PAI-1 mRNA Research Articles

Osteoprotegerin is an Early Marker of the Fibrotic Process and of Antifibrotic Treatment Responses in Ex Vivo Lung Fibrosis

BackgroundLung fibrosis is a chronic lung disease with a high mortality rate with only two approved drugs (pirfenidone and nintedanib) to attenuate its progression. To date, there are no reliable biomarkers to assess fibrosis development and/or treatment effects for these two drugs. Osteoprotegerin (OPG) is used as a serum marker to diagnose liver fibrosis and we have previously shown it associates with lung fibrosis as well.MethodsHere we used murine and human precision-cut lung slices to investigate the regulation of OPG in lung tissue to elucidate whether it tracks with (early) fibrosis development and responds to antifibrotic treatment to assess its potential use as a biomarker.ResultsOPG mRNA expression in murine lung slices was higher after treatment with profibrotic cytokines TGFβ1 or IL13, and closely correlated with Fn and PAI1 mRNA expression. More OPG protein was released from fibrotic human lung slices than from the control human slices and from TGFβ1 and IL13-stimulated murine lung slices compared to control murine slices. This OPG release was inhibited when murine slices were treated with pirfenidone or nintedanib. OPG release from human fibrotic lung slices was inhibited by pirfenidone treatment.ConclusionOPG can already be detected during the early stages of fibrosis development and responds, both in early- and late-stage fibrosis, to treatment with antifibrotic drugs currently on the market for lung fibrosis. Therefore, OPG should be further investigated as a potential biomarker for lung fibrosis and a potential surrogate marker for treatment effect.

Arid5a/IL-6/PAI-1 Signaling Is Involved in the Pathogenesis of Lipopolysaccharide-Induced Kidney Injury.

A systemic inflammatory response leads to widespread organ dysfunction, such as kidney dysfunction. Plasminogen activator inhibitor-1 (PAI-1) is involved in the pathogenesis of inflammatory kidney injury; however, the regulatory mechanism of PAI-1 in injured kidneys remains unclear. PAI-1 is induced by interleukin (IL)-6 in patients with sepsis. In addition, the stabilization of IL-6 is regulated by the adenine-thymine-rich interactive domain-containing protein 5a (Arid5a). Therefore, the aim of the present study was to examine the involvement of Arid5a/IL-6/PAI-1 signaling in lipopolysaccharide (LPS)-induced inflammatory kidney injury. LPS treatment to C57BL/6J mice upregulated Pai-1 mRNA in the kidneys. Enzyme-linked immunosorbent assay (ELISA) revealed that PAI-1 expression was induced in the culture supernatants of LPS-treated human umbilical vein endothelial cells, but not in those of LPS-treated human kidney 2 (HK-2) cells, a tubular cell line. Combined with single-cell analysis, endothelial cells were found to be responsible for PAI-1 elevation in LPS-treated kidneys. Administration of TM5441, a PAI-1 inhibitor, reduced the urinary albumin/creatinine ratio, concomitant with downregulation of Il-6 and Arid5a mRNA expressions. IL-6 treatment in LPS model mice further upregulated Pai-1 mRNA expression compared with LPS alone, accompanied by renal impairment. Furthermore, the expression of Il-6 and Pai-1 mRNA was lower in Arid5a knockout mice than in wild-type mice after LPS treatment. Taken together, the vicious cycle of Arid5a/IL-6/PAI-1 signaling is involved in LPS-induced kidney injury.

Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

Hyper-angiogenesis is a typical feature of glioblastoma (GBM), the most aggressive brain tumor. We have reported the expression of aldehyde dehydrogenase 1A3 (ALDH1A3) in proliferating vasculature in GBM patients. We hypothesized that ALDH1A3 may act as an angiogenesis promoter in GBM. Two GBM cell lines were lentivirally transduced with either ALDH1A3 (ox) or an empty vector (ev). The angiogenesis phenotype was studied in indirect and direct co-culture of endothelial cells (ECs) with oxGBM cells (oxGBMs) and in an angiogenesis model in vivo. Angiogenesis array was performed in oxGBMs. RT2-PCR, Western blot, and double-immunofluorescence staining were performed to confirm the expression of targets identified from the array. A significantly activated angiogenesis phenotype was observed in ECs indirectly and directly co-cultured with oxGBMs and in vivo. Overexpression of ALDH1A3 (oxALDH1A3) led to a marked upregulation of PAI-1 and IL-8 mRNA and protein and a consequential increased release of both proteins. Moreover, oxALDH1A3-induced angiogenesis was abolished by the treatment of the specific inhibitors, respectively, of PAI-1 and IL-8 receptors, CXCR1/2. This study defined ALDH1A3 as a novel angiogenesis promoter. oxALDH1A3 in GBM cells stimulated EC angiogenesis via paracrine upregulation of PAI-1 and IL-8, suggesting ALDH1A3-PAI-1/IL-8 as a novel signaling for future anti-angiogenesis therapy in GBM.

