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Objectives: Cardiovascular diseases are the leading cause of death worldwide, with patients having limited options for treatment. Pigment epithelium-derived factor (PEDF) is an endogenous multifunctional protein with several mechanisms of action. Recently, PEDF has emerged as a potential cardioprotective agent in response to myocardial infarction. However, PEDF is also associated with pro-apoptotic effects, complicating its role in cardioprotection. This review summarises and compares knowledge of PEDF's activity in cardiomyocytes with other cell types and draws links between them. Following this, the review offers a novel perspective of PEDF's therapeutic potential and recommends future directions to understand the clinical potential of PEDF better., Key Findings: PEDF's mechanisms as a pro-apoptotic and pro-survival protein are not well understood, despite PEDF's implication in several physiological and pathological activities. However, recent evidence suggests that PEDF may have significant cardioprotective properties mediated by key regulators dependent on cell type and context., Conclusions: While PEDF's cardioprotective activity shares some key regulators with its apoptotic activity, cellular context and molecular features likely allow manipulation of PEDF's cellular activity, highlighting the importance of further investigation into its activities and its potential to be applied as a therapeutic to mitigate damage from a range of cardiac pathologies., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

SERPINB5 is a mammary serine protease inhibitor, which is involved in various cellular functions. The aberrant expression of SERPINB5 is reported in many cancers along with GBC but limited information is available about its role in genetic predisposition for GBC. We carried out case-control study in 206 cases and 219 controls. Promoter SNPs were genotyped by Sanger's sequencing. In-silico promoter analysis and luciferase reporter assay were done to elucidate the role of promoter variants in regulation of SERPINB5 expression. Out of four SNPs, three SERPINB5 promoter variants showed association with GBC in different models. The 'C' allele of variant rs17071138 was found to be significantly associated with GBC (p = 0.017). The 'T' allele of rs3744940 significantly increased the risk for GBC in dominant (p = 0.035) and additive models (p = 0.005). Also, rs3744941 'T' allele increased the risk for GBC by dominant (p = 0.042) as well as additive models (p = 0.016). In-silico promoter analysis and luciferase reporter assay revealed the probable regulatory role of the SERPINB5 promoter variant rs17071138 on the expression. Overall, our study reveals the genetic association of SERPINB5 promoter variants with GBC and possible role of rs17071138 in the regulation of expression., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Purpose: NK-5962 is a key component of photoelectric dye-coupled polyethylene film, designated Okayama University type-retinal prosthesis (OUReP™). Previously, we found that NK-5962 solution could reduce the number of apoptotic photoreceptors in the eyes of the Royal College of Surgeons (RCS) rats by intravitreal injection under a 12 h light/dark cycle. This study aimed to explore possible molecular mechanisms underlying the anti-apoptotic effect of NK-5962 in the retina of RCS rats., Methods: RCS rats received intravitreal injections of NK-5962 solution in the left eye at the age of 3 and 4 weeks, before the age of 5 weeks when the speed in the apoptotic degeneration of photoreceptors reaches its peak. The vehicle-treated right eyes served as controls. All rats were housed under a 12 h light/dark cycle, and the retinas were dissected out at the age of 5 weeks for RNA sequence (RNA-seq) analysis. For the functional annotation of differentially expressed genes (DEGs), the Metascape and DAVID databases were used., Results: In total, 55 up-regulated DEGs, and one down-regulated gene ( LYVE1 ) were found to be common among samples treated with NK-5962. These DEGs were analyzed using Gene Ontology (GO) term enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway analyses. We focused on the up-regulated DEGs that were enriched in extracellular matrix organization, extracellular exosome, and PI3K-Akt signaling pathways. These terms and pathways may relate to mechanisms to protect photoreceptor cells. Moreover, our analyses suggest that SERPINF1 , which encodes pigment epithelium-derived factor (PEDF), is one of the key regulatory genes involved in the anti-apoptotic effect of NK-5962 in RCS rat retinas., Conclusions: Our findings suggest that photoelectric dye NK-5962 may delay apoptotic death of photoreceptor cells in RCS rats by up-regulating genes related to extracellular matrix organization, extracellular exosome, and PI3K-Akt signaling pathways. Overall, our RNA-seq and bioinformatics analyses provide insights in the transcriptome responses in the dystrophic RCS rat retinas that were induced by NK-5962 intravitreal injection and offer potential target genes for developing new therapeutic strategies for patients with retinitis pigmentosa.

Pigment epithelium derived factor (PEDF), an endogenous inhibitor of angiogenesis, targets the growth of aberrant blood vessels in many tissues, including the eye. In this study we show that PEDF prevented early mitogenic signals of vascular endothelial growth factor (VEGF-A) in primate retinal endothelial cells, blocking proliferation, migration and tube formation. PEDF inhibited the phosphorylation and activation of five major downstream VEGF-A signaling partners, namely phosphoinositide-3-OH Kinase (PI3K), AKT, FAK, Src (Y416), and PLC-γ. It did so by binding to the extracellular domain of VEGF-R2, blocking VEGF-A-induced tyrosine phosphorylation (Tyr 951 and Tyr 1175), and inhibiting VEGF-R2 receptor kinase activity. PEDF had no effect on the transcription or translation of VEGF-R2 in cultured HUVECs. PEDF also bound to the extracellular domain of VEGF-R1. We conclude that PEDF blocks the growth of new blood vessels, in part, by reducing VEGF-A activation of its key mitogenic receptor, VEGF-R2, and by preventing its downstream signals in endothelial cells., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Children with hypoplastic lung disease associated with congenital diaphragmatic hernia (CDH) continue to suffer significant morbidity and mortality secondary to progressive pulmonary disease. Recently published work from our lab demonstrated the potential of Roxadustat (FG-4592), a prolyl hydroxylase inhibitor, as a treatment for CDH-associated pulmonary hypoplasia. Treatment with Roxadustat led to significantly accelerated compensatory lung growth (CLG) through downregulation of pigment epithelium-derived factor (PEDF), an anti-angiogenic factor, rather than upregulation of vascular endothelial growth factor (VEGF). PEDF and its role in pulmonary development is a largely unexplored field. In this study, we sought to further evaluate the role of PEDF in accelerating CLG. PEDF-deficient mice demonstrated significantly increased lung volume, total lung capacity, and alveolarization compared to wild type controls following left pneumonectomy without increased VEGF expression. Furthermore, Roxadustat administration in PEDF-deficient mice did not further accelerate CLG. Human microvascular endothelial lung cells (HMVEC-L) and human pulmonary alveolar epithelial cells (HPAEC) similarly demonstrated decreased PEDF expression with Roxadustat administration. Additionally, downregulation of PEDF in Roxadustat-treated HMVEC-L and HPAEC, a previously unreported finding, speaks to the potential translatability of Roxadustat from small animal studies. Taken together, these findings further suggest that PEDF downregulation is the primary mechanism by which Roxadustat accelerates CLG. More importantly, these data highlight the critical role PEDF may have in pulmonary growth and development, a previously unexplored field., (© 2021 Federation of American Societies for Experimental Biology.)

Serine proteinase inhibitor B9 (serpin B9) is a member of the serine protease inhibitor superfamily, which is widely found in animals, plants and microorganisms. Serpin B9 has been reported to protect cells from the immune‑killing effect of granzyme B (GrB) released by lymphocytes. In recent years, an increasing number of studies have indicated that serpin B9 is involved in tumour apoptosis, immune evasion, tumorigenesis, progression, metastasis, drug resistance and even in maintaining the stemness of cancer stem cells (CSCs). Moreover, according to clinical studies, serpin B9 has been demonstrated to be significantly associated with the development of precancerous lesions, a poor prognosis and ineffective therapies, suggesting that serpin B9 may be a potential target for cancer treatment and an indicator of cancer diagnosis; thus, it has begun to attract increased attention from scholars. The present review concisely described the structure and biological functions of the serpin superfamily and serpin B9. In addition, related research on serpins in cancer is discussed in order to provide a comprehensive understanding of the role of serpin B9 in cancer, as well as its clinical significance for cancer diagnosis and prognosis.

