Cellular architecture of evolving neuroinflammatory lesions and multiple sclerosis pathology.

Multiple sclerosis (MS) is a neurological disease characterized by multifocal lesions and smoldering pathology. Although single-cell analyses provided insights into cytopathology, evolving cellular processes underlying MS remain poorly understood. We investigated the cellular dynamics of MS by modeling temporal and regional rates of disease progression in mouse experimental autoimmune encephalomyelitis (EAE). By performing single-cell spatial expression profiling using in situ sequencing (ISS), we annotated disease neighborhoods and found centrifugal evolution of active lesions. We demonstrated that disease-associated (DA)-glia arise independently of lesions and are dynamically induced and resolved over the disease course. Single-cell spatial mapping of human archival MS spinal cords confirmed the differential distribution of homeostatic and DA-glia, enabled deconvolution of active and inactive lesions into sub-compartments, and identified new lesion areas. By establishing a spatial resource of mouse and human MS neuropathology at a single-cell resolution, our study unveils the intricate cellular dynamics underlying MS.

Regulation of CNS pathology by Serpina3n/SERPINA3: The knowns and the puzzles.

Neuroinflammation, blood-brain barrier (BBB) dysfunction, neuron and glia injury/death and myelin damage are common central nervous system (CNS) pathologies observed in various neurological diseases and injuries. Serine protease inhibitor (Serpin) clade A member 3n (Serpina3n), and its human orthologue SERPINA3, is an acute-phase inflammatory glycoprotein secreted primarily by the liver into the bloodstream in response to systemic inflammation. Clinically, SERPINA3 is dysregulated in brain cells, cerebrospinal fluid and plasma in various neurological conditions. Although it has been widely accepted that Serpina3n/SERPINA3 is a reliable biomarker of reactive astrocytes in diseased CNS, recent data have challenged this well-cited concept, suggesting instead that oligodendrocytes and neurons are the primary sources of Serpina3n/SERPINA3. The debate continues regarding whether Serpina3n/SERPINA3 induction represents a pathogenic or a protective mechanism. Here, we propose possible interpretations for previously controversial data and present perspectives regarding the potential role of Serpina3n/SERPINA3 in CNS pathologies, including demyelinating disorders where oligodendrocytes are the primary targets. We hypothesise that the 'good' or 'bad' aspects of Serpina3n/SERPINA3 depend on its cellular sources, its subcellular distribution (or mis-localisation) and/or disease/injury types. Furthermore, circulating Serpina3n/SERPINA3 may cross the BBB to impact CNS pathologies. Cell-specific genetic tools are critically important to tease out the potential roles of cell type-dependent Serpina3n in CNS diseases/injuries.

rBC2LCN-reactive SERPINA3 is a glycobiomarker candidate for pancreatic ductal adenocarcinoma.

Early detection is urgently needed to improve the patient's pancreatic ductal adenocarcinoma (PDAC) survival. Previously, we identified a novel tumor-associated glycan, H-type3, which is expressed on PDAC cells and is detected by rBC2LCN (recombinant N-terminal domain of BC2L-C identified from Burkholderia cenocepacia) lectin. Here, we identified that SERPINA3 is an rBC2LCN-reactive glycoprotein (BC2-S3) secreted from PDAC cells into the blood in patients with PDAC by liquid chromatography-tandem mass spectrometry analysis and lectin blotting. In immune staining, BC2-S3 was detected specifically in the tumor but not in normal tissues of PDAC. Lectin-ELISA was then developed to measure the serum level of BC2-S3 in healthy control (HC, n = 99) and patients with PDAC (n = 88). BC2-S3 exhibited higher in patients with PDAC than in those with HC. BC2-S3 showed similar diagnostic performance in all stages of PDAC (stages IA-IV, true positive rate = 76.1%, true negative rate = 81.8%) to CA19-9 (72.7%, 75.8%). Remarkably, BC2-S3 showed a significantly higher detection rate (89.7%) for early stage PDAC (IA-IIA) than CA19-9 (62.1%, P = 0.029). The combination of BC2-S3 and CA19-9 further improved the diagnostic ability for all stages of PDAC (81.8%, 87.9%). In conclusion, BC2-S3 is a glycobiomarker candidate for PDAC.

