Site-Specific Conjugation of a Selenopolypeptide to Alpha-1-antitrypsin Enhances Oxidation Resistance and Pharmacological Properties.

Human alpha-1-antitrypsin (A1AT), a native serine-protease inhibitor that protects tissue damage from excessive protease activities, is used as an augmentation therapy to treat A1AT-deficienct patients. However, A1AT is sensitive to oxidation-mediated deactivation and has a short circulating half-life. Currently, there is no method that can effectively protect therapeutic proteins from oxidative damage in vivo. Here we developed a novel biocompatible selenopolypeptide and site-specifically conjugated it with A1AT. The conjugated A1AT fully retained its inhibitory activity on neutrophil elastase, enhanced oxidation resistance, extended the serum half-life, and afforded long-lasting protective efficacy in a mouse model of acute lung injury. These results demonstrated that conjugating A1AT with the designed selenopolymer is a viable strategy to improve its pharmacological properties, which could potentially further be applied to a variety of oxidation sensitive biotherapeutics.

FURIN Inhibition Reduces Vascular Remodeling and Atherosclerotic Lesion Progression in Mice.

Objective- Atherosclerotic coronary artery disease is the leading cause of death worldwide, and current treatment options are insufficient. Using systems-level network cluster analyses on a large coronary artery disease case-control cohort, we previously identified PCSK3 (proprotein convertase subtilisin/kexin family member 3; FURIN) as a member of several coronary artery disease-associated pathways. Thus, our objective is to determine the role of FURIN in atherosclerosis. Approach and Results- In vitro, FURIN inhibitor treatment resulted in reduced monocyte migration and reduced macrophage and vascular endothelial cell inflammatory and cytokine gene expression. In vivo, administration of an irreversible inhibitor of FURIN, α-1-PDX (α1-antitrypsin Portland), to hyperlipidemic Ldlr-/- mice resulted in lower atherosclerotic lesion area and a specific reduction in severe lesions. Significantly lower lesional macrophage and collagen area, as well as systemic inflammatory markers, were observed. MMP2 (matrix metallopeptidase 2), an effector of endothelial function and atherosclerotic lesion progression, and a FURIN substrate was significantly reduced in the aorta of inhibitor-treated mice. To determine FURIN's role in vascular endothelial function, we administered α-1-PDX to Apoe-/- mice harboring a wire injury in the common carotid artery. We observed significantly decreased carotid intimal thickness and lower plaque cellularity, smooth muscle cell, macrophage, and inflammatory marker content, suggesting protection against vascular remodeling. Overexpression of FURIN in this model resulted in a significant 67% increase in intimal plaque thickness, confirming that FURIN levels directly correlate with atherosclerosis. Conclusions- We show that systemic inhibition of FURIN in mice decreases vascular remodeling and atherosclerosis. FURIN-mediated modulation of MMP2 activity may contribute to the atheroprotection observed in these mice.

Alpha-1-anti-trypsin-Fc fusion protein ameliorates gouty arthritis by reducing release and extracellular processing of IL-1ß and by the induction of endogenous IL-1Ra.

OBJECTIVES: In the present study, we generated a new protein, recombinant human alpha-1-anti-trypsin (AAT)-IgG1 Fc fusion protein (AAT-Fc), and evaluated its properties to suppress inflammation and interleukin (IL)-1ß in a mouse model of gouty arthritis. METHODS: A combination of monosodium urate (MSU) crystals and the fatty acid C16.0 (MSU/C16.0) was injected intra-articularly into the knee to induce gouty arthritis. Joint swelling, synovial cytokine production and histopathology were determined after 4 h. AAT-Fc was evaluated for inhibition of MSU/C16.0-induced IL-1ß release from human blood monocytes and for inhibition of extracellular IL-1ß precursor processing. RESULTS: AAT-Fc markedly suppressed MSU/C16.0-induced joint inflammation by 85-91% (p<0.001). Ex vivo production of IL-1ß and IL-6 from cultured synovia were similarly reduced (63% and 65%, respectively). The efficacy of 2.0 mg/kg AAT-Fc in reducing inflammation was comparable to 80 mg/kg of plasma-derived AAT. Injection of AAT-Fc into mice increased circulating levels of endogenous IL-1 receptor antagonist by fourfold. We also observed that joint swelling was reduced by 80%, cellular infiltration by 95% and synovial production of IL-1ß by 60% in transgenic mice expressing low levels of human AAT. In vitro, AAT-Fc reduced MSU/C16.0-induced release of IL-1ß from human blood monocytes and inhibited proteinase-3-mediated extracellular processing of the IL-1ß precursor into active IL-1ß. CONCLUSIONS: A single low dose of AAT-Fc is highly effective in reducing joint inflammation in this model of acute gouty arthritis. Considering the long-term safety of plasma-derived AAT use in humans, subcutaneous AAT-Fc emerges as a promising therapy for gout attacks.

