α1-AT Expression Research Articles

Alpha-1 antitrypsin inhibits pertussis toxin

Pertussis (whooping cough) is a vaccine-preventable but re-emerging, highly infectious respiratory disease caused by Bordetella pertussis. There are currently no effective treatments for pertussis, complicating care for non-vaccinated individuals, especially newborns. Disease manifestations are predominantly caused by pertussis toxin (PT), a pivotal virulence factor classified as an ADP-ribosylating AB-type protein toxin. In this work, an unbiased approach using peptide libraries, bioassay-guided fractionation and mass spectrometry revealed α1-antitrypsin (α1AT) as a potent PT inhibitor. Biochemistry-, cell culture- and molecular modeling-based in vitro experimentation demonstrated that the α1AT mode of action is based on blocking PT-binding to the host target cell surface. In the infant mouse model of severe pertussis, α1AT expression was reduced upon infection. Further, systemic administration of α1AT significantly reduced B. pertussis-induced leukocytosis, which is a hallmark of infant infection and major risk factor for fatal pertussis. Taken together our data demonstrates that α1AT is a novel PT inhibitor and that further evaluation and development of α1AT as a therapeutic agent for pertussis is warranted. Importantly, purified α1AT is already in use clinically as an intravenous augmentation therapy for those with genetic α1AT deficiency and could be repurposed to clinical management of pertussis.

Alpha-1 antitrypsin protects against phosgene-induced acute lung injury by activating the ID1-dependent anti-inflammatory response

Phosgene is widely used as an industrial chemical, and phosgene inhalation causes acute lung injury (ALI), which may further progress into pulmonary edema. Currently, an antidote for phosgene poisoning is not known. Alpha-1 antitrypsin (α1-AT) is a protease inhibitor used to treat patients with emphysema who are deficient in α1-AT. Recent studies have revealed that α1-AT has both anti-inflammatory and anti-SARS-CoV-2 effects. Herein, we aimed to investigate the role of α1-AT in phosgene-induced ALI. We observed a time-dependent increase in α1-AT expression and secretion in the lungs of rats exposed to phosgene. Notably, α1-AT was derived from neutrophils but not from macrophages or alveolar type II cells. Moreover, α1-AT knockdown aggravated phosgene- and lipopolysaccharide (LPS)-induced inflammation and cell death in human bronchial epithelial cells (BEAS-2B). Conversely, α1-AT administration suppressed the inflammatory response and prevented death in LPS- and phosgene-exposed BEAS-2B cells. Furthermore, α1-AT treatment increased the inhibitor of DNA binding 1 (ID1) gene expression, which suppressed NF-κB pathway activation, reduced inflammation, and inhibited cell death. These data demonstrate that neutrophil-derived α1-AT acts as a self-protective mechanism, which protects against phosgene-induced ALI by activating the ID1-dependent anti-inflammatory response. This study may provide novel strategies for the treatment of patients with phosgene-induced ALI.

Post-Transcriptional Regulation of Alpha One Antitrypsin by a Proteasome Inhibitor.

Alpha one antitrypsin (α1AT), a serine proteinase inhibitor primarily produced by the liver, protects pulmonary tissue from neutrophil elastase digestion. Mutations of the SERPINA1 gene results in a misfolded α1AT protein which aggregates inside hepatocytes causing cellular damage. Therefore, inhibition of mutant α1AT production is one practical strategy to alleviate liver damage. Here we show that proteasome inhibitors can selectively downregulate α1AT expression in human hepatocytes by suppressing the translation of α1AT. Translational suppression of α1AT is mediated by phosphorylation of eukaryotic translation initiation factor 2α and increased association of RNA binding proteins, especially stress granule protein Ras GAP SH3 binding protein (G3BP1), with α1AT mRNA. Treatment of human-induced pluripotent stem cell-derived hepatocytes with a proteasome inhibitor also results in translational inhibition of mutant α1AT in a similar manner. Together we revealed a previously undocumented role of proteasome inhibitors in the regulation of α1AT translation.