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a progressive neurodegenerative disease caused by point mutations in the gene for neuroserpin, a serine protease inhibitor of the nervous system. Different mutations are known that are responsible for mutant neuroserpin polymerization and accumulation as inclusion bodies in many cortical and subcortical neurons, thereby leading to cell death, dementia and epilepsy. Many efforts have been undertaken to elucidate the molecular pathways responsible for neuronal death. Most investigations have concentrated on analysis of intracellular mechanisms such as endoplasmic reticulum (ER) stress, ER-associated protein degradation (ERAD) and oxidative stress. We have generated a HEK-293 cell model of FENIB by overexpressing G392E-mutant neuroserpin and in this study we examine trafficking and toxicity of this polymerogenic variant. We observed that a small fraction of mutant neuroserpin is secreted via the ER-to-Golgi pathway, and that this release can be pharmacologically regulated. Overexpression of the mutant form of neuroserpin did not stimulate cell death in the HEK-293 cell model. Finally, when treating primary hippocampal neurons with G392E neuroserpin polymers, we did not detect cytotoxicity or synaptotoxicity. Altogether, we report here that a polymerogenic mutant form of neuroserpin is secreted from cells but is not toxic in the extracellular milieu.

Vaspin is a novel adipokine mainly expressed in visceral adipose tissue and closely related to obesity and insulin-resistance. Currently, data about its ovarian expression are limited to animal models and its role in human reproduction is largely unexplored. Our study's aims were then to characterise vaspin expression in the human ovary and to study in vitro its effects on granulosa cells physiology. Secondly, we assessed vaspin and its receptor GRP78 variations in granulosa cells and follicular fluid of a cohort of 112 infertile women undergoing an in vitro fertilisation procedure and allocated to three groups, each including normal-weight and obese subjects: 34 PCOS patients, 33 women with isolated polycystic ovary morphology (ECHO group) and 45 controls. Vaspin and GRP78 expression in the ovary was assessed by immunohistochemistry, RT-qPCR and Western blot. Granulosa cells and follicular fluid were analysed by RT-qPCR and ELISA, respectively. In vitro, granulosa cells metabolism was studied after stimulation with recombinant human vaspin, with and without a siRNA directed against GRP78. Vaspin was highly expressed in the human ovary and concentration-dependently enhanced granulosa cells steroidogenesis, proliferation and viability through GRP78 (P < 0.0001). Vaspin levels in both granulosa cells and follicular fluid were significantly higher in obese women (P < 0.0001) and in the normal-weight ECHO group (P < 0.001), which also had the highest expression rates of GRP78 (P < 0.05). Although further investigation is needed, vaspin appears as a novel modulator of human granulosa cells physiology and possibly plays a role in PCOS pathogenesis, notably protecting from insulin-resistance induced complications.

Among animals, diploblasts contain two germ layers, endoderm and ectoderm, while triploblasts have a distinct third germ layer called the mesoderm. Spiralians are a group of triploblast animals that have highly conserved development: they share the distinctive spiralian cleavage pattern as well as a unique source of mesoderm, the ectomesoderm. This population of mesoderm is distinct from endomesoderm and is considered a hallmark of spiralian development, but the regulatory network that drives its development is unknown. Here we identified ectomesoderm-specific genes in the mollusc Tritia (aka Ilyanassa) obsoleta through differential gene expression analyses comparing control and ectomesoderm-ablated embryos, followed by in situ hybridization of identified transcripts. We identified a Tritia serpin gene (ToSerpin1) that appears to be specifically expressed in the ectomesoderm of the posterior and head. Ablation of the 3a and 3b cells, which make most of the ectomesoderm, abolishes ToSerpin1 expression, consistent with its expression in these cells. Morpholino knockdown of ToSerpin1 causes ectomesoderm defects, most prominently in the muscle system of the larval head. This is the first gene identified that is specifically implicated in spiralian ectomesoderm development., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pigment epithelium-derived factor (PEDF) plays a role in protecting retinal pigment epithelial (RPE) cells from oxidative stress (OS), a causative factor of RPE cell death. Genetically modified mesenchymal stem cells (MSCs) can be used to treat critical and incurable retinal diseases. Here, we overexpressed PEDF in placenta-derived MSCs (PD-MSCs PEDF , PEDF+) using a nonviral gene delivery system and evaluated the characteristics of PD-MSCs PEDF and their potential regenerative effects on RPE cells damaged by H 2 O 2 -induced OS. PD-MSCs PEDF maintained their stemness, cell surface marker, and differentiation potential characteristics. Compared to naive cells, PD-MSCs PEDF promoted mitochondrial respiration by enhancing biogenesis regulators (e.g., NRF1, PPARGC1A, and TFAM) as well as antioxidant enzymes (e.g., HMOXs, SODs, and GPX1). Compared to OS-damaged RPE cells cocultured with naive cells, OS-damaged RPE cells cocultured with PD-MSCs PEDF showed PEDF upregulation and VEGF downregulation. The expression levels of antioxidant genes and RPE-specific genes, such as RPE65, RGR, and RRH, were significantly increased in RPE cells cocultured with PD-MSCs PEDF . Furthermore, OS-damaged RPE cells cocultured with PD-MSCs PEDF had dramatically enhanced mitochondrial functions, and antiapoptotic effects improved due to cell survival signaling pathways. In the H 2 O 2 -induced retinal degeneration rat model, compared to administration of the naive counterpart, intravitreal administration of PD-MSCs PEDF alleviated proinflammatory cytokines and restored retinal structure and function by increasing PEDF expression and decreasing VEGF expression. Intravitreal administration of PD-MSCs PEDF also protected retinal degeneration against OS by increasing antioxidant gene expression and regulating the mitochondrial ROS levels and biogenesis. Taken together, PEDF overexpression in PD-MSCs improved the mitochondrial activities and induced OS-damaged RPE cell regeneration by regulating the oxidative status and mitochondrial biogenesis in vitro and in vivo. These data suggest that genetic modification of PEDF in PD-MSCs might be a new cell therapy for the treatment of retinal degenerative diseases.

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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Inherited palmoplantar keratodermas refer to a large and heterogeneous group of conditions resulting from abnormal epidermal differentiation and featuring thickening of the skin of the palms and soles. Here, we aimed at delineating the genetic basis of an autosomal recessive form of palmoplantar keratodermas manifesting with erythematous hyperkeratotic plaques over the palms and soles, extending to non-palmoplantar areas. Whole-exome sequencing in affected individuals revealed homozygous nonsense variants in the SERPINA12 gene. SERPINA12 encodes the visceral adipose tissue-derived serpin A12, a serine protease inhibitor. The pathogenic variants were found to result in reduced visceral adipose tissue-derived serpin A12 expression in patients' skin biopsies in comparison to healthy controls. In addition, SERPINA12 downregulation in three-dimensional skin equivalents was associated with marked epidermal acanthosis and hyperkeratosis, replicating the human phenotype. Moreover, decreased SERPINA12 expression resulted in reduced visceral adipose tissue-derived serpin A12-mediated inhibition of kallikrein 7 activity as well as decreased levels of desmoglein-1 and corneodesmosin, two known kallikrein 7 substrates, which are required for normal epidermal differentiation. The present data, taken collectively, demarcate a unique type of autosomal recessive palmoplantar keratodermas, attribute to visceral adipose tissue-derived serpin A12 a role in skin biology, and emphasize the importance of mechanisms regulating proteolytic activity for normal epidermal differentiation., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Cancer cachexia and muscle loss are associated with increased morbidity and mortality. In preclinical animal models, blocking activin receptor (ACVR) ligands has improved survival and prevented muscle wasting in cancer cachexia without an effect on tumour growth. However, the underlying mechanisms are poorly understood. This study aimed to identify cancer cachexia and soluble ACVR (sACVR) administration-evoked changes in muscle proteome., Methods: Healthy and C26 tumour-bearing (TB) mice were treated with recombinant sACVR. The sACVR or PBS control were administered either prior to the tumour formation or by continued administration before and after tumour formation. Muscles were analysed by quantitative proteomics with further examination of mitochondria and nicotinamide adenine dinucleotide (NAD + ) metabolism. To complement the first prophylactic experiment, sACVR (or PBS) was injected as a treatment after tumour cell inoculation., Results: Muscle proteomics in TB cachectic mice revealed downregulated signatures for mitochondrial oxidative phosphorylation (OXPHOS) and increased acute phase response (APR). These were accompanied by muscle NAD + deficiency, alterations in NAD + biosynthesis including downregulation of nicotinamide riboside kinase 2 (Nrk2), and decreased muscle protein synthesis. The disturbances in NAD + metabolism and protein synthesis were rescued by treatment with sACVR. Across the whole proteome and APR, in particular, Serpina3n represented the most upregulated protein and the strongest predictor of cachexia. However, the increase in Serpina3n expression was associated with increased inflammation rather than decreased muscle mass and/or protein synthesis., Conclusions: We present evidence implicating disturbed muscle mitochondrial OXPHOS proteome and NAD + homeostasis in experimental cancer cachexia. Treatment of TB mice with a blocker of activin receptor ligands restores depleted muscle NAD + and Nrk2, as well as decreased muscle protein synthesis. These results indicate putative new treatment therapies for cachexia and that although acute phase protein Serpina3n may serve as a predictor of cachexia, it more likely reflects a condition of elevated inflammation., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