Astrocyte-derived SerpinA3N promotes neuroinflammation and epileptic seizures by activating the NF-κB signaling pathway in mice with temporal lobe epilepsy.

Impaired activation and regulation of the extinction of inflammatory cells and molecules in injured neuronal tissues are key factors in the development of epilepsy. SerpinA3N is mainly associated with the acute phase response and inflammatory response. In our current study, transcriptomics analysis, proteomics analysis, and Western blotting showed that the expression level of Serpin clade A member 3N (SerpinA3N) is significantly increased in the hippocampus of mice with kainic acid (KA)-induced temporal lobe epilepsy, and this molecule is mainly expressed in astrocytes. Notably, in vivo studies using gain- and loss-of-function approaches revealed that SerpinA3N in astrocytes promoted the release of proinflammatory factors and aggravated seizures. Mechanistically, RNA sequencing and Western blotting showed that SerpinA3N promoted KA-induced neuroinflammation by activating the NF-κB signaling pathway. In addition, co-immunoprecipitation revealed that SerpinA3N interacts with ryanodine receptor type 2 (RYR2) and promotes RYR2 phosphorylation. Overall, our study reveals a novel SerpinA3N-mediated mechanism in seizure-induced neuroinflammation and provides a new target for developing neuroinflammation-based strategies to reduce seizure-induced brain injury.

Serpina3c deficiency induced necroptosis promotes non-alcoholic fatty liver disease through ß-catenin/Foxo1/TLR4 signaling.

Non-alcoholic fatty liver disease (NAFLD) is a public health challenge and an increasing cause of chronic liver disease worldwide. However, the underlying molecular mechanism remains unclear. The aim of this study was to determine the precise role of serpina3c in the process of NAFLD. Male Apoe-/- /serpina3c-/- double knockout (DKO) and Apoe-/- mice were fed a high-fat diet (HFD) for 12 weeks. Several markers of steatosis and inflammation were evaluated. In vitro cell models induced by palmitic acid (PA) treatment were used to evaluate the beneficial effect of serpina3c on necroptosis and the underlying molecular mechanism. Compared with Apoe-/- mice, DKO mice exhibited a significantly exacerbated hepatic steatosis, increased hepatic triglyceride content and expression of genes involved in lipid metabolism (SREBP1c and SCD1), promoted hepatic inflammation and fibrosis, promoted necroptosis by increasing expression of receptor-interacting protein 3 (RIP3), phosphorylated mixed lineage kinase domain-like (MLKL) and high mobility group box 1 (HMGB1). Notably, serpina3c deficiency increased ß-catenin, Foxo1, and Toll-like receptor 4 (TLR4) protein expression. In vitro , serpina3c knockdown promoted necroptosis and lipid droplet formation under condition of lipotoxicity. However, these phenomena were reversed by the overexpression of serpina3c. Mechanistically, downregulation of serpina3c expression promoted Foxo1 and ß-catenin colocalized in the nucleus under condition of lipotoxicity, consequently upregulating the expression of TLR4. Conversely, disruption of Foxo1-ß/catenin by Foxo1 and ß-catenin inhibitors decreased TLR4 expression and ameliorated hepatic necroptosis in vitro. This study highlights a novel mechanism that serpina3c modulates NAFLD development by inhibiting necroptosis via ß-catenin/Foxo1/TLR4.

Neurorescuing effect of Cinacalcet against hypercalcemia-induced nerve injury in chronic kidney disease via TRAF2/cIAP1/KLF2/SERPINA3 signal axis.