Alpha1-Antitrypsin in Urinary Extracellular Vesicles: A Potential Biomarker of Diabetic Kidney Disease Prior to Microalbuminuria.

PurposeDiabetic kidney disease (DKD), which is related to inflammation and immune response, is the primary vascular complication of diabetes mellitus and also the leading etiology of end-stage renal disease. Urinary extracellular vesicles (UEVs) are an attractive source for biomarker detection as they involve molecular constituents derived from their parental sections of the nephron. In this study, we aimed to search for a potential biomarker in UEVs for the early diagnosis and prediction of DKD, especially before the emergence of microalbuminuria.Patients and MethodsUEVs were isolated from the urine of healthy subjects, pre-diabetic, and diabetic patients with varying degrees of kidney damage by ultracentrifugation, and the extracted UEVs were used to measure alpha1-antitrypsin (α1-AT) by Western blot. To explore the function of α1-AT in the inflammatory process leading to DKD, we silenced the expression of α1-AT in renal tubular epithelial cells using cell transfection techniques to assess the differential expression of the inflammatory factors such as MCP-1 and TNF-α using qRT-PCR.ResultsThere was no expression of α1-AT in the UEVs of either healthy or pre-diabetic subjects. Its expression was significantly increased in the UEVs of diabetic patients with normoalbuminuria (prior to microalbuminuria), which was more sensitive and more stable than other renal indexes to predict DKD. Additionally, the expression of α1-AT in UEVs was gradually upregulated with the aggravation of DKD and the decline of renal function. In vitro, the mRNA expression of MCP-1 and TNF-α was significantly decreased when the generation of α1-AT in tubular epithelial cells was inhibited under high glucose stimulation.ConclusionOur results suggest that α1-AT derived from UEVs, especially in diabetic patients with normoalbuminuria, might serve as a potential noninvasive biomarker for diagnosis of DKD early in the development of the disease and may predict the future decline of renal function.

Silence of α1-Antitrypsin Inhibits Migration and Proliferation of Triple Negative Breast Cancer Cells.

Backgroundα1-antitrypsin (α1-AT) is highly expressed in many tumors. However, to the best of our knowledge, its relationship to triple negative breast cancer (TNBC) has not yet been studied. Thus, in this research we first explored the influence of α1-AT silencing on the abilities of migration and invasion, and then further study its molecular mechanism in TNBC cells.Material/MethodsThe viability of MDA-MB-231 cells were detected using cell counting kit-8 (CCK-8). The abilities of migration and invasion were examined by Transwell assay. The metastasis-related factors were tested respectively by quantitative real-time PCR (qRT-PCR) and western blot assays.ResultsOur study results showed that α1-AT level in TNBC tissues was higher than non-triple negative breast cancer (n-TNBC) and adjacent normal breast tissues. The high expression of α1-AT was linked to type of cancer, tumor size, TNM stage and metastasis, but was not correlated with α1-AT expression and age. si-α1-AT suppressed the viability, migration, and invasion of cells. While si-α1-AT upregulated E-cadherin and the tissue inhibitor of metalloproteinases-2 (TIMP-2) levels, it downregulated metastasis associated 1 (MTA1), matrix metallopeptidase 2 (MMP2), phosphorylated-mammalian target of rapamycin (p-mTOR), phosphorylated-protein kinase B (p-Akt), and phosphorylated-phosphatidylinositol 3 kinase (p-PI3K) levels. We also found that the PI3K/Akt/mTOR pathway activator reversed the role of si-α1-AT in metastasis-related factors.Conclusionsα1-AT was highly expressed in TNBC tissues, and its silencing suppressed the abilities of migration and invasion in TNBC cells and downregulated the PI3K/Akt/mTOR pathway. Thus, α1-AT may have a potential therapeutic effect on TNBC.