This study aims to investigate how Maspin affects the EMT and angiogenesis of gastric cancer (GC) cells via ITGB1/FAK pathway. Immunohistochemistry was used to evaluate the expressions of Maspin, ITGB1, FAK, E-cadherin, Vimentin, D2-40, and CD34 in GC and adjacent normal tissues from 160 patients. Then, the human GC cells with different degree of differentiation were transfected with Maspin CRISPR activation plasmid, ITGB1 siRNA and/or Maspin siRNA, followed by the following experiments, including qRT-PCR, western blotting, tube formation assay, Transwell assay and wound healing. GC tumor tissues manifested decreased Maspin with the activated ITGB1/FAK pathway. In tumor tissues, Maspin was negatively correlated with the expressions of ITGB1 and FAK, as well as Lauren's classification, differentiation degree, and TNM stage. Besides, Maspin was negatively related with lymphatic vessel density (LVD) and microvessel density (MVD), Vimentin and VEGF, but was positive correlated with E-cadherin. Maspin expression decreased, but ITGB1 and p-FAK expressions increased gradually in MKN-28 (well differentiated), SGC-7901 (moderate differentiated), and MKN-45 (poorly differentiated). Maspin CRISPR and ITGB1 siRNA increased E-cadherin with the decreased Vimentin, VEGF and bFGF, and the reductions of tube length. In comparison with the ITGB1 siRNA group, cells in the Maspin siRNA + ITGB1 siRNA group presented the more evident EMT and angiogenesis. Furthermore, ITGB1 siRNA reduced the malignancies of GC cells, which could be restored by Maspin siRNA. Maspin was downregulated in GC tissues, which could inhibit the EMT and angiogenesis by blocking the ITGB1/FAK pathway, thereby decreasing cell invasion and migration of GC.

Background: Percutaneous coronary intervention represents the most important treatment modality of coronary artery stenosis. In-stent restenosis (ISR) is still a limitation for the long-term outcome despite the introduction of drug eluting stents. It has been shown that adipokines directly influence vessel wall homeostasis by influencing the function of endothelial cells and arterial smooth muscle cells. Visceral adipose tissue-derived serpin vaspin was recently identified as a member of serine protease inhibitor family and serveral studies could demonstrate a relation to metabolic diseases. The aim of this study was to investigate a role of vaspin in the development of in-stent restenosis in vivo and on migration of smooth muscle cells and endothelial cells in vitro., Methods: We studied 85 patients with stable coronary artery disease who underwent elective and successful PCI with implatation of drug eluting stents. Blood samples were taken directly before PCI. Vaspin plasma levels were measured by specific ELISA. ISR was evaluated eight months later by coronary angiography. Human coronary artery smooth muscle cells (HCASMC) and human umbilical vein endothelial cells (HUVEC) migration was analyzed by an in-vitro migration assay with different concentrations (0.004ng/mL up to 40ng/mL) of vaspin as well as by an scratch assay. For proliferation an impedance measurement with specialiced E-Plates was performed., Results: During the follow up period, 14 patients developed ISR. Patients with ISR had significantly lower vaspin plasma levels compared to patients without ISR (0.213 ng/ml vs 0.382 ng/ml; p = 0.001). In patients with plasma vaspin levels above 1.35 ng/ml we could not observe any restenosis. There was also a significant correlation of plasma vaspin levels and late lumen loss in the stented coronary segments. Further we could demonstrate that vaspin nearly abolishes serum induced migration of HCASMC (100% vs. 9%; p<0.001) in a biphasic manner but not migration of HUVEC. Proliferation of HCASMC and HUVEC was not modulated by vaspin treatment., Conclusion: We were able to show that the adipokine vaspin selectively inhibits human coronary SMC migration in vitro and has no effect on HUVEC migration. Vaspin had no effect on proliferation of HUVEC which is an important process of the healing of the stented vessel. In addition, the occurrence of ISR after PCI with implantation of drug eluting stents was significantly associated with low vaspin plasma levels before intervention. Determination of vaspin plasma levels before PCI might be helpful in the identification of patients with high risk for development of ISR after stent implantation. In addition, the selective effects of vaspin on smooth muscle cell migration could potentially be used to reduce ISR without inhibition of re-endothelialization of the stented segment., Competing Interests: The authors have declared that no competing interests exist.

Diapause is a state of arrested growth, which allows insects to adapt to diverse environments. Serine protease inhibitors (serpins) play an important role in various physiological processes, including blood coagulation, fibrinolysis, development, complement activation and extracellular matrix remodeling. We hypothesized that serpin may affect energy metabolism and thereby control diapause of migratory locust (Locusta migratoria) embryos by regulating protease cascades. A total of seven nonredundant serpin genes (named serpin1-serpin7) of L. migratoria were obtained through transcriptomic analysis. We further performed label-free proteomic sequencing and analysis of diapause and nondiapause eggs of L. migratoria, revealing significant differences in serpin7 expression. A significant reduction in diapause rate under the short photoperiod was observed in insects treated with serpin7 double-stranded RNA. Furthermore, knockdown of the serpin7 gene resulted in significant upregulation of the activity of polyphenol oxidase. We therefore propose that the observed serpin7 gene plays a crucial role in diapause, suggesting that control of energy metabolism may have potential as a future strategy for the reproductive control of insect pests., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Alzheimer's disease (AD) is the most common dementia type affecting nearly 44 million people worldwide. Recent findings point to microglia as a significant contributor to neural development, neuroinflammation, and degeneration. Dysregulated immunoactivity in AD has been broadly studied, and current research on animal models enabled us to identify a new cluster of microglia (disease-associated microglia) alongside previously detected glial populations (e.g., plaque-associated microglia, dark microglia, Human Alzheimer's microglia) associated with neuroinflammation and with macrophagic activity. These distinct populations of glia show a spatial distribution within plaques with unique imaging features and distinct gene expression profile. Novel genetic approaches using single-nuclei RNA sequencing (sn-RNA seq) allowed researchers to identify gene expression profiles from fixed human samples. Recent studies, exposing transcriptomic clusters of disease-related cells and analyzing sequenced RNA from sorted myeloid cells, seem to confirm the hypothesis of the central role of glia in the pathogenesis of Alzheimer's disease. These discoveries may shed light on the effects of microglial activation and differences in gene expression profiles, furthering research towards the development of a cell-specific therapy. In this review, we examine recent studies that guide us towards recognizing the role of diverse populations of glial cells and their possible heterogeneous functional states in the pathogenesis of AD in humans., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Porcine pancreatic stem cells (pPSCs) can be induced to insulin-secreting cells and therefore considered the most promising seeding cells for curing human diabetes in future. However, insufficient pPSCs number is one of the bottleneck problems before its clinical application. SerpinB1 is a serine protease inhibitor in neutrophils and can directly promote the proliferation of β cells. Whether SerpinB1 is involved in pPSC proliferation and differentiation remains unknown. The effects of SerpinB1 on pPSCs proliferation were measured by Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine, qRT-PCR, western blot, and flow cytometry assays. We found that pPSCs did not efficiently reach the S phase when SerpinB1 expression was knocked down with short hairpin RNA (sh-SerpinB1), the expression of Cyclin D1, CDK-2, and PCNA also decreased. Meanwhile, cell viability and proliferation ability were both declined. Further analyses showed that the expression level of phosphorylated STAT3/STAT3was downregulated, along with an upregulation of p53 and p21. We used a two-step induction method to induce pPSCs to insulin-secreting cells and found that SerpinB1 expression in insulin-secreting cells was higher than in pPSCs. Meanwhile, the protein expression level of phosphorylated STAT3/STAT3 was increased while p53 and p21 was decreased in induced insulin-secreting cells in comparison with control cells. The insulin-secreting cells derived from the sh-SerpinB1 cells secreted less insulin and showed poor sensitivity to high glucose than control group. However, the insulin-secreting cells derived from the ov-SerpinB1 cells has a quite contrary tendency. In conclusion, this study demonstrates that SerpinB1 promotes the proliferation of pPSCs through the STAT3 signaling pathway, and SerpinB1 is a key factor for maintaining the viability of pPSCs during the transition to insulin-secreting cells., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