Hypercalcemia is a common complication in chronic kidney disease (CKD) and unfortunately contributes to nerve injury. This study aims to investigate the potential role and underlying mechanisms of Cinacalcet (CIN) in hypercalcemia-driven nerve injury in CKD. A CKD mouse model was first established by adenine feeding to identify the therapeutic effects of CIN. Molecules related to CIN and CKD were predicted by bioinformatics analysis and their expression in the kidney tissues of CKD mice was measured by immunochemistry. Gain- and loss-of-functions assays were performed both in vitro and in vivo to evaluate their effects on nerve injury in CKD, as reflected by Scr and BUN, and brain calcium content as well as behavior tests. CIN ameliorated hypercalcemia-driven nerve injury in CKD mice. Interactions among TRAF2, an E3-ubiquitin ligase, KLF2, and SERPINA3 were bioinformatically predicted on CIN effect. CIN restricted the ubiquitin-mediated degradation of KLF2 by downregulating TRAF2. KLF2 targeted and inversely regulated SERPINA3 to repress hypercalcemia-driven nerve injury in CKD. CIN was substantiated in vivo to ameliorate hypercalcemia-driven nerve injury in CKD mice through the TRAF2/KLF2/SERPINA3 regulatory axis. Together, CIN suppresses SERPINA3 expression via TRAF2-mediated inhibition of the ubiquitin-dependent degradation of KLF2, thus repressing hypercalcemia-induced nerve injury in CKD mice.

Association between copy number variation of SERPINA3-1 gene and growth traits in Chinese cattle.

Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 (SERPINA3) belongs to the serine protease inhibitor family A subtype, and contains 8 genes from SERPINA3-1 to SERPINA3-8. Although the regulatory effects of these 8 genes have been revealed one by one in recent years, the related effects of SERPINA3-1 gene on cattle growth is still unclear. This study used quantitative Real time PCR (qPCR) to detect the type of copy number variation (CNV) of SERPINA3-1 gene in a total of 542 Chinese cattle, and expression of SERPINA3-1 gene in different tissues of Qinchuan cattles (adult) on mRNA level. Then association analysis was conducted between the detection results and cattle growth traits. The results showed that the Duplication type in SERPINA3-1 gene performed better on the growth traits and the CNV was significantly correlated with multiple growth traits (p < 0.05). In addition, SERPINA3-1 gene has different expression conditions in different tissues, results showed that SERPINA3-1 gene has a low expression in muscle. In conclusion, we speculate that the SERPINA3-1 gene can be used as a molecular marker and the result of this study could be a basic material for candidate functional genes for beef cattle growth and development.

In order to detect the gene expression diversification of the SERPINA3-1 gene, blood samples were collected from five Chinese cattle breeds, we detected related signal and made an associated analyze with cattle growth traits. We determined the copy number variation distribution of the SERPINA3-1 gene in cattle populations and found that the SERPINA3-1 gene has a certain promoting effect on the growth and development of Chinese cattle. For example, Pinan cattle with Duplication type copy number have a better performance on growth traits. This study has enriched the candidate genes of Chinese cattle molecular breeding and provided basic data for Chinese cattle breeding.

Identification of biomarkers and candidate small-molecule drugs in lipopolysaccharide (LPS)-induced acute lung injury by bioinformatics analysis.