SERPINA1 Hepatocyte-Specific Promoter Polymorphism Associate with Chronic Obstructive Pulmonary Disease in a Study of Kashmiri Ancestry Individuals

Different mutations in coding and non-coding sequences of the SERPINA1 gene have been implicated in the pathogenesis of COPD. However, - 10T/Cmutation in the hepatocyte-directed promoter region has not been associated with COPD pathogenesis so far. Here, we report an increased frequency of - 10C genotype that is associated with decreased levels of serum alpha1-antitrypsin (α1AT) in COPD patients. The quantification of serum α1AT was done by ELISA, the phenol-chloroform method was used for DNA extraction, PCR products were directly sequenced. The IBM SPSS Statistics v21 software was used for statistical analyses of the data. The mean serum α1AT level was found to be 1.203+0.239 and 3.162+0.160 g/L in COPD cases and in control, respectively. The - 10C allele is associated with an increased risk of COPD [OR, 3.50 (95%CI, 1.86-6.58); p < 0.001]. The combined variant genotype (TT+CC) was significantly found associated with an increased risk of COPD [OR, 3.20 (95% CI, 1.47-6.96); p = 0.003]. A significant association of the family history with COPD (overall p value= 0.0331) suggests that genetics may play an important role in the pathogenesis of COPD. The polymorphism associated with hepatocyte-specific promoter region (- 10T/C) is likely to be associated withthe pathogenesis of COPD. It is quite possible that the change of the base in thehepatocyte-specific promoterof the SERPINA1 gene can modulate its strength, thereby driving the reduced expression of α1AT.

Possible Role of α1-Antitrypsin in Endometriosis-Like Grafts From a Mouse Model of Endometriosis.

Previous study indicated that bleeding into the peritoneum may accelerate inflammatory response in endometriosis-like grafts in mice. To identify changes in protein levels in the grafts from mice that underwent unilateral ovariectomy (uOVX), which causes bleeding from ovarian arteries and vein, the grafts were generated by injecting a suspension of human endometrial cells in BALB/c nude female mice, and protein profile changes were compared with non-uOVX control mice. The level of α1-antitrypsin (α1-AT) decreased in grafts from nude mice that underwent uOVX. The levels of phosphorylated Akt, mammalian target of rapamycin, S6K, regulatory factors for cell survival, and of phosphorylated nuclear factor κB, an inflammatory mediator, were higher in endometriosis-like grafts from the uOVX group than from the control. The grafts were mostly comprised of stromal cells. The bioactivity of α1-AT was assessed by investigating cytokine expression in protease-activated receptor (PAR) 1/2 agonists-stimulated stromal cells. The PARs promoted the expression of interleukin 8 (IL-8), but treatment with α1-AT blocked IL-8 expression dose dependently. Knocking down α1-AT expression increased the constitutive IL-6, IL-8, and cyclooxygenase 2 expression as well as PAR1 agonist-stimulated IL-6 expression. These findings support the notion that decreased α1-AT protein in the grafts constituted with human endometrial cells in mice may have exacerbated inflammation in endometriosis-like grafts, suggesting the possible involvement of α1-AT in the pathophysiology of endometriosis.

Proteomics-based identification of α1-antitrypsin and haptoglobin precursors as novel serum markers in infiltrating ductal breast carcinomas

Background The identification of pathological markers of breast cancer for either diagnosis, treatment response or for survival is of critical importance. Methods Serum protein profiling using 2-DE separations coupled to matrix-assisted laser desorption ionization mass spectrometry has been used to explore protein alterations in patients with infiltrating ductal breast carcinomas (IDCA). Sera from 39 breast cancer patients and 40 healthy controls were selected for screening study using 2-DE combined with MS. The protein expression patterns obtained after the depletion of high abundance proteins was determined by coomassie blue G-250 stain after 2-DE electrophoresis. Results Six proteins that expressed differentially in the IDCA group were found. The expression levels of four isoforms corresponding to haptoglobin precursor and two isoforms of α1-antitrypsin precursor (α1-AT) were upregulated in sera from breast cancer patients. There was an increased expression of both proteins in the sera of patients with various tumor stages (I, II, III) in comparison to healthy women. Applying immunohistochemistry, we further validated α1-AT immunoreactivity in 51 formalin-fixed paraffin-embedded sections of breast tumors. Enhanced expression of α1-AT like activity has been found in IDCA breast tumors, as well as, in different histological types of breast cancer. No significant association has been found with lymph node occurrence, while in high tumor categories a tendency to an increased expression of α1-AT has been found, thereby suggesting a possible role of this protein in tumor growth. Conclusions These proteins may constitute new and useful markers of breast cancer that offer a clue to a better understanding of inflammatory pathways and carcinogenesis events linked to breast cancer progression.