SerpinB1, a protease inhibitor and neutrophil survival factor, was recently linked with IL-17-expressing T cells. Here, we show that serpinB1 ( Sb1 ) is dramatically induced in a subset of effector CD4 cells in experimental autoimmune encephalomyelitis (EAE). Despite normal T cell priming, Sb1 -/- mice are resistant to EAE with a paucity of T helper (T H ) cells that produce two or more of the cytokines, IFNγ, GM-CSF, and IL-17. These multiple cytokine-producing CD4 cells proliferate extremely rapidly; highly express the cytolytic granule proteins perforin-A, granzyme C (GzmC), and GzmA and surface receptors IL-23R, IL-7Rα, and IL-1R1; and can be identified by the surface marker CXCR6. In Sb1 -/- mice, CXCR6 + T H cells are generated but fail to expand due to enhanced granule protease-mediated mitochondrial damage leading to suicidal cell death. Finally, anti-CXCR6 antibody treatment, like Sb1 deletion, dramatically reverts EAE, strongly indicating that the CXCR6 + T cells are the drivers of encephalitis., Competing Interests: Conflict of interest statement: L.H. and E.R.-O. filed a provisional patent related to the findings of this study and have equity ownership and are on the Scientific Advisory Board of the recently launched Edelweiss Immune, Inc. The other authors declare no conflict of interest.

Ovalbumin-related protein X (OVAX) is a 50 kDa egg-white protein which has heparin-binding affinity. In this study, migration of OVAX and its heparin-binding performance during embryogenesis of fertilized egg were investigated to explore a possible involvement in chick embryo development. Western blotting of egg yolks at different stages using an anti-OVAX antibody showed that OVAX accumulates in yolk during incubation of fertilized egg. Immunohistochemical analysis of embryo resided in 10-day-incubated eggs showed that OVAX existed in almost all tissues of the embryo. These suggest that OVAX is incorporated from egg white into the embryo through yolk sac. Heparin-sepharose chromatography, isothermal titrating calorimetry using fondaparinux as a ligand, and zeta potential measurement indicated that OVAX retained the heparin-binding affinity (Kd = 0.185 ± 0.037) even after 10 D incubation of fertilized egg, although the affinity was slightly decreased during egg incubation because of acidification of molecular surface charge. In conclusion, although heparin-binding ability of egg-white OVAX slightly decreases during embryogenesis, OVAX incorporated into embryo can retain heparin-binding affinity. Our findings provide a new insight that OVAX participates in the functions of heparin during embryogenesis., (© 2019 Poultry Science Association Inc.)

The tumor suppressor activity of maspin (mammary serine protease inhibitor) has been associated with its nuclear localization. In this study we explore the regulation of maspin nuclear translocation. An in vitro nuclear import assay suggested that maspin can passively enter the nucleus. However, in silico analysis identified a putative maspin nuclear localization signal (NLS), which was able to mediate the nuclear translocation of a chimeric protein containing this NLS fused to five green fluorescent protein molecules in tandem (5GFP). Dominant-negative Ran-GTPase mutants RanQ69L or RanT24N suppressed this process. Unexpectedly, the full-length maspin fused to 5GFP failed to enter the nucleus. As maspin's putative NLS is partially hidden in its three-dimensional structure, we suggest that maspin nuclear transport could be conformationally regulated. Our results suggest that maspin nuclear translocation involves both passive and active mechanisms., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Neutrophil granule serine proteases contribute to immune responses through cleavage of microbial toxins and structural proteins. They induce tissue damage and modulate inflammation if levels exceed their inhibitors. Here, we show that the intracellular protease inhibitors Serpinb1a and Serpinb6a contribute to monocyte and neutrophil survival in steady-state and inflammatory settings by inhibiting cathepsin G (CatG). Importantly, we found that CatG efficiently cleaved gasdermin D (GSDMD) to generate the signature N-terminal domain GSDMD-p30 known to induce pyroptosis. Yet GSDMD deletion did not rescue neutrophil survival in Sb1a.Sb6a -/- mice. Furthermore, Sb1a.Sb6a -/- mice released high levels of pro-inflammatory cytokines upon endotoxin challenge in vivo in a CatG-dependent manner. Canonical inflammasome activation in Sb1a.Sb6a -/- macrophages showed increased IL-1β release that was dependent on CatG and GSDMD. Together, our findings demonstrate that cytosolic serpins expressed in myeloid cells prevent cell death and regulate inflammatory responses by inhibiting CatG and alternative activation of GSDMD., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pigment epithelium-derived factor (PEDF) is involved in signal transduction cascades necessary for protection of the retina. The interaction between PEDF and retinal cells elicits neuroprotective effects in vitro and in vivo. The direct substrates and signaling mechanisms involved in the survival response derived from such interaction are beginning to be revealed. It is of interest to elucidate cell death pathways that are crucial for the retinoprotective response of PEDF for the identification of targets that interfere with retina degeneration with potential therapeutic value. Here we review the molecular pathways triggered by PEDF that are involved in retinal survival activity.

Objectives: Pigment epithelium-derived factor (PEDF), a multifunctional endogenous glycoprotein, has a very wide range of biological actions, notably in bone homeostasis. The question has been raised regarding the place of PEDF in the treatment of bone disorders and osteosarcoma, and its potential for tumour growth suppression., Methods: The PubMed database was used to compile this review., Key Findings: Pigment epithelium-derived factor's actions in osteoid tissues include promoting mesenchymal stem cell commitment to osteoblasts, increasing matrix mineralisation, and promoting osteoblast proliferation. It shows potential to improve therapeutic outcomes in treatment of multiple cancer types and regrowth of bone after trauma or resection in animal studies. PEDF may possibly have a reduced adverse effect profile compared with current osteo-regenerative treatments; however, there is currently very limited evidence regarding the safety or efficacy in human models., Summary: Pigment epithelium-derived factor is very active within the body, particularly in osseous tissue, and its physiological actions give it potential for treatment of both bone disorders and multiple tumour types. Further research is needed to ascertain the adverse effects and safety profile of PEDF as a therapeutic agent., (© 2018 Royal Pharmaceutical Society.)

Pigment epithelium-derived factor (PEDF) is a secreted glycoprotein that has anti-angiogenic, anti-proliferative, neurotrophic and immunomodulatory properties. PEDF has recently emerged as a critical metabolic regulatory protein since the discovery of its modulatory activities in the lipolytic pathway by binding to adipose triglyceride lipase (ATGL). Despite being beneficial in maintaining the homeostasis of hepatic lipid accumulation, PEDF has been uncovered an unfavorable role associated with insulin resistance. The molecular events that connect these two apparent distinct observations have been controversial and remained largely unknown. Therefore in this short review, we attempt to summarize the current findings of PEDF regarding its lipid metabolic functions and provide perspectives in identifying PEDF as a potential therapeutic target in lipid disorders., (Copyright © 2018 Chang Gung University. Published by Elsevier B.V. All rights reserved.)