BACKGROUND/OBJECTIVE: Acute lung injury (ALI) is a critical clinical syndrome with high rates of incidence and mortality. However, its molecular mechanism remains unclear. The current work aimed to explore the molecular mechanisms of ALI by identifying different expression genes (DEGs) and candidate drugs using a combination of chip analysis and experimental validation. METHODS: Three microarray datasets were downloaded from Gene Expression Omnibus (GEO) database to obtain DEGs. We conducted a Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway-enrichment analyses of overlapping DEGs among three databases. The expression level of key gene was verified by Western blotting analysis in LPS-treated ALI cell models. Finally, we predicted the candidate drugs targeting the key gene that might be effective for ALI treatment, and the role of candidate drug in treating ALI was verified by investigation. RESULTS: A total 29 overlapping DEGs were up-regulated in LPS-induced ALI groups. They were enriched in inflammation and inflammation-related pathways. Serpin family A member 3 (SERPINA3) was defined as a key gene because it was associated with inflammation pathway and up-regulated in microarray datasets in LPS-induced ALI. In LPS-induced human bronchial epithelial cells transformed with Ad12-SV40-2B (BEAS-2B) cells, SERPINA3 was enhanced. Pyridoxal phosphate as an upstream drug of SERPINA3 could improve cell viability and reduce expression inflammatory factors in LPS-treated BEAS-2B cells. CONCLUSION: Our study suggested that pyridoxal phosphate could be a candidate drug targeting SERPINA3 gene in LPS-induced ALI. It has protective and anti-inflammatory effects in BEAS-2B cells, and may become a potential novel treatment for ALI.

HOXA-AS2 enhances GBM cell malignancy by suppressing miR-2116-3p thereby upregulating SERPINA3.

BACKGROUND: Glioblastoma (GBM) is malignant, demanding more attention to the improvement of the diagnosis and therapy. LncRNAs have been implicated in the malignancy of GBM cells. METHODS: HOXA-AS2, miR-2116-3p and SERPINA3 expression levels in GBM tissues and cell lines were detected by qRT-PCR. Western blotting was performed to detect the protein levels of Bax and Bcl-2. Dual-luciferase reporter assay was for detection of relationship among these factors, together with RIP and RNA pull-down. CCK-8, EdU, wound healing and transwell assays were for detection of the role of HOXA-AS2, miR-2116-3p and SERPINA3 in cell viability, proliferation, migration and invasion in GBM, respectively. RESULTS: HOXA-AS2 and SERPINA3 showed higher level in GBM tissues and cell lines. Low level of HOXA-AS2 attenuated GBM cell growth in vitro. Moreover, the anti-tumor impact of silenced HOXA-AS2 was restored by miR-2116-3p inhibitor, but its tumor-promotional effect could be reversed by silenced SERPINA3. CONCLUSION: HOXA-AS2 enhanced GBM cell malignancy through sponging miR-2116-3p and releasing SERPINA3, which might shed light on the diagnosis and therapy for GBM in the future.

DNA methylation status of SERPINA3 gene involved in mRNA expression in three cattle breeds.

DNA methylation could take part in the gene expression and acts an important role in muscle development. In this study, DNA methylation and expression in adipose and muscle tissues were examined at the same time to evaluate the extent of epigenetic modifications and gene expression on the differentially methylated region (DMR) in SERPINA3. Chain reaction of bisulfite sequencing polymerase (BSP) was used to compared difference among DNA methylation patterns. The result of quantitative real-time PCR (qPCR) analysis showed that there was an extensive expression of SERPINA3 gene in tissue and there was a significant difference existing in muscle and adipose between Jiaxian cattle and individual of other breeds with increasing hybridization (p < 0.05). The statistic analyses indicated that DNA methylation patterns had a significant influence to the level of mRNA in tissue of fat and muscle. This study may be an important reference for investigating development of muscle tissue in cattle, and may promote the process of cattle molecular breeding.

Differential expression of tau species and the association with cognitive decline and synaptic loss in Alzheimer's disease.

Pathological tau proteins in patients with Alzheimer's disease (AD) mainly accumulate in the form of neurofibrillary tangles (NFTs) and neuritic plaques (NPs). However, the molecular properties of tau species present in NFTs and NPs are not known. We tested the hypothesis that tau species within NFT-predominant tissue (NFT_AD) are distinct and more toxic than those in NP-predominant tissue (NP_AD). We analyzed the tau species from post mortem prefrontal cortical brains of NFT_AD and NP_AD. Compared to NP_AD, NFT_AD displayed highly phosphorylated tau oligomers, possessed tau oligomers in extracellular vesicles, and the 3-repeat (3R) and 4-repeat (4R) isoforms were differentially expressed between the groups. Comparison of tau proteins isolated from NFT- versus NP-AD subjects demonstrated higher tau seeding activity in NFT subjects and a greater degree of inducing synaptic loss in cultured neurons. We propose that tau species from NFT-predominant tissues possess greater levels of degenerative properties, thereby causing synaptic loss and cognitive decline.