High Levels of Persistent Expression of α1-Antitrypsin Mediated by the Nonhuman Primate Serotype rh.10 Adeno-associated Virus Despite Preexisting Immunity to Common Human Adeno-associated Viruses

α1-Antitrypsin (α1AT) deficiency is a genetic disorder causing emphysema if serum α1AT levels are <570 μg/ml. We have shown that intrapleural administration of an AAV5α1AT vector yielded persistent therapeutic α1AT serum levels. Since anti-AAV2 and -AAV5 antibodies prevalent in humans may limit the use of these common serotypes in gene therapy, we screened 25 AAV vectors derived from humans and nonhuman primates for α1AT expression following intrapleural administration to mice. The rhesus AAVrh.10 serotype yielded the highest levels and was chosen for further study. Following intrapleural administration, 77% of total body transgene expression was in the chest wall, diaphragm, lung, and heart. Intrapleural administration of AAVrh.10α1AT provided long-term, therapeutic α1AT expression in mice, although higher doses were required to achieve therapeutic levels in female mice than in male mice. Intrapleural administration of AAVrh.10α1AT produced the same levels in AAV2/AAV5-preimmune and naive mice. In mice administered with AAV5α1AT and subsequently “boosted” with the AAVrh.10α1AT vector, serum levels were increased by 300%. These data indicate that AAVrh.10 is the most effective known AAV vector for intrapleural gene delivery and has the advantage of circumventing human immunity to AAV.

338. Therapeutic Levels for α1-Antitrypsin Following Intrapleural Administration of a Non-Human Primate Serotype rh10 AAV Vector Expressing α1-Antitrypsin

Alpha 1-antitrypsin (α1AT), a serine protease inhibitor synthesized and secreted by the liver, protects the lung from degradation by neutrophil proteases. α1AT deficiency is a common autosomal recessive disorder associated with the accelerated development of emphysema if serum α1AT levels are <11 μM (570 μg/ml). In this study, we test the hypothesis that persistent expression of α1AT at therapeutic levels can be achieved in mice by intrapleural administration of AAV vectors expressing the cDNA for α1AT at vector doses that can be safely scaled up to humans. In initial studies, mice were injected with 1011 genome copies (gc) of an adeno-associated virus seroytpe 2 (AAV2) based vector expressing human cDNA for α1AT from a cytomegalovirus-chicken β-actin hybrid promoter. Serum levels of 80 ± 19 μg/ml (mean ± SD) were achieved 8 wk following intrapleural administration, a route chosen to minimize safety issues while providing proximity to the lung. Since this fell short of the target expression level, an alternate approach was developed using the same vector construct pseudotyped by the capsid of AAVrh.10. AAVrh.10 is derived from rhesus macaque and utilizes cell surface receptor(s) distinct from that of AAV2 and is not recognized by preexisting anti-AAV2 neutralizing antibodies. The AAVrh.10α1AT vector was prepared by triple transfection with a yield of 15,000 ± 5,000 gc/cell and purified by iodixanol gradient and ion exchange chromatography. The AAVrh.10α1AT vector was administered in C57Bl/6 mice (n=4/group) at a dose of 5 × 1010 gc, via intrapleural, intramuscular, and intratracheal route and serum α1AT levels measured by ELISA. At 2 wk post-injection, the level of α1AT in intrapleural injected animals was 200 ± 10 μg/ml, compared to 1.2 ± 0.6 μg/ml for intratracheal injected animals and 60 ± 15 μg/ml for intramuscular injected animals (p < 0.01 all pairwise comparisons). Intrapleural administration of AAVrh.1α1AT vector at a higher dose (1011 gc) showed serum α1AT levels of 2,200 ± 300 μg/ml after 8 wk, higher than the target for therapy. To determine the relative distribution of the vector in lung following intrapleural administration, an AAVrh.10 vector expressing luciferase was injected intrapleurally in C57Bl/6 mice (n = 4/group) and after 8 wk, luciferase activity was measured in tissue homogenates. The luciferase activity in the diaphragm was 7 × 106 RLU/mg, in the left lung 7.5 × 106 RLU/mg, in the right lung 1.7 × 106 RLU/mg whereas in liver and kidney luciferase activities were 0.8 × 106 RLU/mg and 0.02 × 106 RLU/mg, respectively, indicating that diaphragm and lung are the major sites of gene transfer. These data indicate that intrapleural administration of an AAVrh.10 vector may be a more efficient and readily scalable strategy for delivery of α1AT to the lung than by other routes including direct intratracheal administration.