Vaspin (visceral adipose tissue-derived serine protease inhibitor) is a recently discovered adipokine that has been implicated in diabetes mellitus and other metabolic disorders. However, the effects of vaspin on pancreatic β cell function and related mechanisms are not fully understood. Thus, the present study was performed to investigate the effects of vaspin on pancreatic β cell function and the potential underlying mechanisms. Both in vitro (rat insulinoma cells, INS-1) and in vivo (high fat diet fed rats) experiments were conducted. The results showed that vaspin significantly increased INS-1 cell secretory function. Potential mechanisms were explored using inhibitors, western blot and real-time PCR techniques. We found that vaspin increased the levels of IRS-2 mRNA and IRS-2 total protein, while decreased the serine phosphorylation level of IRS-2 protein. Moreover, vaspin increased the Akt phosphorylation protein level which was reversed by PI3K inhibitor ly294002. In addition, vaspin increased the phosphorylation levels of mTOR and p70S6K, which was inhibited by rapamycin. Meanwhile, we found that the NF-κB mRNA and protein levels were reduced after vaspin treatment, similar to the effect of NF-κB inhibitor TPCK. Furthermore, vaspin increased the glucose stimulated insulin secretion (GSIS) level, lowered blood glucose level and improved the glucose tolerance and insulin sensitivity of high fat diet fed rats. Hyperglycemic clamp test manifested that vaspin improved islet β cell function. Together, these findings provide a new understanding of the function of vaspin on pancreatic β cell and suggest that it may serve as a potential agent for the prevention and treatment of type 2 diabetes.

Backgroud: Widespread endothelial injury contributes to the occurrence of preeclampsia. Maspin, first identified as a tumor suppressor, plays a critical role in cell invasion and angiogenesis. Our previous studies found that the expression of maspin was increased in preeclampsic placenta. In this research, we studied the function of human umbilical vein endothelial cells (HUVECs) to explore the role and possible mechanism of maspin gene in the pathogenesis of preeclampsia., Methods: HUVECs were treated with different concentration of recombinant human maspin protein (r-maspin) during normoxia and hypoxia, we detected the proliferation, apoptosis, migration and tube formation of HUVECs. We also assessed nitride oxide (NO) synthesis and the expression of matrix metalloproteinase 2 (MMP2) to further explore the underlying molecular mechanism., Results: There was only slight maspin expression at mRNA level in HUVECs. Treated HUVECs with r-maspin, the proliferation of HUVECs was significantly promoted both under normoxia and hypoxia. The tubes formed by HUVECs were significantly inhibited and NO synthesis was significantly reduced by r-maspin. Meantime, r-maspin also inhibited MMP2 expression and activity in HUVECs. However, there was no significant change in the migration and apoptosis of HUVECs., Conclusions: Maspin may be an important participant for mediating endothelial function and ultimately leads to the occurence of preeclamsia.

We discovered that pigment epithelium-derived factor (PEDF)-null mice have endometrial hyperplasia, the precursor to human type I endometrial cancer (ECA), which is etiologically linked to unopposed estrogen (E2), suggesting that this potent antiangiogenic factor might contribute to dysregulated growth and the development of type I ECA. Treatment of both ECA cell lines and primary ECA cells with recombinant PEDF dose dependently decreased cellular proliferation via an autocrine mechanism by blocking cells in G1 and G2 phases of the cell cycle. Consistent with the known opposing effects of E2 and progesterone (Pg) on endometrial proliferation, Pg increases PEDF protein synthesis and release, whereas E2 has the converse effect. Using PEDF luciferase promoter constructs containing two Pg and one E2 response elements, E2 reduced and Pg increased promoter activity due to distal response elements. Furthermore, E2 decreases and Pg increases PEDF secretion into conditioned media (CM) by both normal endometrial stromal fibroblasts (ESFs) and cancer-associated fibroblasts (CAFs), but only CM from ESFs mediated growth-inhibitory activity of primary endometrial epithelial cells (EECs). In addition, in cocultures with primary EECs, Pg-induced growth inhibition is mediated by ESFs, but not CAFs. This is consistent with reduced levels of Pg receptors on CAFs surrounding human malignant glands in vivo. Taken together, the data suggest that PEDF is a hormone-regulated negative autocrine mediator of endometrial proliferation, and that paracrine growth inhibition by soluble factors, possibly PEDF, released by ESFs in response to Pg, but not CAFs, exemplifies a tumor microenvironment that contributes to the pathogenesis of ECA., (Copyright © 2017 Endocrine Society.)

Angiogenesis contributes to the pathogenesis of many diseases including exudative age-related macular degeneration (AMD). It is normally kept in check by a tightly balanced production of pro- and anti-angiogenic factors. The up-regulation of the pro-angiogenic factor, vascular endothelial growth factor (VEGF), is intimately linked to the pathogenesis of exudative AMD, and its antagonism has been effectively targeted for treatment. However, very little is known about potential changes in expression of anti-angiogenic factors and the role they play in choroidal vascular homeostasis and neovascularization associated with AMD. Here, we will discuss the important role of thrombospondins and pigment epithelium-derived factor, two major endogenous inhibitors of angiogenesis, in retinal and choroidal vascular homeostasis and their potential alterations during AMD and choroidal neovascularization (CNV). We will review the cell autonomous function of these proteins in retinal and choroidal vascular cells. We will also discuss the potential targeting of these molecules and use of their mimetic peptides for therapeutic development for exudative AMD., (© 2017 The Author(s), published by Portland Press Limited on behalf of the Biochemical Society.)

The publication of the first tick sialome (salivary gland transcriptome) heralded a new era of research of tick protease inhibitors, which represent important constituents of the proteins secreted via tick saliva into the host. Three major groups of protease inhibitors are secreted into saliva: Kunitz inhibitors, serpins, and cystatins. Kunitz inhibitors are anti-hemostatic agents and tens of proteins with one or more Kunitz domains are known to block host coagulation and/or platelet aggregation. Serpins and cystatins are also anti-hemostatic effectors, but intriguingly, from the translational perspective, also act as pluripotent modulators of the host immune system. Here we focus especially on this latter aspect of protease inhibition by ticks and describe the current knowledge and data on secreted salivary serpins and cystatins and their role in tick-host-pathogen interaction triad. We also discuss the potential therapeutic use of tick protease inhibitors.

Adipokines are peptides that signal the functional status of adipose tissue to the brain and other target organs. In adipose tissue dysfunction, adipokine secretion is altered, and this can contribute to a spectrum of obesity-associated conditions including cardiovascular disease. Some adipokines have anti-inflammatory and cardioprotective effects (omentin, apelin, adiponectin). Others are pro-inflammatory with negative impact on cardiovascular function (leptin, visfatin, resistin, adipocyte fatty-acid-binding protein). In the first part, this article reviews the endocrine functions of adipose tissue in general, effects of the distribution and composition of fat tissue, and the roles of cortisol and the renin-angiotensin-aldosterone system in the development of the inflammatory state of addipose tissue. In the second part, the known cardiovascular effects of different adipokines and their clinical potential are discussed in detail.

Maspin (SerpinB5) is an epithelial-specific tumor suppressor gene product that displays context-dependent cellular functions. Maspin-deficient mouse models created to date have not definitively established maspin functions critical for cancer suppression. In this study, we generated a mouse strain in which exon 4 of the Maspin gene was deleted, confirming its essential role in development but also enabling a breeding scheme to bypass embryonic lethality. Phenotypic characterization of this viable strain established that maspin deficiency was associated with a reduction in maximum body weight and a variety of context-dependent epithelial abnormalities. Specifically, maspin-deficient mice exhibited pulmonary adenocarcinoma, myoepithelial hyperplasia of the mammary gland, hyperplasia of luminal cells of dorsolateral and anterior prostate, and atrophy of luminal cells of ventral prostate and stratum spinosum of epidermis. These cancer phenotypes were accompanied by increased inflammatory stroma. These mice also displayed the autoimmune disorder alopecia aerate. Overall, our findings defined context-specific tumor suppressor roles for maspin in a clinically relevant model to study maspin functions in cancer and other pathologies. Cancer Res; 77(4); 886-96. ©2017 AACR ., (©2016 American Association for Cancer Research.)