Effects of knockdown ACC1 on glioma U251 cell migration and its mechanisms

Objective: To investigate the effects of ACC1 knockdown on human glioma U251 cell migration and its molecular mechanisms. Methods: Human glioma U251 cell line was used. The experiment was carried out in three steps. Experiment 1: knockdown of ACC1 in U251 cells (shACC1) and its control (NC) U251 cells were established by transfection of shACC1 lentivirus and negative control virus. The cell migration was detected by Transwell migration assay and scratch test. Western blot (WB) was performed to detect the levels of ACC1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug proteins. Experiment 2: RT-qPCR and WB were performed to verify the RNA-seq result, upregulation effect of ACC1 knockdown on PAI-1 in U251 cells. The cells then were treated with PAI-1 inhibitor PAI-039, and the cell migration was detected by Transwell migration assay and scratch assay. The protein levels of ACC1, PAI-1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug were examined by WB. Experiment 3: the molecular mechanisms of knocking down ACC1 to increase PAI-1 were explored. The cells were treated with acetyltransferase inhibitor C646, and cell migration was examined by Transwell migration assay and scratch assay. WB was conducted to test the levels of ACC1, H3K9ac, PAI-1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug proteins. Each experiment was repeated three times. Results: Experiment 1: lentivirus transfection was performed on glioma U251 cells. Compared with NC group, the expression level of ACC1 in shACC1 group was decreased significantly, indicating that lentivirus transfection was successful (P＜0.01), and the number of migrated cells in shACC1 group was increased significantly (P＜0.01). Migration-related proteins Vimentin, Fibronectin, N-cadherin and Slug were up-regulated, while E-cadherin was down-regulated (P＜0.01). Experiment 2: Compared with NC group, PAI-1 mRNA level in shACC1 group was up-regulated. Compared with control group, cell migration in shACC1+PAI-039 group was decreased (P＜0.01), and migration-related proteins Vimentin, Fibronectin, N-cadherin, and Slug were up-regulated. E-cadherin expression was down-regulated (P＜0.01). Experiment 3: Compared with NC group, the concentration of acetyl-coA and the expression level of H3K9ac in shACC1 group were increased significantly (P＜0.01); After further treatment with histone acetyl transferase inhibitor C646, PAI-1 mRNA level was decreased, cell migration number and H3K9ac expression level were decreased in shACC1+C646 group compared with control group (P＜0.01). Migration-related proteins Vimentin, Fibronectin, N-cadherin and Slug were up-regulated, while E-cadherin was down-regulated (P＜0.01). Conclusion: Knockdown of ACC1 promotes the migration of human glioma U251 cells by increasing histone acetylation which elevates the level of PAI-1.

SARS-CoV-2 modulates inflammatory responses of alveolar epithelial type II cells via PI3K/AKT pathway.

SARS-CoV-2 infects through the respiratory route and triggers inflammatory response by affecting multiple cell types including type II alveolar epithelial cells. SARS-CoV-2 triggers signals via its Spike (S) protein, which have been shown to participate in the pathogenesis of COVID19. Aim of the present study was to investigate the effect of SARS-CoV2 on type II alveolar epithelial cells, focusing on signals initiated by its S protein and their impact on the expression of inflammatory mediators. For this purpose A549 alveolar type II epithelial cells were exposed to SARS CoV2 S recombinant protein and the expression of inflammatory mediators was measured. The results showed that SARS-CoV-2 S protein decreased the expression and secretion of IL8, IL6 and TNFα, 6 hours following stimulation, while it had no effect on IFNα, CXCL5 and PAI-1 expression. We further examined whether SARS-CoV-2 S protein, when combined with TLR2 signals, which are also triggered by SARS-CoV2 and its envelope protein, exerts a different effect in type II alveolar epithelial cells. Simultaneous treatment of A549 cells with SARS-CoV-2 S protein and the TLR2 ligand PAM3csk4 decreased secretion of IL8, IL6 and TNFα, while it significantly increased IFNα, CXCL5 and PAI-1 mRNA expression. To investigate the molecular pathway through which SARS-CoV-2 S protein exerted this immunomodulatory action in alveolar epithelial cells, we measured the induction of MAPK/ERK and PI3K/AKT pathways and found that SARS-CoV-2 S protein induced the activation of the serine threonine kinase AKT. Treatment with the Akt inhibitor MK-2206, abolished the inhibitory effect of SARS-CoV-2 S protein on IL8, IL6 and TNFα expression, suggesting that SARS-CoV-2 S protein mediated its action via AKT kinases. The findings of our study, showed that SARS-CoV-2 S protein suppressed inflammatory responses in alveolar epithelial type II cells at early stages of infection through activation of the PI3K/AKT pathway. Thus, our results suggest that at early stages SARS-CoV-2 S protein signals inhibit immune responses to the virus allowing it to propagate the infection while in combination with TLR2 signals enhances PAI-1 expression, potentially affecting the local coagulation cascade.