α-1-Antitrypsin (AAT)-modified donor cells suppress GVHD but enhance the GVL effect: a role for mitochondrial bioenergetics.

Hematopoietic cell transplantation is curative in many patients. However, graft-versus-host disease (GVHD), triggered by alloreactive donor cells, has remained a major complication. Here, we show an inverse correlation between plasma α-1-antitrypsin (AAT) levels in human donors and the development of acute GVHD in the recipients (n = 111; P = .0006). In murine models, treatment of transplant donors with human AAT resulted in an increase in interleukin-10 messenger RNA and CD8(+)CD11c(+)CD205(+) major histocompatibility complex class II(+) dendritic cells (DCs), and the prevention or attenuation of acute GVHD in the recipients. Ablation of DCs (in AAT-treated CD11c-DTR donors) decreased CD4(+)CD25(+)FoxP3(+) regulatory T cells to one-third and abrogated the anti-GVHD effect. The graft-versus-leukemia (GVL) effect of donor cells (against A20 tumor cells) was maintained or even enhanced with AAT treatment of the donor, mediated by an expanded population of NK1.1(+), CD49B(+), CD122(+), CD335(+) NKG2D-expressing natural killer (NK) cells. Blockade of NKG2D significantly suppressed the GVL effect. Metabolic analysis showed a high glycolysis-high oxidative phosphorylation profile for NK1.1(+) cells, CD4(+)CD25(+)FoxP3(+) T cells, and CD11c(+) DCs but not for effector T cells, suggesting a cell type-specific effect of AAT. Thus, via altered metabolism, AAT exerts effective GVHD protection while enhancing GVL effects.

Novel peptides as potential treatment of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a loss of immunologic tolerance, production of auto-antibodies, and inflammatory damage in multiple organs. We have tested the effect of anti-inflammatory peptide, a H2A histone fragment, termed IIIM1, on MRL/lpr mice, animal model of SLE. Oral administration of IIIM1 at early stage of disease caused reduction in proteinuria and serum anti-dsDNA antibodies. Starting the treatment at advanced stage of disease resulted in prolonged animal survival, decreased lymphadenosis and reduced levels of pathogenic or abnormal double negative CD4(-)CD8(-) cells and B220(+) cells in lymph nodes and spleen. We discovered that IIIM1 induces the production of an additional peptide, a fragment of alpha-1-antitrypsin, termed UBE. A relatively low dose (1 µg/kg) of UBE reduced proteinuria and hematuria in MRL/lpr mice. The beneficial effect of the peptide was corroborated by histological examination. Furthermore a significant reduction in serum IL17, IL12 and anti dsDNA antibodies was observed in the UBE-treated mice. Isolated CD4 cells incubated with the peptide showed a similar cytokine profile. Decreased levels of double negative CD4(-)CD8(-) and B220(+) cells were determined in lymph organs of UBE-treated animals. The beneficial effects of both UBE and IIIM1 suggest these peptides as potential drugs for SLE.

α 1-antitrypsin enhances insulin secretion and prevents cytokine-mediated apoptosis in pancreatic ß-cells.

α1-antitrypsin (AAT) is a serine protease inhibitor, which recently has been shown to prevent type 1 diabetes (T1D) development, to prolong islet allograft survival and to inhibit ß-cell apoptosis in vivo. It has also been reported that T1D patients have significantly lower plasma concentrations of AAT suggesting the potential role of AAT in the pathogenesis of T1D. We have investigated whether plasma-purified AAT can affect ß-cell function in vitro. INS-1E cells or primary rat pancreatic islets were used to study the effect of AAT on insulin secretion after glucose, glucagon-like peptide-1 (GLP-1) and forskolin stimulation and on cytokine-mediated apoptosis. The secreted insulin and total cyclic AMP (cAMP) were determined using radioimmunoassay and apoptosis was evaluated by propidium iodide staining followed by FACS analysis. We found that AAT increases insulin secretion in a glucose-dependent manner, potentiates the effect of GLP-1 and forskolin and neutralizes the inhibitory effect of clonidine on insulin secretion. The effect of AAT on insulin secretion was accompanied by an increase in cAMP levels. In addition, AAT protected INS-1E cells from cytokine-induced apoptosis. Our findings show that AAT stimulates insulin secretion and protects ß-cells against cytokine-induced apoptosis, and these effects of AAT seem to be mediated through the cAMP pathway. In view of these novel findings we suggest that AAT may represent a novel anti-inflammatory compound to protect ß-cells under the immunological attack in T1D but also therapeutic strategy to potentiate insulin secretion in type 2 diabetes (T2D).