Maspin is a non-inhibitory member of the serpin family that affects cell behaviours related to migration and survival. We have previously shown that peptides of the isolated G α-helix (G-helix) domain of maspin show bioactivity. Migration, invasion, adhesion and proliferation of vascular smooth muscle cells (VSMC) are important processes that contribute to the build-up of atherosclerotic plaques. Here we report the use of functional assays of these behaviours to investigate whether other maspin-derived peptides impact directly on VSMC; focusing on potential anti-atherogenic properties. We designed 18 new peptides from the structural moieties of maspin above ten amino acid residues in length and considered them beside the existing G-helix peptides. Of the novel peptides screened those with the sequences of maspin strand 4 and 5 of beta sheet B (S4B and S5B) reduced VSMC migration, invasion and proliferation, as well as increasing cell adhesion. A longer peptide combining these consecutive sequences showed a potentiation of responses, and a 7-mer contained all essential elements for functionality. This is the first time that these parts of maspin have been highlighted as having key roles affecting cell function. We present evidence for a mechanism whereby S4B and S5B act through ERK1/2 and AMP-activated protein kinase (AMPK) to influence VSMC responses., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Pro-coagulant, anti-coagulant and fibrinolytic pathways are responsible for maintaining hemostatic balance under physiological conditions. Any deviation from these pathways would result in hypercoagulability leading to life threatening diseases like myocardial infarction, stroke, portal vein thrombosis, deep vein thrombosis (DVT) and pulmonary embolism (PE). Angiogenesis is the process of sprouting of new blood vessels from pre-existing ones and plays a critical role in vascular repair, diabetic retinopathy, chronic inflammation and cancer progression. Serpins; a superfamily of protease inhibitors, play a key role in regulating both angiogenesis and coagulation. They are characterized by the presence of highly conserved secondary structure comprising of 3 β-sheets and 7-9 α-helices. Inhibitory role of serpins is modulated by binding to cofactors, specially heparin and heparan sulfate proteoglycans (HSPGs) present on cell surfaces and extracellular matrix. Heparin and HSPGs are the mainstay of anti-coagulant therapy and also have therapeutic potential as anti-angiogenic inhibitors. Many of the heparin binding serpins that regulate coagulation cascade are also potent inhibitors of angiogenesis. Understanding the molecular mechanism of the switch between their specific anti-coagulant and anti-angiogenic role during inflammation, stress and regular hemostasis is important. In this review, we have tried to integrate the role of different serpins, their interaction with cofactors and their interplay in regulating coagulation and angiogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Serpins are a widely distributed family of high molecular mass protein proteinase inhibitors that can inhibit both serine and cysteine proteinases by a remarkable mechanism-based kinetic trapping of an acyl or thioacyl enzyme intermediate that involves massive conformational transformation. The trapping is based on distortion of the proteinase in the complex, with energy derived from the unique metastability of the active serpin. Serpins are the favoured inhibitors for regulation of proteinases in complex proteolytic cascades, such as are involved in blood coagulation, fibrinolysis and complement activation, by virtue of the ability to modulate their specificity and reactivity. Given their prominence as inhibitors, much work has been carried out to understand not only the mechanism of inhibition, but how it is fine-tuned, both spatially and temporally. The metastability of the active state raises the question of how serpins fold, whereas the misfolding of some serpin variants that leads to polymerization and pathologies of liver disease, emphysema and dementia makes it clinically important to understand how such polymerization might occur. Finally, since binding of serpins and their proteinase complexes, particularly plasminogen activator inhibitor-1 (PAI-1), to the clearance and signalling receptor LRP1 (low density lipoprotein receptor-related protein 1), may affect pathways linked to cell migration, angiogenesis, and tumour progression, it is important to understand the nature and specificity of binding. The current state of understanding of these areas is addressed here., (© 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Serine protease inhibitors, known as serpins, are pleiotropic regulators of endogenous and exogenous proteases, and molecule transporters. They have been documented in animals, plants, fungi, bacteria, and viruses; here, we characterize a serpin from the trematode platyhelminth Schistosoma mansoni. At least eight serpins have been found in the genome of S. mansoni, but only two have characterized molecular properties and functions. Here, the function of S. mansoni serpin isoform 3 (SmSPI) was analyzed, using both computational and molecular biological approaches. Phylogenetic analysis showed that SmSPI was closely related to Schistosoma haematobium serpin and Schistosoma japonicum serpin B10. Structure determined in silico confirmed that SmSPI belonged to the serpin superfamily, containing nine α-helices, three β-sheets, and a reactive central loop. SmSPI was highly expressed in schistosomules, predominantly in the head gland, and in adult male and female with intensive accumulation on the spines, which suggests that it may have a role in facilitating intradermal and intravenous survival. Recombinant SmSPI was overexpressed in Escherichia coli; the recombinant protein was of the same size (46 kDa) as the native protein. Immunological analysis suggested that mice infected with S. mansoni responded to rSmSPI at 8 weeks postinfection (wpi) but not earlier. The inhibitory activity of rSmSPI was specific to chymotrypsin but not trypsin, neutrophil elastase, and porcine pancreatic elastase. Elucidating the biological and physiological functions of SmSPI as well as other serpins will lead to further understanding of host-parasite interaction machinery that may provide novel strategies to prevent and control schistosomiasis in the future.

Background: Pigment epithelium-derived factor (PEDF), which belongs to the noninhibitory serpin family, has shown the ability to stimulate several physiological processes, such as antiangiogenesis, anti-inflammation, and antioxidation. In the present study, the effects of PEDF on contractility and calcium handling of rat ventricular myocytes were investigated., Methods and Results: Adult Sprague-Dawley rat models of acute myocardial infarction (AMI) were surgically established. PEDF-lentivirus was delivered into the myocardium along and away from the infarction border to overexpress PEDF. Video edge detection was used to measure myocyte shortening in vitro. Intracellular Ca(2+) was measured in cells loaded with the Ca(2+) sensitive fluorescent indicator, Fura-2-acetoxymethyl ester. PEDF local overexpression enhanced cardiac functional reserve in AMI rats and reduced myocardial contracture bordering the infracted area. Exogenous PEDF treatment (10 nmol/L) caused a significant decrease in amplitudes of isoproterenol-stimulated myocyte shortening, Ca(2+) transients, and caffeine-evoked Ca(2+) transients in vitro. We then tested a potential role for PEDF receptor-mediated effects on upregulation of protein kinase C (PKC) and found evidence of signaling through the diacylglycerol/PKCα pathway. We also confirmed that pretreatment of cardiomyocytes with PEDF exhibited dephosphorylation of phospholamban at Ser(16), which could be attenuated with PKC inhibition., Conclusions: The results suggest that PEDF depresses myocyte contractility by suppressing phosphorylation of phospholamban and Ca(2+) transients in a PKCα-dependent manner through its receptor, PEDF receptor, therefore improving cardiac functional reserve during AMI., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

The rapidly expanding feto-placental vasculature needs tight control by paracrine and endocrine mechanisms. Here, we focused on paracrine influence by trophoblast, the placental epithelium. We aimed to identify differences in regulation of feto-placental angiogenesis in early versus late pregnancy. To this end, the effect of conditioned media (CM) from early and late pregnancy human trophoblast was tested on network formation, migration and proliferation of human feto-placental endothelial cells. Only CM of late pregnancy trophoblast reduced network formation and migration. Screening of trophoblast transcriptome for anti-angiogenic candidates identified pigment epithelium-derived factor (PEDF) with higher expression and protein secretion in late pregnancy trophoblast. Addition of a PEDF-neutralizing antibody restored the anti-angiogenic effect of CM from late pregnancy trophoblast. Notably, human recombinant PEDF reduced network formation only in combination with VEGF. Also in the CAM assay, the combination of PEDF with VEGF reduced branching of vessels below control levels. Analysis of phosphorylation of ERK1/2 and FAK, two key players in VEGF-induced proliferation and migration, revealed that PEDF altered VEGF signaling, while PEDF alone did not affect phosphorylation of ERK1/2 and FAK. These data suggest that the trophoblast-derived anti-angiogenic molecule PEDF is involved in restricting growth and expansion of the feto-placental endothelium predominantly in late pregnancy and targets to modulate the intracellular effect of VEGF.