Melatonin exerts anti-fibrinolytic effects by regulating IL-1β-induced changes in uPA, uPAR, and PAI-1 expression/production in human dental pulp cells

Interleukin-1b (IL-1β) is a pro-inflammatory cytokine and its expression is increased in inflamed dental pulp. IL-1β affects plasminogen activation system molecules, which are crucial for tissue inflammation, fibrinolysis, matrix turnover, and cell adhesion and migration. Melatonin, which provides circadian and seasonal signals, is a physiological endocrine generated by the pineal gland. It has anti-oxidant and anti-inflammatory properties. Studies are warranted to determine whether melatonin prevents IL-1β-induced expression/production of plasminogen system molecules. Human dental pulp cells (HDPCs) were exposed to IL-1β or melatonin alone or to IL-1β with/without pretreatment with melatonin or other inhibitors. The mRNA expression of uPA, uPAR, and PAI-1 was quantified using real-time polymerase chain reaction analysis. The cellular uPA, PAI-1, and soluble uPAR (suPAR) production was determined using an enzyme- linked immunosorbent assay. Signaling molecules’ protein expression was analyzed by immunofluorescent staining. We found that IL-1β (0.1–10 ng/mL) stimulated uPA and uPAR expression/production but inhibited PAI-1 expression/production of HDPCs. Melatonin inhibited uPA but stimulated uPAR/suPAR and PAI-1 expression/production. Intriguingly, melatonin prevented IL-1β-induced uPA mRNA expression/production. Conversely, melatonin enhanced the IL-1β-induced uPAR and PAI-1 mRNA expression/protein production of HDPCs. IL-1β-induced suPAR production was attenuated by U0126 (a MEK/ERK inhibitor), SB203580 (a p38 inhibitor), and 5Z- 7oxozeaenol (a TAK1 inhibitor), whereas SB203580 prevented an IL-1β-induced decline of PAI-1 production. Moreover, melatonin attenuated the IL-1b-induced p-ERK, p-p38, p-Akt and p-TAK1. These results revealed the crucial role of IL- 1β in the pathogenesis of pulpal inflammation/repair via stimulation of uPA and uPAR and inhibition of PAI-1, which can be differentially regulated by p38, Akt, MEK/ERK, and TAK1. Melatonin exerts an anti-fibrinolytic effect on IL-1β- induced changes in uPA, uPAR, and PAI-1 in HDPCs. Clinically, the melatonin levels of patients may affect pulpal inflammatory response. Melatonin and signal transduction inhibitors may be administered concomitantly for the prevention and treatment of pulpal inflammatory diseases

Increased TGFβ1 and SMAD3 Contribute to Age-Related Aortic Valve Calcification.

AimsCalcific aortic valve disease (CAVD) is a progressive heart disease that is particularly prevalent in elderly patients. The current treatment of CAVD is surgical valve replacement, but this is not a permanent solution, and it is very challenging for elderly patients. Thus, a pharmacological intervention for CAVD may be beneficial. In this study, we intended to rescue aortic valve (AV) calcification through inhibition of TGFβ1 and SMAD3 signaling pathways.Methods and ResultsThe klotho gene, which was discovered as an aging-suppressor gene, has been observed to play a crucial role in AV calcification. The klotho knockout (Kl–/–) mice have shorter life span (8–12 weeks) and develop severe AV calcification. Here, we showed that increased TGFβ1 and TGFβ-dependent SMAD3 signaling were associated with AV calcification in Kl–/– mice. Next, we generated Tgfb1- and Smad3-haploinsufficient Kl–/– mice to determine the contribution of TGFβ1 and SMAD3 to the AV calcification in Kl–/– mice. The histological and morphometric evaluation suggested a significant reduction of AV calcification in Kl–/–; Tgfb1± mice compared to Kl–/– mice. Smad3 heterozygous deletion was observed to be more potent in reducing AV calcification in Kl–/– mice compared to the Kl–/–; Tgfb1± mice. We observed significant inhibition of Tgfb1, Pai1, Bmp2, Alk2, Spp1, and Runx2 mRNA expression in Kl–/–; Tgfb1± and Kl–/–; Smad3± mice compared to Kl–/– mice. Western blot analysis confirmed that the inhibition of TGFβ canonical and non-canonical signaling pathways were associated with the rescue of AV calcification of both Kl–/–; Tgfb1± and Kl–/–; Smad3± mice.ConclusionOverall, inhibition of the TGFβ1-dependent SMAD3 signaling pathway significantly blocks the development of AV calcification in Kl–/– mice. This information is useful in understanding the signaling mechanisms involved in CAVD.