Plasma Proteomics Enable Differentiation of Lung Adenocarcinoma from Chronic Obstructive Pulmonary Disease (COPD).

Chronic obstructive pulmonary disease (COPD) is a major risk factor for the development of lung adenocarcinoma (AC). AC often develops on underlying COPD; thus, the differentiation of both entities by biomarker is challenging. Although survival of AC patients strongly depends on early diagnosis, a biomarker panel for AC detection and differentiation from COPD is still missing. Plasma samples from 176 patients with AC with or without underlying COPD, COPD patients, and hospital controls were analyzed using mass-spectrometry-based proteomics. We performed univariate statistics and additionally evaluated machine learning algorithms regarding the differentiation of AC vs. COPD and AC with COPD vs. COPD. Univariate statistics revealed significantly regulated proteins that were significantly regulated between the patient groups. Furthermore, random forest classification yielded the best performance for differentiation of AC vs. COPD (area under the curve (AUC) 0.935) and AC with COPD vs. COPD (AUC 0.916). The most influential proteins were identified by permutation feature importance and compared to those identified by univariate testing. We demonstrate the great potential of machine learning for differentiation of highly similar disease entities and present a panel of biomarker candidates that should be considered for the development of a future biomarker panel.

Nr1d1 Mediated Cell Senescence in Mouse Heart-Derived Sca-1+CD31- Cells.

AIM: Sca-1+CD31- cells are resident cardiac progenitor cells, found in many mammalian tissues including the heart, and able to differentiate into cardiomyocytes in vitro and in vivo. Our previous work indicated that heart-derived Sca-1+CD31- cells increased the Nr1d1 mRNA level of Nr1d1 with aging. However, how Nr1d1 affects the senescence of Sca-1+CD31- cells. METHODS: Overexpression and knockdown of Nr1d1 in Sca-1+CD31- cells and mouse cardiac myocyte (MCM) cell lines were performed by lentiviral transduction. The effects of Nr1d1 abundance on cell differentiation, proliferation, apoptosis, cell cycle, and transcriptomics were evaluated. Moreover, binding of Nr1d1 to the promoter region of Nr4a3 and Serpina3 was examined by a luciferase reporter assay. RESULTS AND CONCLUSIONS: Upregulation Nr1d1 in young Sca-1+CD31- cells inhibited cell proliferation and promoted apoptosis. However, depletion of Nr1d1 in aged Sca-1+CD31- cells promoted cell proliferation and inhibited apoptosis. Furthermore, Nr1d1 was negatively associated with cell proliferation, promoting apoptosis and senescence-associated beta-galactosidase production in MCMs. Our findings show that Nr1d1 stimulates Serpina3 expression through its interaction with Nr4a3. Nr1d1 may therefore act as a potent anti-aging receptor that can be a therapeutic target for aging-related diseases.

Serpin-A3 is Associated with Persistent Albuminuria in Adolescents with Secondary Podocytopathy, in a Region with High Prevalence of Chronic Kidney Disease of Unknown Origin.