155. Non-Viral Gene Therapy for Type 1 Diabetes by Electrotransfer of Insulin and Glucokinase Genes to Skeletal Muscle

Alpha1-antitrypsin (α1-AT) deficiency is caused by mutations in the α1-AT gene, a protease inhibitor, and can result in emphysema and/or liver disease. The lung disease is caused by a decrease in secreted α1-AT enzyme, a loss-of-function which results in tissue damage from unchecked leukocyte proteases. In contrast, the liver disease occurs by a gain-of-function, whereby mutant protein polymers are retained in the endoplasmic reticulum of hepatocytes. In persuit of an alternative to the only current treatment for the liver disease, to transplant the organ, we are investigating a genetic therapy that would combine the technologies of RNA interference (RNAi) with the Sleeping Beauty transposon system. The RNAi pathway consists of endogenous enzymes that destroy mRNAs selectively in response to small RNAs that are complimentary in sequence. Small inhibitory RNAs (siRNAs), which can be transcribed from plasmid constructs introduced into cells, have been shown to mediate RNAi in vitro and in vivo. Moreover, siRNAs can mediate long-term repression of a target gene if they are constitutively expressed from the genome. The Sleeping Beauty (SB) transposon system can integrate DNA transgenes into the genome for long-term expression in vitro and in vivo, and is being developed as a novel, non-viral gene therapy tool. A SB transposon vector was constructed to express siRNAs that target human α1-AT mRNA. The results demonstrate that siRNA transposons can be used to reduce wildtype and mutant α1-AT expression in cultured cells, whereas the house-keeping gene GAPDH was not affected. Next, we plan to test these siRNA transposons in vivo, in the transgenic mouse model of α1-AT liver disease. This gene therapy model combines two technologies, RNAi and the SB transposon, for a potential new therapeutic for the liver disease associated with mutant α1-AT.

Cell-specific involvement of HNF-1beta in alpha(1)-antitrypsin gene expression in human respiratory epithelial cells.

The synergistic action of hepatocyte nuclear factor (HNF)-1alpha and HNF-4 plays an important role in expression of the alpha(1)-antitrypsin (alpha(1)-AT) gene in human hepatic and intestinal epithelial cells. Recent studies have indicated that the alpha(1)-AT gene is also expressed in human pulmonary alveolar epithelial cells, a potentially important local site of the lung antiprotease defense. In this study, we examined the possibility that alpha(1)-AT gene expression in a human pulmonary epithelial cell line H441 was also directed by the synergistic action of HNF-1alpha and HNF-4 and/or by the action of HNF-3, which has been shown to play a dominant role in gene expression in H441 cells. The results show that alpha(1)-AT gene expression in H441 cells is predominantly driven by HNF-1beta, even though HNF-1beta has no effect on alpha(1)-AT gene expression in human hepatic Hep G2 and human intestinal epithelial Caco-2 cell lines. Expression of alpha(1)-AT and HNF-1beta was also demonstrated in primary cultures of human respiratory epithelial cells. HNF-4 has no effect on alpha(1)-AT gene expression in H441 cells, even when it is cotransfected with HNF-1beta or HNF-1alpha. HNF-3 by itself has little effect on alpha(1)-AT gene expression in H441, Hep G2, or Caco-2 cells but tends to have an upregulating effect when cotransfected with HNF-1 in Hep G2 and Caco-2 cells. These results indicate the unique involvement of HNF-1beta in alpha(1)-AT gene expression in a cell line and primary cultures derived from human respiratory epithelium.