Effect of DPP-4 inhibitors on erectile dysfunction caused by vincristine in rats

ABSTRACT Introduction Various chemotherapeutic agents are administered to cancer patients worldwide. Previously, an analysis of the US Food and Drug Administration (FDA) Adverse Event Reporting System (AERS) database showed that vincristine (VCR) increased the risk of erectile dysfunction (ED), as reported in the world meeting on Sexual Medicine 2018. As such, countermeasures for late complications after anticancer drug treatment are required. Neuropathy is a well-known side effect of VCR; however, in recent years, it has been reported that DPP-4 inhibitors improve neuropathy. Objective To investigate the effect of DPP-4 inhibitors to ED caused by VCR in rats. Methods Twelve-week-old male Wistar ST rats were separated into Control, VCR, and VCR+DPP-4i groups. VCR (0.1 mg/kg) was intravenously administered on days 1, 8, 15, and 22 in the VCR and VCR+DPP-4i groups. Two types of long-term preparations, toleragliptin (TGP; 3 mg/kg/week, po.) and omaligliptin (OGP; 3 mg/kg/week, po.), were examined for DPP-4i. Erectile function was tested using intracavernous pharmacotherapy (ICP) measurements, and endothelial function was tested through an isometric tension study. Additionally, mRNA expressions were tested using real-time PCR. Results After the 4-week observation period, the ICP/mean arterial pressure (MAP) ratio in the VCR group (0.42 ± 0.04) was significantly decreased compared to the Control group (0.72 ± 0.05) (P <0.01). The ICP/MAP ratio in the VCR+TGP group (0.64 ± 0.02) increased significantly compared to the VCR group (P <0.05), however the ICP/MAP ratio in the VCR+OGP (0.50 ± 0.08) did not increase significantly compared to the VCR group (P > 0.05). Real-time PCR analysis showed that the catalase, VEGF, MCP-1, and PAI-1 mRNA expressions were increased, while the IL-6 mRNA expression decreased significantly in the VCR+TGP and VCR+OGP groups. In contrast, the nNOS mRNA expression level was higher in the VCR+TGP group than in the VCR+OGP group. Conclusions In this study, we determined that DPP-4 inhibitors may improve ED by VCR administration. For ED associated with VCR administration, long-term DPP-4 inhibitor TGP administration suggested a significant improvement in erectile function, but OGP did not show a significant improvement effect. An increase in antioxidant activity and a decrease in inflammatory cytokines were observed for both drugs, including a surge in vascular increase factors. In contrast, the nNOS expression level was higher in TGP, suggesting that there may be a difference in the amount of NO production via nerves. Therefore, TGP may be useful for male sexual dysfunction associated with vincristine administration. Disclosure Work supported by industry: no.

Histomorphological changes and molecular mechanisms underlying the ameliorative effect of resveratrol on the liver of silver nanoparticles-exposed rats

ABSTRACT Exposure to the deleterious effects of silver nanoparticles (AgNPs) is inevitable due to their wide use in medicine and daily life. The current study aimed to delineate the histomorphological changes and the molecular mechanisms underlying the ameliorative effect of Resveratrol (RSV) on rats’ livers exposed to AgNPs. Fifty healthy adult male Wistar albino rats were divided into four groups: control, AgNPs-exposed, RSV-treated after AgNPs exposure, and recovery groups. Liver sections were examined by light and electron microscopes, and immunohistochemistry was performed for detection of activated caspase3 and TNFα. Serum ALT and AST, plasma levels of TNFα, IL-6, GSH and SOD were measured. mRNA expression of SIRT1, ADORA3, PAI, CDK1, Nrf2 and NFκB genes in liver tissue homogenate was performed using qRT-PCR. AgNPs-exposure for 28 days caused marked liver tissue damage with trapping in hepatocytes and Kupffer cells, while RSV treatment ameliorated liver ultrastructure and function. Our results clarified the molecular basis of RSV ameliorative effect on liver tissue by significant upregulation of SIRT1-NrF2 signaling pathway with increased levels of the antioxidant GSH and SOD, which represent the antioxidant effect of RSV. Significant upregulation of the protective ADORA3 with downregulation of the proinflammatory PAI-1 and NFκB mRNA expression levels besides decreased plasma levels of TNFα, IL-6 and decreased immunoexpression of TNFα in liver tissue, represent the anti-inflammatory effect of RSV. In addition, decreased immunoexpression of caspase3 and downregulation of CDK1 expression, represent its antiapoptotic effect. In conclusion: RSV ameliorates AgNPs-induced liver damage by antioxidant, anti-inflammatory and antiapoptotic effects. Abbreviations: AgNPs: Silver nanoparticles, RSV: Resveratrol, ROS: Reactive oxygen species, ESR: Electron spin resonance, DMPO: 5,5-Dimethyl-1-pyrroline-N-oxide, H2O2: Hydrogen peroxide, SOD: Superoxide dismutase, CAT: Catalase, GPx: Glutathione peroxidase, MPTP: Methyl-4-phenyl-1.2.3.6-tetrahydropyridine, MDA: Malondialdehyde, TNF: Tumor necrosis factor, GSH: Glutathione, Nrf2: Nuclear factor-erythroid 2-related factor 2, ARE: Antioxidant response elements, KEAP1: Kelch-1ike ECH-associated protein l, AMPK: AMP-activated protein kinase, HO-1: Heme oxygenase-1, NF-κB: Nuclear factor-kappa B, SIRT1: Sirtuins, FOXO: Forkhead box, UCP2: Uncoupling protein 2, STZ: Streptozotocin nicotinamide, HSC: hepatic stellate cells, ECM: extracellular matrix.

The role of CDH2 and MCP-1 mRNAs of blood extracellular vesicles in predicting early-stage diabetic nephropathy.

