors. PDGF, HGF and bFGF stimulated cell migration activity and enhanced the expression of heparanase mRNA in the human lung adenocarcinoma cell line A549. Via different mechanisms, EM and CAM modulate the induction by these factors of heparanase mRNA expression on A549 cells. EM also significantly suppressed A549 cell migration induced by PDGF and HGF, and CAM significantly suppressed A549cell migration induced by bFGF. The results suggest that the growth factors PDGF, HGF and bFGF are important inducers of heparanase in potentially invasive and metastatic cancer cells. The suppressive effect of heparanase mRNA expression by EM and CAM may have interestingtherapeutic applications in the prevention of metastasis.     

Airway epithelium controls lung inflammation and injury through the NF-kappa B pathway

Although airway epithelial cells provide important barrier and host defense functions, a crucial role for these cells in development of acute lung inflammation and injury has not been elucidated. We investigated whether NF-kappaB pathway signaling in airway epithelium could decisively impact inflammatory phenotypes in the lungs by using a tetracycline-inducible system to achieve selective NF-kappaB activation or inhibition in vivo. In transgenic mice that express a constitutively active form of IkappaB kinase 2 under control of the epithelial-specific CC10 promoter, treatment with doxycycline induced NF-kappaB activation with consequent production of a variety of proinflammatory cytokines, high-protein pulmonary edema, and neutrophilic lung inflammation. Continued treatment with doxycycline caused progressive lung injury and hypoxemia with a high mortality rate. In contrast, inducible expression of a dominant inhibitor of NF-kappaB in airway epithelium prevented lung inflammation and injury resulting from expression of constitutively active form of IkappaB kinase 2 or Escherichia coli LPS delivered directly to the airways or systemically via an osmotic pump implanted in the peritoneal cavity. Our findings indicate that the NF-kappaB pathway in airway epithelial cells is critical for generation of lung inflammation and injury in response to local and systemic stimuli; therefore, targeting inflammatory pathways in airway epithelium could prove to be an effective therapeutic strategy for inflammatory lung diseases.     

PG545, a Heparan Sulfate Mimetic,Reduces Heparanase Expression In Vivo,Blocks Spontaneous Metastases and Enhances Overall Survival in the 4T1 Breast Carcinoma Model

PG545 is a clinically relevant heparan sulfate (HS) mimetic which, in addition to possessing anti-angiogenic properties, also acts as a heparanase inhibitor which may differentiate its mechanism(s) of action from approved angiogenesis inhibitors. The degradation of HS by heparanase has been strongly implicated in cell dissemination and the metastatic process. Thus, the anti-metastatic activity of PG545 has been linked to the enzymatic function of heparanase – the only endoglycosidase known to cleave HS, an important component of the extracellular matrix (ECM) which represents a potential avenue for therapeutic intervention for certain metastatic cancer indications. Recent concerns raised about the paucity of overall survival as an endpoint in mouse models of clinically relevant metastasis led us to examine the effect of PG545 on the progression of both primary tumor growth and the spontaneously metastasizing disease in the 4T1 syngeneic breast carcinoma model in a non-surgical and surgical (mastectomy) setting. PG545 significantly inhibited primary tumor growth but importantly also inhibited lung metastasis in treated mice, an effect not observed with the tyrosine kinase inhibitor sorafenib. Importantly, PG545 significantly enhanced overall survival compared to vehicle control and the sorafenib group, suggesting PG545’s inhibitory effect on heparanase is indeed a critical attribute to induce anti-metastatic activity. In addition to blocking a common angiogenic signalling pathway in tumor cells, the expression of heparanase in the primary tumor and lung was also significantly reduced by PG545 treatment. These results support the ongoing development of PG545 and highlight the potential utility in metastatic disease settings.     

The Synthetic Antimicrobial Peptide 19-2.5 Interacts with Heparanase and Heparan Sulfate in Murine and Human Sepsis

Heparanase is an endo-β-glucuronidase that cleaves heparan sulfate side chains from their proteoglycans. Thereby, heparanase liberates highly potent circulating heparan sulfate-fragments (HS-fragments) and triggers the fatal and excessive inflammatory response in sepsis. As a potential anti-inflammatory agent for sepsis therapy, peptide 19–2.5 belongs to the class of synthetic anti-lipopolysaccharide peptides; however, its activity is not restricted to Gram-negative bacterial infection. We hypothesized that peptide 19–2.5 interacts with heparanase and/or HS, thereby reducing the levels of circulating HS-fragments in murine and human sepsis. Our data indicate that the treatment of septic mice with peptide 19–2.5 compared to untreated control animals lowers levels of plasma heparanase and circulating HS-fragments and reduces heparanase activity. Additionally, mRNA levels of heparanase in heart, liver, lung, kidney and spleen are downregulated in septic mice treated with peptide 19–2.5 compared to untreated control animals. In humans, plasma heparanase level and activity are elevated in septic shock. The ex vivo addition of peptide 19–2.5 to plasma of septic shock patients decreases heparanase activity but not heparanase level. Isothermal titration calorimetry revealed a strong exothermic reaction between peptide 19–2.5 and heparanase and HS-fragments. However, a saturation character has been identified only in the peptide 19–2.5 and HS interaction. In conclusion, the findings of our current study indicate that peptide 19–2.5 interacts with heparanase, which is elevated in murine and human sepsis and consecutively attenuates the generation of circulating HS-fragments in systemic inflammation. Thus, peptide 19–2.5 seems to be a potential anti-inflammatory agent in sepsis.     

