Type B Niemann-Pick disease (NPD) is a multiorgan system disorder caused by a genetic deficiency of acid sphingomyelinase (ASM), for which lung is an important and challenging therapeutic target. In this study, we designed and evaluated new delivery vehicles for enzyme replacement therapy of type B NPD, consisting of polystyrene and poly(lactic-coglycolic) acid polymer nanocarriers targeted to intercellular adhesion molecule (ICAM)-1, an endothelial surface protein up-regulated in many pathologies, including type B NPD. Real-time vascular imaging using intravital microscopy and postmortem imaging of mouse organs showed rapid, uniform, and efficient binding of fluorescently labeled ICAM-1-targeted ASM nanocarriers (anti-ICAM/ASM nanocarriers) to endothelium after i.v. injection in mice. Fluorescence microscopy of lung alveoli actin, tissue histology, and 125

Recent studies have suggested that macrophage-derived metalloproteases are the critical mediators of cigarette smoke-induced emphysema, in contrast to earlier hypotheses that this process was mediated by neutrophil elastase. To determine whether smoke can acutely induce connective tissue breakdown in the lung and to examine the mediators of this process, we exposed C57-BL/6 mice to whole cigarette smoke and used high-performance liquid chromatography to examine lavage fluid levels of desmosine (DES), a marker of elastin breakdown, and hydroxyproline (HP), a marker of collagen breakdown. Smoke produced a dose-response increase in lavage neutrophils, DES, and HP, but not lavage macrophages (MACs). This effect was evident by 6 h after exposure to two cigarettes. Pretreatment with an antibody against polymorphonuclear leukocytes (PMNs) reduced lavage PMNs to undetectable levels after smoke exposure, did not affect MAC numbers, and prevented increases in lavage DES and HP. Intraperitoneal injection of a commercial human alpha1-antitrypsin (alpha1AT) 24 h before smoke exposure increased serum alpha1AT levels approximately 3-fold and completely abolished smoke-induced connective tissue breakdown as well as the increase in lavage PMNs, again without affecting MAC numbers. We conclude that in this model cigarette smoke can acutely induce connective tissue breakdown and that this effect is mediated by neutrophil-derived serine proteases, most likely neutrophil elastase. Exogenous alpha1AT is protective and appears to inhibit both matrix degradation and PMN influx, suggesting that alpha1AT has anti-inflammatory as well as antiproteolytic effects in this system.

Progressive accumulation of lipid-laden macrophages is a hallmark of the acid sphingomyelinase (ASM)-deficient forms of Niemann-Pick disease (i.e. Types A and B NPD). To investigate the mechanisms underlying enzyme replacement therapy for this disorder, we studied the uptake of recombinant, human ASM (rhASM) by alveolar macrophages from ASM knock-out (ASMKO) mice. The recombinant enzyme used for these studies was produced in Chinese hamster ovary cells and contained complex type, N-linked oligosaccharides. Binding of radiolabeled, rhASM to the ASMKO macrophages was enhanced as compared with normal macrophages, consistent with their larger size and increased surface area. However, internalization of the enzyme by the ASMKO cells was markedly reduced when compared with normal cells. Studies using receptor-specific ligands to inhibit enzyme uptake revealed that in normal cells rhASM was taken up by a combination of mannose and mannose 6-phosphate receptors (MR and M6PR, respectively), whereas in the ASMKO cells the M6PR had a minimal role in rhASM uptake. Expression of M6PR mRNA was normal in the ASMKO cells, although Western blotting revealed more receptors in these cells when compared with normal. We therefore hypothesized that lipid accumulation in ASMKO macrophages led to abnormalities in M6PR trafficking and/or degradation, resulting in reduced enzyme uptake. Consistent with this hypothesis, we also found that, when rhASM was modified to expose terminal mannose residues and target mannose receptors, the uptake of this modified enzyme form by ASMKO cells was ϳ10-fold greater when compared with the "complex" type rhASM. These findings have important implications for NPD enzyme replacement therapy, particularly in the lung.Types A and B Niemann-Pick disease (NPD) 1 are lysosomal storage disorders resulting from an inherited deficiency of acid sphingomyelinase (ASM) activity (EC 3.1.4.12) (1). ASM is responsible for hydrolyzing the lipid, sphingomyelin, to ceramide and phosphocholine, and both forms of the disease are characterized by extensive lipid storage in various cells and tissues. Patients with Type A NPD follow a rapid, neurodegenerative course that leads to death by about 3 years of age, whereas Type B NPD patients have a less severe form of the disease with little or no neurological involvement. This latter form is characterized mainly by visceral organ complications, including hepatosplenomegaly and progressive pulmonary compromise (1, 2). Intermediate forms also have been described (e.g. Ref.3), revealing that ASM deficiency can lead to a wide spectrum of disease.Enzyme replacement therapy was first suggested as an approach for the treatment of lysosomal storage disorders over 30 years ago (4), although difficulties in purifying the human enzymes limited its early evaluation (5-7). However, during the past decade the isolation of genes encoding most lysosomal enzymes and the development of expression systems that produced large quantities of recombinant proteins led to the successful treatment of sev...