Background: The state of Aguascalientes, Mexico, has been recognized as a chronic kidney disease hotspot. Screening studies have revealed a high prevalence of persistent albuminuria (pA), histologically characterized by glomerulomegaly, and incomplete podocyte fusion, probably associated with oligonephrony. To date, urinary biomarkers have not been explored in this population. Objective: The aim of the study was to identify the presence of potential biomarkers of early renal injury in patients with pA (pACR) and that correspond with the characteristic nephropathy profile that prevails in this entity. Methods: This is a cross-sectional, analytical, and comparative study. Four groups were recruited: adolescents aged 10-17 years with pACR, isolated albuminuria (iACR), no albuminuria (negative control), and adults with biopsy-confirmed glomerulopathy (positive control). Urinary excretion of SerpinA3, heat-shock protein-72 (HSP-72), podocalyxin (PCX), and nephrin was evaluated in urine samples. SerpinA3 and HSP-72 were analyzed by Western blot, and PCX and nephrin were quantified by enzyme-linked immunosorbent assay. Results: The mean GFR in the pACR group was 113.4 mL/min/1.73m2 and differed significantly only from that of the positive control group (65.1 mL/min/1.73m2). The mean albuminuria value in the pACR group was 48.9 mg/g. SerpinA3 concentration differed between groups (0.08 vs. 0.25 ng/mL, p < 0.001): it was significantly higher in the pACR group compared to the negative controls (p = 0.037). Conclusion: SerpinA3 was significantly associated with pA and could become a biomarker of early kidney injury. Further investigations are required to determine whether SerpinA3 precedes the development of albuminuria and its pathogenic role.

Inflammatory resolution and vascular barrier restoration after retinal ischemia reperfusion injury.

BACKGROUND: Several retinal pathologies exhibit both inflammation and breakdown of the inner blood-retinal barrier (iBRB) resulting in vascular permeability, suggesting that treatments that trigger resolution of inflammation may also promote iBRB restoration. METHODS: Using the mouse retinal ischemia-reperfusion (IR) injury model, we followed the time course of neurodegeneration, inflammation, and iBRB disruption and repair to examine the relationship between resolution of inflammation and iBRB restoration and to determine if minocycline, a tetracycline derivative shown to reverse microglial activation, can hasten these processes. RESULTS: A 90-min ischemic insult followed by reperfusion in the retina induced cell apoptosis and inner retina thinning that progressed for approximately 2 weeks. IR increased vascular permeability within hours, which resolved between 3 and 4 weeks after injury. Increased vascular permeability coincided with alteration and loss of endothelial cell tight junction (TJ) protein content and disorganization of TJ protein complexes. Shunting of blood flow away from leaky vessels and dropout of leaky capillaries were eliminated as possible mechanisms for restoring the iBRB. Repletion of TJ protein contents occurred within 2 days after injury, long before restoration of the iBRB. In contrast, the eventual re-organization of TJ complexes at the cell border coincided with restoration of the barrier. A robust inflammatory response was evident a 1 day after IR and progressed to resolution over the 4-week time course. The inflammatory response included a rapid and transient infiltration of granulocytes and Ly6C+ classical inflammatory monocytes, a slow accumulation of Ly6Cneg monocyte/macrophages, and activation, proliferation, and mobilization of resident microglia. Extravasation of the majority of CD45+ leukocytes occurred from the superficial plexus. The presence of monocyte/macrophages and increased numbers of microglia were sustained until the iBRB was eventually restored. Intervention with minocycline to reverse microglial activation at 1 week after injury promoted early restoration of the iBRB coinciding with decreased expression of mRNAs for the microglial M1 markers TNF-α, IL-1ß, and Ptgs2 (Cox-2) and increased expression of secreted serine protease inhibitor Serpina3n mRNA. CONCLUSIONS: These results suggest that iBRB restoration occurs as TJ complexes are reorganized and that resolution of inflammation and restoration of the iBRB following retinal IR injury are functionally linked.

CircSERPINA3 regulates SERPINA3-mediated apoptosis, autophagy and aerobic glycolysis of prostate cancer cells by competitively binding to MiR-653-5p and recruiting BUD13.