Comparison of the effects of serpin A1, a recombinant serpin A1-IGF chimera and serpin A1 C-terminal peptide on wound healing.

Serpin A1 (alpha1-antitrypsin, alpha1-proteinase inhibitor), a potent neutrophil elastase inhibitor, has therapeutic potential as a wound-healing agent. We compared the in vitro wound-healing action of serpin A1-IGF, a recombinant fusion protein of serpin A1(M351E-M358L) and insulin-like growth factor I with that observed in the presence of natural serpin A1 or A1-C26, the synthetic C-terminal 26 residue peptide of serpin A1, previously shown to have mitogenic and antiviral activities. All agents reduced wound sizes in monolayers of the kidney epithelial cell line LLC-PK1 and in primary cultures of human skin fibroblasts. Wound reduction in primary human keratinocytes was only observed with the serpin A1-IGF chimera. None of the factors stimulated cell proliferation using a colorimetric assay, with the exception of the serpin A1-IGF chimera, which caused a significant increase of cell proliferation and thymidine incorporation in human skin fibroblasts. However, wound healing by the A1-IGF chimera was reduced in keratinocytes in the presence of mitomycin C, suggesting a role of cell proliferation in wound reduction. The hydrophobic A1-C26 peptide significantly increased the production of collagen I in skin fibroblasts, an appealing asset for skin care applications.

[Effect of Dansen injection on experimental emphysema in rabbits].

OBJECTIVE: To observe the effect of Dansen injection on the experimental emphysema in rabbits. METHOD: Thirty-six rabbits were randomized into emphysema group (n = 12), Dansen injection treated group (n = 12) and alpha1-antitrypsin(alpha1-AT) treated group (n = 12). The animal model of emphysema was induced by intratracheal instillation of porcine pancreatic elastase. Dansen injection and alpha1-ATwere instilled intratracheal in two treated group after 14 days with porcine pancreatic elastase, respectively, once a week, to continue for four weeks. The level of alpha1-AT in serum and in bronchoalveolar lavage fluid (BALF) were analyzed in different times. The mean linear intercept value (MLIV) and the numbers of alveolar per square (NAPS) of all groups were compared after eight weeks with porcine pancreatic elastase. RESULT: The levels of alpha1-AT in BALF were significantly different between treated groups and emphysema group after two weeks treatment, alpha1-AT levels of treated groups were more increased than those of emphysema group (P < 0.01). The levels of alpha1-AT in serum were similar at same times in different groups (P > 0.05), but were great different in different times. The MLIV and the NAPS were significantly different from emphysema group to treated groups in sixth and eighth weeks (P < 0.01), there is no difference between dancen group and alpha1-AT group. CONCLUSION: The contents of alpha1-AT in local pulmonary tissue could be improved by Dansen injection through intratracheal instillation during the information of emphysema in rabbits. The effect of Dansen injection and alpha1-AT on preventing formation of emphysema is similar.

Effect of recombinant human DNase on alpha1-proteinase inhibitor function: an experimental approach to the combined clinical use of rhDNase and alpha1-PI in CF patients.

Chronic inflammation in cystic fibrosis (CF) airways leads to high concentrations of deoxyribonucleic acid (DNA) and neutrophil elastase (NE). Both play a major role in CF lung pathophysiology and are aims of new therapeutic approaches: rhDNase degrades highly viscosic DNA and alpha1-proteinase inhibitor (alpha1-PI) inhibits NE activity and thereby pulmonary inflammation and hypersecretion. Given the reports on increased sputum NE concentrations upon rhDNase inhalation, there is a rationale for a combined rhDNase/alpha1-PI treatment. With the question of whether a combined therapy is feasible, we first investigated in vitro whether incubation of CF sputum with rhDNase changes proteolytic and secretagogue activity of sputum supernatants and its inhibition by alpha1-PI. Next, we studied whether incubation of alpha1-PI with rhDNase impairs the inhibitory effect of alpha1-PI on proteolytic activity of NE and the inhibitory effect of alpha1-PI on NE-induced secretion from a human mucoepidermoid cell line. Incubation of CF sputum with rhDNase led to a twofold increase in sputum NE activity. Correspondingly, the inhibitory effect of alpha1-PI on sputum NE activity and on secretion induced by these sputum samples was significantly reduced by rhDNase. Preincubation of alpha1-PI with rhDNase significantly reduced the inhibitory effect of alpha1-PI on purified NE activity and on NE-induced secretion. However, this effect was limited to alpha1-PI concentrations lower than those achievable after inhalation. Therefore, impairment of alpha1-PI function by rhDNase is not likely to be relevant in vivo, provided that a sufficient dosage of alpha1-PI is inhaled.

Injury to murine airway epithelial cells by pollen enzymes.