Extracellular vesicles (EVs), including exosomes and microvesicles, are involved in intercellular communication by transferring biomolecules such as mRNA, which has been shown to be as essential biomarkers for many physiological and pathological conditions such as diabetic nephropathy (DN). This study aimed to investigate the expression of CDH1, CDH2, MCP-1, and PAI-1 mRNAs in blood EVs of DN patients and to determine their accuracy in predicting early-stage DN. We recruited 196 participants, including 35 overt DN patients, 53 incipient DN patients, 62 diabetic patients (DM), and 46 healthy individuals. Quantification of the mRNA profile of blood EVs was performed using the qRT-PCR method. The diagnostic performance of mRNA was evaluated using receiver operating characteristic analysis. The mRNA expression of CDH2 and MCP-1 was downregulated in overt DN group (0.22-fold change and 0.15-fold change, respectively) and incipient DN group (0.60-fold change and 0.43-fold change, respectively) compared to DM group (1.72-fold change and 2.77-fold change, respectively), while PAI-1 mRNA expression decreased in incipient DN group (0.70-fold change) and DM group (0.58-fold change) compared to control. However, the expression level of CDH1 mRNA was not significantly different among the four groups (p = 0.408). Moreover, CDH2 and MCP-1 mRNAs inversely correlated with creatinine (r = -0.370 and r = -0.361, p<0.001) and Alb/Cr ratio (r = -0.355 and r = -0.297, p<0.001). 1/CDH2 mRNA also predicted overt DN with an accuracy of 0.75 (95%CI: 0.65-0.85) and incipient DN with an accuracy of 0.61 (95%CI: 0.50-0.71) while 1/MCP-1 mRNA had an accuracy of 0.66 (95%CI: 0.55-0.77) for overt DN prediction and an accuracy of 0.61 (95%CI: 0.51-0.71) for incipient DN prediction. CDH2 and MCP-1 mRNAs expression in blood EVs was decreased with the development of DN, suggesting the renoprotective effect of these mRNAs in diabetic individuals. Moreover, their quantifications could serve as diagnostic biomarkers for early-stage DN.

Lipopolysaccharide acting via toll-like receptor 4 transactivates the TGF-\u03b2 receptor in vascular smooth muscle cells

Toll-like receptors (TLRs) recognise pathogen‑associated molecular patterns, which allow the detection of microbial infection by host cells. Bacterial-derived toxin lipopolysaccharide activates TLR4 and leads to the activation of the Smad2 transcription factor. The phosphorylation of the Smad2 transcription factor is the result of the activation of the transforming growth factor-β receptor 1 (TGFBR1). Therefore, we sought to investigate LPS via TLR4-mediated Smad2 carboxy terminal phosphorylation dependent on the transactivation of the TGFBR1. The in vitro model used human aortic vascular smooth muscle cells to assess the implications of TLR4 transactivation of the TGFBR1 in vascular pathophysiology. We show that LPS-mediated Smad2 carboxy terminal phosphorylation is inhibited in the presence of TGFBR1 inhibitor, SB431542. Treatment with MyD88 and TRIF pathway antagonists does not affect LPS-mediated phosphorylation of Smad2 carboxy terminal; however, LPS-mediated Smad2 phosphorylation was inhibited in the presence of MMP inhibitor, GM6001, and unaffected in the presence of ROCK inhibitor Y27632 or ROS/NOX inhibitor DPI. LPS via transactivation of the TGFBR1 stimulates PAI-1 mRNA expression. TLRs are first in line to respond to exogenous invading substances and endogenous molecules; our findings characterise a novel signalling pathway in the context of cell biology. Identifying TLR transactivation of the TGFBR1 may provide future insight into the detrimental implications of pathogens in pathophysiology.

TM5614, an Inhibitor of Plasminogen Activator Inhibitor-1, Exerts an Antitumor Effect on Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is triggered by t(9;22)(q34;q11.2) translocation, leading to the formation of the BCR-ABL1 fusion gene. Although the development of BCR-ABL1 tyrosine kinase inhibitors (TKIs) has dramatically improved the prognosis of CML, the disease could often relapse, presumably because leukemic stem cell fraction of CML (CML-LSC) may reside in specific niches, and also acquire an ability to resist the cytotoxic agents. Recently a study indicated that pharmacological inhibition of plasminogen activator inhibitor-1 (PAI-1, also known as SERPINE1) would cause detachment of CML-LSCs from their niche by inducing maturation of membrane-type matrix metalloprotease-1 (MT1-MMP), leading to increased susceptibility of CML-LSCs against TKIs. However, the direct antitumor effect of PAI-1 inhibition in CML remains unclear. Because PAI-1 mRNA expression was lower in CML cell line (K562) than bone marrow mononuclear cells derived from CML patients, we established K562 cell clones stably expressing exogenous PAI-1 (K562/PAI-1). We found that TM5614 treatment significantly suppressed cell proliferation and induced apoptosis in K562/PAI-1 cells, accompanied by increased activity of Furin protease, which is a known target of PAI-1. Besides processing mature MT1-MMP, Furin is in charge of cleaving the NOTCH receptor to form a heterodimer before exporting it to the cell surface membrane. In K562/PAI-1 cells, TM5614 treatment increased NOTCH1 intracellular domain (NICD) protein expression as well as NOTCH1 target of HEY1 mRNA levels. Finally, forced expression of either Furin or NICD in K562/PAI-1 cells significantly inhibited cell proliferation and induced apoptosis. Collectively, PAI-1 inhibition may have an antitumor effect by modulating the Furin/NICD pathway.