BACKGROUND: Prostate cancer (PCa) belongs to an epithelial malignancy that occurs in the prostate gland and is the most common malignancy of the male genitourinary system. Referring to related literature, circSERPINA3 has been reported to be up-regulated in PCa. However, its biological function remains unclear. PURPOSE: This study aimed to reveal the specific role and relevant molecular mechanism of circSERPINA3 in PCa. METHODS: RT-qPCR was used to examine gene expression and functional analyses were conducted to verify the effect of circSERPINA3 on cell apoptosis, autophagy and aerobic glycolysis in PCa cells. Mechanism assays were applied to evaluate the relationship among circSERPINA3/miR-653-5p/SERPINA3/BUD13. RESULTS: CircSERPINA3 was verified to be up-regulated in PCa cells and to inhibit cell apoptosis while promoting aerobic glycolysis and autophagy in PCa cells. CircSERPINA3 and SERPINA3 were also testified to bind to miR-653-5p through a line of mechanism experiments. Moreover, it was discovered that circSERPINA3 could stabilize SERPINA3 mRNA via recruiting BUD13. Additionally, SERPINA3 was verified to inhibit cell apoptosis, while promoting aerobic glycolysis and autophagy in PCa cells. CONCLUSIONS: Our study suggested that circSERPINA3 regulated apoptosis, autophagy and aerobic glycolysis of PCa cells by competitively binding to miR-653-5p and recruiting BUD13.

Protective role of serpina3c as a novel thrombin inhibitor against atherosclerosis in mice.

Abnormal vascular smooth muscle cell (VSMC) proliferation is a critical step in the development of atherosclerosis. Serpina3c is a serine protease inhibitor (serpin) that plays a key role in metabolic diseases. The present study aimed to investigate the role of serpina3c in atherosclerosis and regulation of VSMC proliferation and possible mechanisms. Serpina3c is down-regulated during high-fat diet (HFD)-induced atherosclerosis. An Apoe-/-/serpina3c-/--double-knockout mouse model was used to determine the role of serpina3c in atherosclerosis after HFD for 12 weeks. Compared with Apoe-/- mice, the Apoe-/-/serpina3c-/- mice developed more severe atherosclerosis, and the number of VSMCs and macrophages in aortic plaques was significantly increased. The present study revealed serpina3c as a novel thrombin inhibitor that suppressed thrombin activity. In circulating plasma, thrombin activity was high in the Apoe-/-/serpina3c-/- mice, compared with Apoe-/- mice. Immunofluorescence staining showed thrombin and serpina3c colocalization in the liver and aortic cusp. In addition, inhibition of thrombin by dabigatran in serpina3c-/- mice reduced neointima lesion formation due to partial carotid artery ligation. Moreover, an in vitro study confirmed that thrombin activity was also decreased by serpina3c protein, supernatant and cell lysate that overexpressed serpina3c. The results of experiments showed that serpina3c negatively regulated VSMC proliferation in culture. The possible mechanism may involve serpina3c inhibition of ERK1/2 and JNK signaling in thrombin/PAR-1 system-mediated VSMC proliferation. Our results highlight a protective role for serpina3c as a novel thrombin inhibitor in the development of atherosclerosis, with serpina3c conferring protection through the thrombin/PAR-1 system to negatively regulate VSMC proliferation through ERK1/2 and JNK signaling.

Differential gene expression in peritumoral brain zone of glioblastoma: role of SERPINA3 in promoting invasion, stemness and radioresistance of glioma cells and association with poor patient prognosis and recurrence.