Pigment epithelium-derived factor (PEDF) is a multifunctional glycoprotein, associated with lipid catabolism and insulin resistance. In the present study, PEDF increased chronic and acute insulin secretion in a clonal rat β-cell line BRIN-BD11, without alteration of glucose consumption. PEDF also stimulated insulin secretion from primary mouse islets. Seahorse flux analysis demonstrated that PEDF did not change mitochondrial respiration and glycolytic function. The cytosolic presence of the putative PEDF receptor - adipose triglyceride lipase (ATGL) - was identified, and ATGL associated stimulation of glycerol release was robustly enhanced by PEDF, while intracellular ATP levels increased. Addition of palmitate or ex vivo stimulation with inflammatory mediators induced β-cell dysfunction, effects not altered by the addition of PEDF. In conclusion, PEDF increased insulin secretion in BRIN-BD11 and islet cells, but had no impact on glucose metabolism. Thus elevated lipolysis and enhanced fatty acid availability may impact insulin secretion following PEDF receptor (ATGL) stimulation., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background and Study Aim: Although some hypotheses have been postulated on the genesis of gastric cancer (GC), the origin of this disease remains unclear. The aim of this study was to develop a hypothesis about gastric carcinogenesis based on our experience in the field of GC and on published reports on about 28 studies in the field of subcellular maspin expression in GC. In 180 cases of GC, the clinicopathologic features were correlated with the results obtained after paired immunohistochemical stains (tumor/normal mucosa) with 15 antibodies: E-cadherin, HER-2, VEGF, CD31, CD105, COX-2, maspin, bax, bcl-2, p53, Ki67, MLH-1, MSH-2, Mena protein, and vimentin., Results: Cytoplasmic maspin was observed in foveolar cells with intestinal metaplasia, whereas mixed (combined nuclear-cytoplasmic) expressions were more characteristic of the intramucosal foci of signet-ring cells and dysplastic cells. The tumor cells that expressed cytoplasmic maspin were mostly intestinal type bax/COX-2/Mena/E-cadherin-positive differentiated adenocarcinomas with nodular growth and more superficial invasion. The nuclear shift of maspin was more frequent in HER-2/p53-positive intestinal type adenocarcinomas with diffuse architecture at the invasion front, as well as for node-positive poorly cohesive carcinomas. Loss of maspin expression induced a higher risk of distant metastases, without differences in the survival rate., Conclusions: In GC with associated metaplasia, cytoplasmic maspin is predominant; the nuclear shift induces local aggressiveness and risk of node metastases, whereas total loss can indicate a risk of distant metastases. In GC without associated metaplasia, nuclear expression of maspin is retained, indicating a more aggressive behavior.

Androgen deprivation therapy has constituted the main treatment for prostate cancer; however, tumors ultimately progress to hormone-independent prostate cancer (HIPC), and suitable therapeutic strategies for HIPC are not available. Maspin, which is also known as mammary serine protease inhibitor, has been suggested to be a valuable focus for targeted cancer therapy. Specifically, maspin has been shown to be upregulated after androgen ablation therapy. Gemcitabine is used as a first-line therapy for metastatic castration-resistant prostate cancer, but its disease control rate is low. Furthermore, the role of maspin in the therapeutic efficacy of gemcitabine for HIPC remains unclear. The expression levels of maspin in PC-3 and DU145 cells were determined by real-time PCR and Western blotting. Furthermore, the expression of maspin was silenced using shRNA technology to generate maspin-KD cells. The cytotoxicity of gemcitabine to prostate cancer cells was assessed using 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyl tetrazolium bromide (MTT) assays, whereas flow cytometry analyses and annexin V-propidium iodide (PI) apoptosis assays were used to assess the ability of gemcitabine to induce apoptosis in maspin-KD and control cells. Additionally, the expression patterns of anti-apoptosis proteins (myeloid cell leukemia 1 (Mcl-1) and B cell lymphoma 2 (Bcl-2)) and pro-apoptosis proteins (Bcl-2-associated death promoter (Bad) and Bcl-2-associated X protein (Bax)) were determined by Western blotting. In this study, PC-3 cells were more resistant to gemcitabine administration than DU145 cells, which correlated with the higher expression levels of maspin observed in PC-3 cells. Furthermore, maspin knockdown enhanced gemcitabine-induced cell death, as evidenced by the increased number of apoptotic cells. Gemcitabine treatment upregulated the levels of anti-apoptosis proteins (Mcl-2 and Bcl-2) in both scrambled control and maspin-KD cells; however, the fold changes in Mcl-1 and Bcl-2 expression were larger in gemcitabine-treated scrambled control cells than in maspin-KD cells. Finally, our findings indicate for the first time that maspin may mediate the therapeutic efficacy of gemcitabine in HIPC. Our results demonstrate that maspin knockdown enhanced the sensitivity of androgen-independent prostate cancer cells to gemcitabine. Therefore, combining gemcitabine with a drug that targets maspin might constitute a valuable strategy for prostate cancer treatment.

Kallistatin is a serine protease inhibitor with anti-inflammatory, anti-angiogenic, and anti-oxidative properties. Since oxidative stress plays a critical role in the pathogenesis of diabetic nephropathy, we studied the effect and mechanisms of action of kallistatin superinduction. Using ultrasound-microbubble-mediated gene transfer, kallistatin overexpression was induced in kidney tubules. In db/db mice, kallistatin overexpression reduced serum creatinine and BUN levels, ameliorated glomerulosclerosis and tubulointerstitial injury, and attenuated renal fibrosis by inhibiting TGF-β signaling. Additionally, downstream PAI-1 and collagens I and IV expression were reduced and kallistatin partially suppressed renal inflammation by inhibiting NF-κB signaling and decreasing tissue kallikrein activity. Kallistatin lowered blood pressure and attenuated oxidative stress as evidenced by suppressed levels of NADPH oxidase 4, and oxidative markers (nitrotyrosine, 8-hydroxydeoxyguanosine, and malondialdehyde) in diabetic renal tissue. Kallistatin also inhibited RAGE expression in the diabetic kidney and AGE-stimulated cultured proximal tubular cells. Reduced AGE-induced reactive oxygen species generation reflected an anti-oxidative mechanism via the AGE-RAGE-reactive oxygen species axis. These results indicate a renoprotective role of kallistatin against diabetic nephropathy by multiple mechanisms including suppression of oxidative stress, anti-fibrotic and anti-inflammatory actions, and blood pressure lowering.