BACKGROUND: Pollens are important triggers for asthma but the mechanism of sensitisation to their proteins remains poorly understood. The intrinsic protease activity of some allergens may contribute to sensitisation by disrupting the integrity of the airway epithelial barrier. Pollens release a variety of enzymes, including proteases, upon hydration. The hypothesis that such enzymes might be able to damage airway epithelial cells was therefore tested. METHODS: Diffusates from pollens of Lolium perenne (ryegrass), Poa pratensis (Kentucky bluegrass), Acacia longifolia (Sydney golden wattle), or Casuarina distyla (she-oak) were incubated with mouse tracheal epithelial cells in culture and cellular detachment was quantified using a methylene blue dye binding assay. RESULTS: Diffusates prepared using 100 mg/ml of pollen caused detachment of 30-90% of airway epithelial cells in separate experiments. Within each experiment comparable detachment was observed with all diffusates tested, although total protein in the diffusates varied markedly between species. Viability of the cells recovered after exposure to Acacia diffusate was higher than after detachment by exposure to Lolium diffusate. Cellular detachment by all of the diffusates could be almost completely inhibited by addition of 10% serum. Aprotinin, an inhibitor of serine proteases, partially blocked activity in diffusates of Lolium pollen but not of Acacia pollen. In contrast, alpha 1-protease inhibitor and secretory leucocyte protease inhibitor (SLPI) were not able to block the activity of either diffusate at concentrations which inhibited cellular detachment by trypsin. CONCLUSIONS: Proteases released by pollens are able to cause detachment of airway epithelial cells from their substratum in vitro and may not be effectively inhibited by endogenous antiproteases.

Alpha 1-proteinase inhibitor abrogates proteolytic and secretagogue activity of cystic fibrosis sputum.

Airway disease in cystic fibrosis (CF) is characterized by neutrophil-dominated chronic inflammation with an excess of uninhibited neutrophil elastase (NE), which is regarded as an important factor in progressive lung destruction. Therefore, inhalation of alpha 1-proteinase inhibitor (alpha 1-PI) seems to be a reasonable therapeutic approach. To estimate its therapeutic potential, we quantitatively investigated the in vitro interactions of exogenous alpha 1-PI with CF sputum samples (n = 28). High NE and alpha 1-PI concentrations were detected in CF sputum (6.03 +/- 0.78 and 2.56 +/- 0.16 mumol/l, respectively). There was significant NE activity (2.6 +/- 0.4 U/l) due to both the surplus of NE and proteolytic degradation of alpha 1-PI. Addition of exogenous alpha 1-PI resulted in a dose-dependent inhibition of NE activity in CF sputum; > 90% inhibition was achieved at 10 micrograms/ml alpha 1-PI. Purified NE as well as CF sputum potently induced secretion from porcine tracheal glands. Corresponding to inhibition of NE activity, CF sputum-induced secretion was also inhibited by exogenous alpha 1-PI; > 90% inhibition was also achieved at 10 micrograms/ml alpha 1-PI. Incubation of exogenous alpha 1-PI with CF sputum for 24 h did not reduce the inhibitory effects. From our in vitro results we conclude that inhalation of alpha 1-PI might effectively inhibit both NE activity and airway gland hypersecretion in CF airways.

Administration of alpha 1-proteinase inhibitor ameliorates bleomycin-induced pulmonary fibrosis in hamsters.

The effect of alpha 1-proteinase inhibitor (alpha 1Pi) administration on the acute lung injury and subsequent fibrosis induced by bleomycin (BLM) was examined in hamsters. Pulmonary lesions were quantitatively reduced in alpha 1Pi-administered BLM-treated (BLM-alpha 1Pi) animals compared with animals treated by BLM alone (BLM-control) at both 7 days (acute stage) and 30 days (fibrotic stage) after BLM treatment. Analysis of intraalveolar cells from bronchoalveolar lavage (BAL) fluid revealed that neutrophils and lymphocytes were significantly decreased in the BLM-alpha 1Pi animals at 7 days after BLM treatment and that 30 days after BLM treatment macrophages as well as neutrophils and lymphocytes were remarkably decreased in the BLM-alpha 1Pi animals. The elastase activity in supernatants of BAL fluid during 7 days following BLM treatment was detected, but there was no difference between the two groups. In vitro studies on neutrophil responsiveness to stimulation of BAL fluid at 3 days after BLM treatment revealed noticeable chemotaxis and generation of superoxide anion of isolated neutrophils, but alpha 1Pi did not show any inhibitory effects on neutrophil responsiveness. We suggest that alpha 1Pi administration ameliorates pulmonary fibrosis preceded by acute lung injury induced by BLM treatment in hamsters and that the inhibitory effects of alpha 1Pi on lung injury may not be brought about by altered elastase activity, chemotaxis, or superoxide generation in neutrophils. Alternative mechanisms are discussed.