PURPOSE: Glioblastoma (GBM) is a highly invasive tumor. Despite advances in treatment modalities, tumor recurrence is common, seen mainly in the peritumoral brain zone (PBZ). We aimed to molecularly characterize PBZ, to understand the pathobiology of tumor recurrence. METHODS/PATIENTS: We selected eight differentially regulated genes from our previous transcriptome profiling study on tumor core and PBZ. Expression of selected genes were validated in GBM (tumor core and PBZ, n = 37) and control (n = 22) samples by real time quantitative polymerase chain reaction (qPCR). Serine protease inhibitor clade A, member 3 (SERPINA3) was selected for further functional characterization in vitro by gene knockdown approach in glioma cells. Its protein expression by immunohistochemistry (IHC) was correlated with other clinically relevant GBM markers, patient prognosis and tumor recurrence. RESULTS: The mRNA expression of selected genes from the microarray data validated in tumor core and PBZ and was similar to publicly available databases. SERPINA3 knock down in vitro showed decreased tumor cell proliferation, invasion, migration, transition to mesenchymal phenotype, stemness and radioresistance. SERPINA3 protein expression was higher in PBZ compared to tumor core and also was higher in older patients, IDH wild type and recurrent tumors. Finally, its expression showed positive correlation with poor patient prognosis. CONCLUSIONS: SERPINA3 expression contributes to aggressive GBM phenotype by regulating pro-tumorigenic actions in vitro and is associated with adverse clinical outcome.

Polymorphic SERPINA3-R124C reduces pathogenesis of its wild type by shortening the lifetime of oligomeric Aß.

Amyloid beta (Aß) 42 peptide accumulated in Alzheimer disease (AD) patients' brain, often colocalized with serine protease inhibitor family A member 3 (SERPINA3). Being a chaperon, SERPINA3 accelerated Aß42 fibrillization. While analyzing chaperon activity of human SERPINA3 polymorphisms, we found SERPINA3-R124C played a role in protecting cells from Aß42 cytotoxicity. SH-SY5Y cells exposed to Aß42 preincubated with wild-type SERPINA3 (SERPINA3-WT) resulted in extended toxicity leading cell death whereas Aß42 with SERPINA3-R124C resulted in less cytotoxicity. Transmission electron microscope and thioflavin T assay revealed that SERPINA3-R124C shortened lifetime of small soluble oligomer and maintained ß-sheet rich protofibril-like aggregates for longer time compared to that of with SERPINA3-WT. Western blot assay confirmed that SERPINA3-R124C converted Aß42 mostly into high molecular aggregates. Here, we demonstrate first time that polymorphic SERPINA3 acts as a benign chaperon by modulating the transition states of Aß42, which may contribute to the reduction of AD risk.

Serpina3n is closely associated with fibrotic procession and knockdown ameliorates bleomycin-induced pulmonary fibrosis.

OBJECTIVE: Pulmonary fibrosis is a fatal interstitial lung disease that is characterized by excessive accumulation of extracellular matrix (ECM) and remodeling of lung. The precise mechanisms underlying pulmonary fibrosis still remain unclear. In the current study, we aimed to investigate the alteration and function of serine (or cysteine) peptidase inhibitor, clade A, member 3 N (Serpina3n) in pulmonary fibrotic models and explore the potential mechanisms. METHODS: We induced pulmonary fibrosis in mice by silica and bleomycin respectively and determined Serpina3n in lung tissues, and then verified the expression of Serpina3n and its correlation with pulmonary fibrosis at seven time points in a bleomycin longstanding model. Moreover, adeno-associated virus type 9 (AAV9)-mediated Serpina3n knockdown was used to treat pulmonary fibrosis in the bleomycin model, whose possible mechanisms would be preliminarily explored by detecting chymotrypsin C as an example. RESULTS: Serpina3n was up-regulated significantly in lungs of both models at mRNA and protein levels relative to control. Notably, the expression of Serpina3n peaked during the 3rd week and then decreased until nearly normal levels during the 10th week, which was closely related to fibrotic procession in bleomycin-treated mice. AAV-mediated Serpina3n knockdown in the lung tissues alleviated bleomycin-induced fibrotic symptoms at various levels and disinhibit chymotrypsin C. CONCLUSIONS: Our study revealed that Serpina3n is a critical regulator in pulmonary fibrosis and suggested Serpina3n inhibition as a potential therapeutic strategy in chronic pulmonary injuries.