BFGF stimulated plasminogen activation factors, but inhibited alkaline phosphatase and SPARC in stem cells from apical Papilla: Involvement of MEK/ERK, TAK1 and p38 signaling

IntroductionBasic fibroblast growth factor (bFGF) plays a critical role in odontoblast differentiation and dentin matrix deposition, thereby aiding pulpo-dentin repair and regeneration. ObjectivesThe purpose of this study was to clarify the effects of bFGF on plasminogen activation factors, TIMP-1), ALP; and SPARC (osteonectin) expression/production of stem cells from apical papilla (SCAP) in vitro; and the involvement of MEK/ERK, p38, Akt, and TAK1 signaling. MethodsSCAP were exposed to bFGF with/without pretreatment and co-incubation with various signal transduction inhibitors (U0126, SB203580, LY294002, and 5Z-7-oxozeaenol). The expression of FGF receptors (FGFRs), PAI-1, uPA, p-ERK, p-TAK1, and p-p38 was analyzed via immunofluorescent staining. The gene expression and protein secretion of SCAP were determined via real-time PCR and ELISA. ALP activity was evaluated via ALP staining. ResultsSCAP expressed FGFR1, 2, 3, and 4. bFGF stimulated the PAI-1, uPA, uPAR, and TIMP-1 mRNA expression (p < 0.05). bFGF induced PAI-1, uPA, and soluble uPAR production (p < 0.05) but suppressed the ALP activity and SPARC production (p < 0.05) of SCAP. bFGF stimulated ERK, TAK1, and p38 phosphorylation of SCAP. U0126 (a MEK/ERK inhibitor) and 5Z-7-oxozeaenol (a TAK1 inhibitor) attenuated the bFGF-induced PAI-1, uPA, uPAR, and TIMP-1 expression and production of SCAP, but SB203580 (a p38 inhibitor) did not. LY294002, SB203580, and 5Z-7oxozeaenol could not reverse the inhibition of ALP activity caused by bFGF. Interestingly, U0126 and 5Z-7-oxozeaenol prevented the bFGF-induced decline of SPARC production (p < 0.05). ConclusionbFGF may regulate fibrinolysis and matrix turnover via modulation of PAI-1, uPA, uPAR, and TIMP-1, but bFGF inhibited the differentiation (ALP, SPARC) of SCAP. These events are mainly regulated by MEK/ERK, p38, and TAK1. Combined use of bFGF and SCAP may facilitate pulpal/root repair and regeneration via regulation of the plasminogen activation system, migration, matrix turnover, and differentiation of SCAP.

Fat-1 Transgenic Mice With Augmented n3-Polyunsaturated Fatty Acids Are Protected From Liver Injury Caused by Acute-On-Chronic Ethanol Administration.

Alcohol-associated liver disease (ALD) is the leading cause of liver disease worldwide, and alcohol-associated hepatitis (AH), a severe form of ALD, is a major contributor to the mortality and morbidity due to ALD. Many factors modulate susceptibility to ALD development and progression, including nutritional factors such as dietary fatty acids. Recent work from our group and others showed that modulation of dietary or endogenous levels of n6-and n3-polyunsaturated fatty acids (PUFAs) can exacerbate or attenuate experimental ALD, respectively. In the current study, we interrogated the effects of endogenous n3-PUFA enrichment in a mouse model which recapitulates features of early human AH using transgenic fat-1 mice which endogenously convert n6-PUFAs to n3-PUFAs. Male wild type (WT) and fat-1 littermates were provided an ethanol (EtOH, 5% v/v)-containing liquid diet for 10 days, then administered a binge of EtOH (5 g/kg) by oral gavage on the 11th day, 9 h prior to sacrifice. In WT mice, EtOH treatment resulted in liver injury as determined by significantly elevated plasma ALT levels, whereas in fat-1 mice, EtOH caused no increase in this biomarker. Compared to their pair-fed controls, a significant EtOH-mediated increase in liver neutrophil infiltration was observed also in WT, but not fat-1 mice. The hepatic expression of several cytokines and chemokines, including Pai-1, was significantly lower in fat-1 vs WT EtOH-challenged mice. Cultured bone marrow-derived macrophages isolated from fat-1 mice expressed less Pai-1 and Cxcl2 (a canonical neutrophil chemoattractant) mRNA compared to WT when stimulated with lipopolysaccharide. Further, we observed decreased pro-inflammatory M1 liver tissue-resident macrophages (Kupffer cells, KCs), as well as increased liver T regulatory cells in fat-1 vs WT EtOH-fed mice. Taken together, our data demonstrated protective effects of endogenous n3-PUFA enrichment on liver injury caused by an acute-on-chronic EtOH exposure, a paradigm which recapitulates human AH, suggesting that n3-PUFAs may be a viable nutritional adjuvant therapy for this disease.