Although compensatory islet hyperplasia in response to insulin resistance is a recognized feature in diabetes, the factor(s) that promote β cell proliferation have been elusive. We previously reported that the liver is a source for such factors in the liver insulin receptor knockout (LIRKO) mouse, an insulin resistance model that manifests islet hyperplasia. Using proteomics we show that serpinB1, a protease inhibitor, which is abundant in the hepatocyte secretome and sera derived from LIRKO mice, is the liver-derived secretory protein that regulates β cell proliferation in humans, mice, and zebrafish. Small-molecule compounds, that partially mimic serpinB1 effects of inhibiting elastase activity, enhanced proliferation of β cells, and mice lacking serpinB1 exhibit attenuated β cell compensation in response to insulin resistance. Finally, SerpinB1 treatment of islets modulated proteins in growth/survival pathways. Together, these data implicate serpinB1 as an endogenous protein that can potentially be harnessed to enhance functional β cell mass in patients with diabetes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Future curative cancer chemotherapies have to overcome tumor cell heterogeneity and plasticity. To test the hypothesis that the tumor suppressor maspin may reduce microenvironment-dependent prostate tumor cell plasticity and thereby modulate drug sensitivity, we established a new schematic combination of two-dimensional (2D), three-dimensional (3D), and suspension cultures to enrich prostate cancer cell subpopulations with distinct differentiation potentials. We report here that depending on the level of maspin expression, tumor cells in suspension and 3D collagen I manifest the phenotypes of stem-like and dormant tumor cell populations, respectively. In suspension, the surviving maspin-expressing tumor cells lost the self-renewal capacity, underwent senescence, lost the ability to dedifferentiate in vitro, and failed to generate tumors in vivo. Maspin-nonexpressing tumor cells that survived the suspension culture in compact tumorspheres displayed a higher level of stem cell marker expression, maintained the self-renewal capacity, formed tumorspheres in 3D matrices in vitro, and were tumorigenic in vivo. The drug sensitivities of the distinct cell subpopulations depend on the drug target and the differentiation state of the cells. In 2D, docetaxel, MS275, and salinomycin were all cytotoxic. In suspension, while MS275 and salinomycin were toxic, docetaxel showed no effect. Interestingly, cells adapted to 3D collagen I were only responsive to salinomycin. Maspin expression correlated with higher sensitivity to MS275 in both 2D and suspension and to salinomycin in 2D and 3D collagen I. Our data suggest that maspin reduces prostate tumor cell plasticity and enhances tumor sensitivity to salinomycin, which may hold promise in overcoming tumor cell heterogeneity and plasticity., (©2015 American Association for Cancer Research.)

Kallistatin, an endogenous protein, consists of two structural elements: active site and heparin-binding domain. Kallistatin exerts beneficial effects on fibrosis by suppressing transforming growth factor (TGF)-β synthesis in animal models. TGF-β is the most potent inducer of endothelial-mesenchymal transition (EndMT), which contributes to fibrosis and cancer. MicroRNA (miR)-21 is an important player in organ fibrosis and tumor invasion. Here we investigated the potential role of kallistatin in EndMT via modulation of miR-21 in endothelial cells. Human kallistatin treatment blocked TGF-β-induced EndMT, as evidenced by morphological changes as well as increased endothelial and reduced mesenchymal marker expression. Kallistatin also inhibited TGF-β-mediated reactive oxygen species (ROS) formation and NADPH oxidase expression and activity. Moreover, kallistatin antagonized TGF-β-induced miR-21 and Snail1 synthesis, Akt phosphorylation, NF-κB activation, and matrix metalloproteinase 2 (MMP2) synthesis and activation. Kallistatin via its heparin-binding site blocked TGF-β-induced miR-21, Snail1 expression, and ROS formation, as wild-type kallistatin, but not heparin-binding site mutant kallistatin, exerted the effect. Conversely, kallistatin through its active site stimulated the synthesis of endothelial nitric oxide synthase (eNOS), sirtuin 1 (Sirt1) and forkhead box O1 (FoxO1); however, these effects were blocked by genistein, a tyrosine kinase inhibitor. This is the first study to demonstrate that kallistatin's heparin-binding site is crucial for preventing TGF-β-induced miR-21 and oxidative stress, while its active site is key for stimulating the expression of antioxidant genes via interaction with an endothelial surface tyrosine kinase. These findings reveal novel mechanisms of kallistatin in protection against fibrosis and cancer by suppressing EndMT., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The ovalbumin gene family in Gallus gallus is composed of three homologous genes located within a 46 kb locus on chromosome 2: ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX) genes. The expression of these genes in hen oviduct is under estrogen control, but their relative hormonal responsiveness and subsequent protein concentration in egg, is distinctive. Interestingly, all three proteins lack the classical signal peptide for secretion. Ovalbumin, OVAX, and OVAY belong to the serine protease inhibitor (serpin) family whose members share a common tertiary structure. Ovalbumin and OVAX are one of the few members of this family that do not express any protease inhibition activity whereas OVAY has been predicted to be inhibitory, by comparison with the consensus sequence for inhibitory serpins. In contrast to ovalbumin and OVAY, OVAX interacts with heparin, a negatively charged glycosaminoglycan, via a positively charged domain exposed at the surface of the molecule. Ovalbumin is the major egg white protein and might be a source of amino acids for the developing embryo. The physiological function of OVAY is not known, but recent data have revealed a possible role of this protein in early embryonic development. Considering the antibacterial activities of OVAX, this protein might play a role in egg defense. This review sheds light on the expression, biochemistry, and structural specificities of these three highly similar paralogs. It gives new clues in favor of diverging functions, which are likely to have arisen by duplication events from a common ancestral gene., (© 2015 by the Society for the Study of Reproduction, Inc.)

Pigment epithelium-derived factor (PEDF) is a broadly expressed multifunctional member of the serine proteinase inhibitor (serpin) family. This widely studied protein plays critical roles in many physiological and pathophysiological processes, including neuroprotection, angiogenesis, fibrogenesis and inflammation. The present review summarizes the temporal and spatial distribution patterns of PEDF in a variety of developing and adult organs, and discusses its functions in maintaining physiological homoeostasis. The major focus of the present review is to discuss the implication of PEDF in diabetic and hypoxia-induced angiogenesis, and the pathways mediating PEDF's effects under these conditions. Furthermore, the regulatory mechanisms of PEDF expression, function and degradation are also reviewed. Finally, the therapeutic potential of PEDF as an anti-angiogenic drug is briefly summarized.

Diabetic retinopathy (DR), a major microvascular complication of diabetes, has emerged as a leading cause of visual impairment and blindness among adults worldwide. However, aside from pathological damage, the traditional laser and multi-needle operation treatments required for more advanced disease can cause further damage to the visual field and increase the operation risk. Therefore, the development of new therapeutic strategies for the prevention and treatment of DR is essential. Some emerging evidence now indicates that pigment epithelium-derived factor (PEDF), a multifunctional protein, can target multiple pathways to exert neurotropic, neuropro- tective, anti-angiogenic, anti-vasopermeability, anti-inflammation, anti-thrombogenic, and anti-oxidative effects against DR. This review addresses the functions of PEDF in different pathways that could lead to potential therapeutics for the treatment of DR.

Neuroserpin (NSP) reportedly exerts neuroprotective effects in cerebral ischemic animal models and patients; however, the mechanism of protection is poorly understood. We thus attempted to confirm neuroprotective effects of NSP on astrocytes in the ischemic state and then explored the relative mechanisms. Astrocytes from neonatal rats were treated with oxygen-glucose deprivation (OGD) followed by reoxygenation (OGD/R). To confirm the neuroprotective effects of NSP, we measured the cell survival rate, relative lactate dehydrogenase (LDH) release; we also performed morphological methods, namely Hoechst 33342 staining and Annexin V assay. To explore the potential mechanisms of NSP, the release of nitric oxide (NO) and TNF-α related to NSP administration were measured by enzyme-linked immunosorbent assay. The proteins related to the NF-κB, ERK1/2, and PI3K/Akt pathways were investigated by Western blotting. To verify the cause-and-effect relationship between neuroprotection and the NF-κB pathway, a NF-κB pathway inhibitor sc3060 was employed to observe the effects of NSP-induced neuroprotection. We found that NSP significantly increased the cell survival rate and reduced LDH release in OGD/R-treated astrocytes. It also reduced NO/TNF-α release. Western blotting showed that the protein levels of p-IKKBα/β and P65 were upregulated by the OGD/R treatment and such effects were significantly inhibited by NSP administration. The NSP-induced inhibition could be significantly reversed by administration of the NF-κB pathway inhibitor sc3060, whereas, expressions of p-ERK1, p-ERK2, and p-AKT were upregulated by the OGD/R treatment; however, their levels were unchanged by NSP administration. Our results thus verified the neuroprotective effects of NSP in ischemic astrocytes. The potential mechanisms include inhibition of the release of NO/TNF-α and repression of the NF-κB signaling pathways. Our data also indicated that NSP has little influence on the MAPK and PI3K/Akt pathways.