SUMMARY:Types A and B Niemann-Pick disease (NPD) are lipid storage diseases caused by the deficient activity of the lysosomal enzyme, acid sphingomyelinase (ASM). Type B NPD is associated with progressive pulmonary function decline and frequent respiratory infections. ASM knock-out (ASMKO) mice are available as a model for NPD, but the lung pathology in these mice has not been adequately characterized. This study shows that by 10 weeks of age ASMKO mice have a significantly higher number of cells in their pulmonary airspaces than normal mice, consisting primarily of enlarged and often multinucleated macrophages. These mice also have much higher levels of sphingomyelin in their airspaces at 10 weeks of age, and both cell numbers and sphingomyelin concentrations remain elevated until 26 weeks of age. In these older mice an increased number of neutrophils is also seen. The alveolar cell population in the ASMKO mice produces less superoxide when stimulated, but this can be corrected by providing recombinant ASM to the culture media. Elevated levels of the chemokines macrophage inflammatory protein-2 and macrophage inflammatory protein-1␣ were also present in the bronchoalveolar lavage fluid of ASMKO mice, and this correlated with increased production of these chemokines by cultured macrophages and enhanced immunostaining in situ. Also, lung histology showed increased cellularity in the alveolar walls of ASMKO mice, but no evidence of fibrosis. Ultrastructural analysis of the lungs showed that the ASMKO mice have similar pathologic features to human NPD patients, with variable lipid storage evident in type I pneumocytes, endothelial cells, and airway ciliated epithelia. The alveolar macrophage, however, was the most dramatically affected cell type in both mice and humans. These studies indicate that the ASMKO mice can be used as a model to study the lung pathology associated with NPD, and demonstrate that the cellular and biochemical analysis of pulmonary airspaces may be a useful approach to monitoring disease progression and/or treatment. (Lab Invest 2001, 81:987-999).

Background/Aims: The sphingomyelin/ceramide signaling pathway is an important component of many cellular processes implicated in the pathogenesis of lung disease. Acid sphingomyelinase (ASM) is a key mediator of this pathway, but its specific role in pulmonary fibrosis has not been previously investigated. Here we used the bleomycin model of pulmonary fibrosis to investigate fibrotic responses in normal and ASM knockout (ASM^-/-) mice, and in NIH3T3 fibroblasts with and without ASM siRNA treatment. Methods: Mice and cells with and without ASM activity were treated with bleomycin, and the effects on lung inflammation, formation of collagen producing myofibroblasts, and apoptosis were assessed. Results: The development of bleomycin-induced inflammation and fibrosis in wildtype mice correlated with the rapid activation of ASM, and was markedly attenuated in the absence of ASM activity. Along with the elevated ASM activity, there also was an elevation of acid ceramidase (AC) activity, which was sustained for up to 14 days post-bleomycin treatment. Studies in NIH3T3 fibroblasts confirmed these findings, and revealed a direct effect of ASM/AC activation on the formation of myofibroblasts. Cell studies also showed that a downstream effect of bleomycin treatment was the production of sphingosine-1-phosphate. Conclusions: These data demonstrate that the sphingomyelin/ceramide signaling pathway is involved in the pathogenesis of bleomycin-induced pulmonary fibrosis, and suggest that inhibition of ASM may potentially slow the fibrotic process in the lung.