Adeno-associated virus as a vector for liver-directed gene therapy.

Factors relevant to the successful application of adeno-associated virus (AAV) vectors for liver-directed gene therapy were evaluated. Vectors with different promoters driving expression of human alpha-1-antitrypsin (alpha-1AT) were injected into the portal circulation of immunodeficient mice. alpha-1AT expression was stable but dependent on the promoter. Southern analysis of liver DNA revealed approximately 0.1 to 2.0 provirus copies/diploid genome in presumed head-to-tail concatamers. In situ hybridization and immunohistochemical analysis revealed expression in approximately 5% of hepatocytes clustered in the pericentral region. These results support the use of AAV as a vector for diseases treatable by targeting of hepatocytes.

Expression of Human Alpha1Antitrypsin in Murine Hematopoietic Cellsin Vivoafter Retrovirus-Mediated Gene Transfer

For patients with alpha1antitrypsin (α1AT) deficiency, the expression of α1AT in hematopoietic cells may result in a number of benefits not provided by gene transfer strategies involving local modification of the respiratory epithelium or liver-directed gene transfer. We investigated the expression of α1AT in murine hematopoietic cells after retrovirus-mediated gene transfer. For this purpose we constructed an LNL-6-derived recombinant retrovirus vector (Lα1ED) that expresses the α1AT cDNA from the Moloney murine leukemia virus (MoMuLV) U3 promoter/enhancer and coexpresses the cDNA for a mutant form of the murine dihydrofolate reductase molecule (*DHFR) from the encephalomyocarditis virus (emc) internal ribosome entry site (IRES). All of the mice transplanted with bone marrow transduced with the Lα1ED vector expressed the α1AT protein at the 3-week time point after transplantation. By the 6-week time point the α1AT levels declined to a lower level, where they generally remained for the duration of the experiment. This study demonstrates the potential utility of hematopoietic cell gene transfer for gene therapy of α1AT deficiency.

Effect of Pseudomonas Elastase on Human Mononuclear Phagocyte α1-Antitrypsin Expression

The net balance of neutrophil elastase and its inhibitor, alpha 1-antitrypsin (alpha 1-AT), is a critical determinant of connective tissue turnover during homeostasis and in disease states. In addition to liver-derived alpha 1-AT, which translocates from blood to tissues, this elastase-alpha 1-AT balance is maintained by expression of alpha 1-AT at the local tissue level in resident mononuclear phagocytes. Our previous studies have shown that this elastase-alpha 1-AT balance is also tightly controlled at a cellular level in that addition of exogenous neutrophil elastase (serpine-type elastase) to cultured mononuclear phagocytes is associated with an increase in expression of the alpha 1-AT gene. Subsequent studies have demonstrated that this novel regulatory loop involves interaction between exogenous neutrophil elastase and endogenous alpha 1-AT inducing a structural rearrangement in the alpha 1-AT molecule and exposing highly conserved conformation-specific domain of alpha 1-AT, which can then be recognized by a specific cell surface receptor, the serpine-enzyme complex receptor. In the following study, we examined the effect of a bacterial metalloelastase, Pseudomonas aeruginosa elastase, on expression of alpha 1-AT in human mononuclear phagocytes. We show that pseudomonas elastase inactivates monocyte-derived alpha 1-AT by limited proteolysis but, in so doing, alpha 1-AT becomes recognized by the serpine-enzyme complex receptor and mediates an increase in de novo synthesis of alpha 1-AT in these cells. However, the concentrations of pseudomonas elastase needed to proteolytically inactivate alpha 1-AT in monocyte culture fluid are higher than those required for inactivation of purified plasma alpha 1-AT.(ABSTRACT TRUNCATED AT 250 WORDS)