Understanding SARS-CoV-2-Mediated Inflammatory Responses: From Mechanisms to Potential Therapeutic Tools

Currently there is no effective antiviral therapy for SARS-CoV-2 infection, which frequently leads to fatal inflammatory responses and acute lung injury. Here, we discuss the various mechanisms of SARS-CoV-mediated inflammation. We also assume that SARS-CoV-2 likely shares similar inflammatory responses. Potential therapeutic tools to reduce SARS-CoV-2-induced inflammatory responses include various methods to block FcR activation. In the absence of a proven clinical FcR blocker, the use of intravenous immunoglobulin to block FcR activation may be a viable option for the urgent treatment of pulmonary inflammation to prevent severe lung injury. Such treatment may also be combined with systemic anti-inflammatory drugs or corticosteroids. However, these strategies, as proposed here, remain to be clinically tested for effectiveness.     

Transgenic or tumor-induced expression of heparanase upregulates sulfation of heparan sulfate

Heparan sulfate proteoglycans (HSPGs) interact with numerous proteins of importance in animal development and homeostasis. Heparanase, which is expressed in normal tissues and upregulated in angiogenesis, cancer and inflammation, selectively cleaves beta-glucuronidic linkages in HS chains. In a previous study, we transgenically overexpressed heparanase in mice to assess the overall effects of heparanase on HS metabolism. Metabolic labeling confirmed extensive fragmentation of HS in vivo. In the current study we found that in liver showing excessive heparanase overexpression, HSPG turnover is accelerated along with upregulation of HS N- and O-sulfation, thus yielding heparin-like chains without the domain structure typical of HS. Heparanase overexpression in other mouse organs and in human tumors correlated with increased 6-O-sulfation of HS, whereas the domain structure was conserved. The heavily sulfated HS fragments strongly promoted formation of ternary complexes with fibroblast growth factor 1 (FGF1) or FGF2 and FGF receptor 1. Heparanase thus contributes to regulation of HS biosynthesis in a way that may promote growth factor action in tumor angiogenesis and metastasis.     

Metalloproteinase and growth factor interactions: do they play a role in pulmonary fibrosis?

Chronic lung disease due to interstitial fibrosis can be a consequence of acute lung injury and inflammation. The inflammatory response is mediated through the migration of inflammatory cells, actions of proinflammatory cytokines, and the secretion of matrix-degrading proteinases. After the initial inflammatory insult, successful healing of the lung may occur, or alternatively, dysregulated tissue repair can result in scarring and fibrosis. On the basis of recent insights into the mechanisms underlying acute lung injury and its long-term consequences, data suggest that proteinases, such as the matrix metalloproteinases (MMPs), may not only be involved in the breakdown and remodeling that occurs during the injury but may also cause the release of growth factors and cytokines known to influence growth and differentiation of target cells within the lung. Through the release of and activation of fibrosis-promoting cytokines and growth factors such as transforming growth factor-beta1, tumor necrosis factor-alpha, and insulin-like growth factors by MMPs, we propose that these metalloproteinases may be integral to the initiation and progression of pulmonary fibrosis.     

Eicosanoids and the endogenous control of acute inflammatory resolution

Inflammation is a formidable ally in the constant battle against infection, cancer and tissue injury. It is a primordial response that protects against injury and restores damaged tissue to its normal physiological functioning. In fact, our wellbeing and survival depends upon its efficiency and carefully balanced control. In general, the innate inflammatory response initiates within minutes and, if all is well, resolves within hours. In contrast, chronic inflammation persists for weeks, months or even years. Here, we are going to discuss the key endogenous checkpoints necessary for mounting an effective yet limited inflammatory response and the crucial biochemical pathways necessary to prevent its persistence. In this setting, the biochemical synthesis of key pro-resolution eicosanoids as well as their mode of action in self-limiting inflammation will be discussed.     

Molecular properties and involvement of heparanase in cancer progression and normal development

Heparan sulfate proteoglycans (HSPGs) play a key role in the self-assembly, insolubility and barrier properties of basement membranes and extracellular matrices. Hence, cleavage of heparan sulfate (HS) affects the integrity and functional state of tissues and thereby fundamental normal and pathological phenomena involving cell migration and response to changes in the extracellular microenvironment. Here, we describe the molecular properties, expression and function of a human heparanase, degrading HS at specific intrachain sites. The enzyme is synthesized as a latent approximately 65 kDa protein that is processed at the N-terminus into a highly active approximately 50 kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and human tumor tissues. Overexpression of the heparanase cDNA in low-metastatic tumor cells conferred a high metastatic potential in experimental animals, resulting in an increased rate of mortality. The heparanase enzyme also releases ECM-resident angiogenic factors in vitro and its overexpression induces an angiogenic response in vivo. Heparanase may thus facilitate both tumor cell invasion and neovascularization, both critical steps in cancer progression. The enzyme is also involved in cell migration associated with inflammation and autoimmunity. The unexpected identification of a single predominant functional heparanase suggests that the enzyme is a promising target for drug development. In fact, treatment with heparanase inhibitors markedly reduces tumor growth, metastasis and autoimmune disorders in animal models. Studies are underway to elucidate the involvement of heparanase in normal processes such as implantation, embryonic development, morphogenesis, tissue repair, inflammation and HSPG turnover. Heparanase is the first functional mammalian HS-degrading enzyme that has been cloned, expressed and characterized. This may lead to identification and cloning of other glycosaminoglycan degrading enzymes, toward a better understanding of their involvement and significance in normal and pathological processes.