Human acid ceramidase was overexpressed in Chinese hamster ovary cells by amplification of the transfected, full-length cDNA. The majority of the overexpressed enzyme was secreted into the culture media and purified to apparent homogeneity. The purified protein contained the same 13-(␣) and 40 (␤)؊kDa subunits as human acid ceramidase from natural sources, had an acidic pH optimum (4.5), and followed normal MichaelisMenten kinetics using 14 C-and BODIPY-labeled C 12 -ceramide as substrates. Deglycosylation studies showed that the recombinant enzyme contained mostly "high mannose" type oligosaccharides and that two distinct ␤-subunits were present. Amino acid sequencing of these subunit polypeptides revealed a single N terminus, suggesting that the ϳ2-4-kDa molecular mass difference was likely due to C-terminal processing. The purified enzyme also catalyzed ceramide synthesis in vitro using 14 C-labeled C12 fatty acid and sphingosine as substrates. Surprisingly, we found that media from the overexpressing hamster cells had increased acid sphingomyelinase activity and that this activity could be coprecipitated with acid ceramidase using anti-ceramidase antibodies. Overexpression of acid ceramidase in normal human skin fibroblasts also led to enhanced acid sphingomyelinase secretion, but this was not observed in Niemann-Pick disease cells. RNA studies showed that this increased activity was not due to overexpression of the endogenous acid sphingomyelinase gene. Uptake studies using mouse macrophages revealed rapid internalization of the acid ceramidase activity from the hamster cell media but not acid sphingomyelinase. These studies provide new insights into acid ceramidase and the related lipid hydrolase, acid sphingomyelinase.

Types A and B Niemann-Pick disease (NPD) are lipid storage disorders caused by the deficient activity of acid sphingomyelinase (ASM). In humans, NPD is associated with the dysfunction of numerous organs including the lung. Gene targeting of the ASM gene in transgenic mice produced an animal model with features typical of NPD, including pulmonary inflammation. To assess mechanisms by which ASM perturbed lung function, we studied lung morphology, surfactant content, and metabolism in ASM-deficient mice in vivo. Pulmonary inflammation, with increased cellular infiltrates and the accumulation of alveolar material, was associated with alterations in surfactant content. Saturated phosphatidylcholine (SatPC) content was increased twofold, and sphingomyelin content was increased 5.5-fold in lungs of the ASM knockout (ASMKO) mice. Additional sphingomyelin enhanced the sensitivity of surfactant inhibition by plasma proteins. Clearance of SatPC from the lungs of ASMKO mice was decreased. Catabolism of SatPC by alveolar macrophages from the ASMKO mouse was significantly decreased, likely accounting for decreased pulmonary SatPC in vivo. In summary, ASM is required for normal surfactant catabolism by alveolar macrophages in vivo. Alterations in surfactant composition, including increased sphingomyelin content, contributed to the abnormal surfactant function observed in the ASM-deficient mouse.

Acid sphingomyelinase deficiency in type B Niemann-Pick disease leads to lysosomal sphingomyelin storage, principally affecting lungs, liver, and spleen. Infused recombinant enzyme is beneficial, yet its delivery to the lungs is limited and requires higher dosing than liver and spleen, leading to potentially adverse reactions. Previous studies showed increased enzyme pulmonary uptake by nanocarriers targeted to ICAM-1, a protein overexpressed during inflammation. Here, using polystyrene and poly(lactic-co-glycolic acid) nanocarriers, we optimized lung delivery by varying enzyme dose and nanocarrier concentration, verified endocytosis and lysosomal trafficking in vivo, and evaluated delivered activity and effects. Raising the enzyme load of nanocarriers progressively increased absolute enzyme delivery to all lung, liver, and spleen, over the naked enzyme. Varying nanocarrier concentration inversely impacted lung versus liver and spleen uptake. Mouse intravital and postmortem examination verified endocytosis, transcytosis, and lysosomal trafficking using nanocarriers. Compared to naked enzyme, nanocarriers increased enzyme activity in organs and reduced lung sphingomyelin storage and macrophage infiltration. Although old mice with advanced disease showed reactivity (pulmonary leukocyte infiltration) to injections, including buffer without carriers, antibody, or enzyme, younger mice with mild disease did not. We conclude that anti-ICAM nanocarriers may result in effective lung enzyme therapy using low enzyme doses.