Anti-thrombotic activity of phenolic acids obtained from Salvia miltiorrhiza f. alba in TNF-α-stimulated endothelial cells via the NF-κB/JNK/p38 MAPK signaling pathway.

Over the past 100years, Salvia miltiorrhiza f. alba (Lamiaceae) (RSMA) roots have been used to cure thromboangiitis obliterans (TAO) in local clinics. This study aimed to confirm the anti-thrombotic efficacy of 12 phenolic acids obtained from RSMA and to clarify the possible underlying mechanisms. The results of quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that most of the phenolic acids markedly inhibited PAI-1 protein and mRNA levels but increased t-PA protein and mRNA levels in TNF-α-induced EA.hy926 cells (P < 0.05 or 0.001), with lithospermic acid displaying the strongest effect. In vitro anticoagulation and antiplatelet aggregation assays showed that lithospermic acid and salvianolic acid B significantly prolonged prothrombin time (PT), activated partial thromboplastin time (APTT), decreased fibrinogen concentration (FIB), and inhibited platelet aggregation induced by adenosine diphosphate (ADP) in rat blood. Both lithospermic acid and salvianolic acid B markedly down-regulated the expression of factor Xa and factor IIa on the external surface of EA.hy926 cells and demonstrated significant anti-factor IIa and anti-factor Xa activity using chromogenic substrates in vitro. Western blot results revealed that both lithospermic acid and salvianolic acid B also significantly inhibited the expression of TF, p-p65, p-p38, and pJNK proteins induced by TNF-α. These results indicated that all of the phenolic acids appeared to have some anti-thrombotic activity, with salvianolic acid B and lithospermic acid markedly decreasing the chance of thrombosis by regulating the NF-κB/JNK/p38 MAPK signaling pathway in response to TNF-α.

Modulation of the inflammatory environment by spermatozoa through regulation of transforming growth factor beta in porcine uterine epithelial cells

This study investigated the changes in the mRNA expression of transforming growth factor beta (TGF-β), plasminogen activators (PAs), and interleukin (IL) caused by sperm, as well as the regulatory mechanism of PA activity through TGF-β, in porcine uterine epithelial cells. The cells were isolated from the uterine horn of pig and co-incubated with Percoll-separated boar sperm (45% or 90%), or TGF-β for 24 h. The mRNA expression of TGF-β isoforms (TGF-β1, 2 and 3) and their receptors (TGF-β R1 and R2), PAs (urokinase-type, uPA; tissue-type, tPA; uPA receptor, uPAR; type 1 PA inhibitor, PAI-1), IL-6 and IL-8 was analyzed using real-time PCR. Supernatant was used to measure PA activity. Co-incubation with sperm from the 90% Percoll layer increased TGF-β1 mRNA, whereas TGF-β2 and TGF-β3 were decreased (P < 0.05). However, both TGF-βRs were not changed by the presence of the spermatozoa. Expression of tPA, PAI-1, IL-6, and IL-8 mRNA was down-regulated by 90% Percoll-separated sperm (P < 0.05), and sperm from 45% Percoll increased uPA expression (P < 0.05). TGF-β decreased tPA and IL-8 mRNA expression, and increased uPAR and PAI-1 mRNA (P < 0.05). The suppressive effect of TGF-β on PA activity was blocked by Smad2/3 and JNK1/2 signaling inhibitors (P < 0.05). In conclusion, sperm separated in 90% in porcine uterus could suppressed inflammation via modulation of TGF-β and down-regulation of PAs and ILs. Therefore, the regulatory mechanism of inflammation by sperm in the porcine uterus could be associated with interactions between numerous cytokines including TGF-β.

Remodeling the homeostasis of pro- and anti-angiogenic factors by Shenmai injection to normalize tumor vasculature for enhanced cancer chemotherapy

Ethnopharmacological relevanceNormalization of the tumor vasculature can enhance tumor perfusion and the microenvironment, leading to chemotherapy potentiation. Shenmai injection (SMI) is a widely used traditional Chinese herbal medicine for the combination treatment of cancer in China. Aim of this studyThis study aimed to investigate whether SMI can regulate tumor vasculature to improve chemotherapy efficacy and identify the underlying mechanism. Materials and methodsThe antitumor effect of SMI combined with 5-florouracil (5-FU) was investigated in xenograft tumor mice. Two-photon microscopy, laser speckle contrast imaging and immunofluorescence staining were used to investigate the effects of SMI on tumor vasculature in vivo. The mRNA and protein expression of pro- and anti-angiogenic factors were measured by Q-PCR and ELISA. Histone acetylation and transcriptional regulation were detected by Western blot and ChIP assay. ResultsSMI promoted normalization of tumor microvessels within a certain time window, which was accompanied by enhanced blood perfusion and 5-FU distribution in tumors. SMI significantly increased the expression of antiangiogenic factor angiostatin and decreased the pro-angiogenic factors VEGF, FGF and PAI-1 by day 10. SMI combined with neoadjuvant chemotherapy in colorectal cancer patients also showed a significant increase in angiostatin and decrease in VEGF and FGF in surgically resected tumors when compared to the neoadjuvant chemotherapy group. Further in vitro and in vivo studies revealed that SMI downregulated VEGF, FGF and PAI-1 mRNA expression by inhibiting histone H3 acetylation at the promoter regions. The enhanced production of angiostatin was attributed to the regulation of the plasminogen proteolysis system via SMI-induced PAI-1 inhibition. ConclusionSMI can remodel the homeostasis of pro- and anti-angiogenic factors to promote tumor vessel normalization, and thus enhance drug delivery and anti-tumor effect. This study provides additional insights into the pharmacological mechanisms of SMI on tumors from the perspective of vascular regulation.

Regulatory Mechanism of MicroRNA-30b on Neonatal Hypoxic-Ischemic Encephalopathy (HIE)

ObjectiveThis study is to investigate the role of microRNA (miR)-30b in the pathogenesis of hypoxic-ischemic encephalopathy (HIE) in neonates. MethodsTotally 26 cases of neonatal HIE were included in this study. The protein expression levels of CD26P and PAI-1 were detected with ELISA. Serum levels of miR-30b and PAI-1 mRNA was measured by quantitative real-time PCR. Human brain microvascular endothelial cells (HBMECs) were cultured under hypoxic condition, and the intracellular expression levels of miR-30b and PAI-1 were evaluated. Dual-luciferase reporter assay was performed to confirm the interaction between miR-30b and PAI-1. ResultsCompared with the control group, both the mRNA and protein expression levels of PAI-1 in the serum were up-regulated in the neonates with HIE, together with up-regulated serum CD26P levels. However, the serum expression level of miR-30b was down-regulated in neonatal HIE. In hypoxia-induced HBMECs, the mRNA and protein expression levels of PAI-1 were significantly up-regulated, while the miR-30b expression level was significantly down-regulated. Dual-luciferase reporter assay showed that PAI-1 was the direct target of miR-30b. ConclusionNeonatal HIE is accompanied with abnormal platelet activation, significantly up-regulated serum PAI-1 expression levels, and down-regulated miR-30b expression. MiR-30b might regulate the disease pathogenesis and immune responses via modulating PAI-1.

The phosphodiesterase 5 inhibitor tadalafil has renoprotective effects in a rat model of chronic kidney disease.

Phosphodiesterase 5 inhibitors are widely used to treat erectile dysfunction and lower urinary tract symptoms with benign prostatic hyperplasia. Recent studies have indicated the renoprotective effects of this class of compounds. Whether renoprotection depends on blood pressure reduction remains controversial. In this study, we investigated the renoprotective effects of the phosphodiesterase 5 inhibitor, tadalafil, in a rat model of high‐salt induced kidney injury with hypertension. Dahl salt‐sensitive rats were fed a normal diet, high‐salt (8% sodium chloride) diet, or high‐salt diet with oral administration of either low‐ or high‐dose tadalafil (1 and 10 mg kg−1 day−1, respectively). Serum creatinine, urinary protein, and blood pressure were measured at baseline and after 8 weeks, at which point the rats were examined for glomerular injury and fibrosis. PAI1 mRNA levels were also evaluated. After 8 weeks, blood pressure, serum creatinine, and urinary protein levels were significantly higher in the high‐salt group than those in the normal‐salt group. Serum creatinine and urinary protein were significantly lower in both tadalafil groups than those in the high‐salt group, while only high‐dose tadalafil affected blood pressure. In addition, glomerulosclerosis and α‐smooth muscle actin expression significantly decreased in both tadalafil treatment groups. PAI1 mRNA increased significantly in the high‐salt group but decreased in both tadalafil‐treated groups. Our results indicated that both low‐ and high‐dose tadalafil prevented fibrosis and glomerular injury in a chronic kidney disease rat model. Mechanistically, these effects may be associated with PAI1 expression and glomerular structure protection.

Galectin-1-Related Modulation of Trophoblast Endothelial Interactions by Integrins α1 and β1.

During normal trophoblast invasion, integrins α6β4 are downregulated, and α1β1 are upregulated in invasive cytotrophoblast cells. In preeclampsia both interstitial and endovascular invasion are shallow and cytotrophoblasts fail to upregulate α1β1 and downregulate α6β4. This study aims to investigate the role of integrins α1β1 and α6β4 on cellular pathways influencing trophoblast integration into endothelial cellular networks in vitro. Red fluorescent-labeled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on Matrigel to form endothelial networks. Green fluorescent-labeled trophoblastic HTR-8/SVneo cells pre-incubated with 20μg/ml of neutralizing antibodies (anti-α1, β1, α6, β4, α1+β1, or α6+β4) for 1h were then co-cultured with endothelial networks with the neutralizing antibodies for 24h. Fluorescent images were captured, and quantified utilizing Image J. Cells were retrieved to analyze mRNA expression of galectin-1, TIMP-1, and PAI-1 by quantitative PCR. MMP-2, MMP-9, free sFlt-1, and PlGF from conditioned media were measured by ELISA. The integration of trophoblast cells into endothelial cellular networks was inhibited by anti-β1(- 28± 3%, p< 0.0001), and increased by anti-α6(+ 19± 5%, p< 0.01). Galectin-1 mRNA expression was decreased by anti-α1(- 35± 7%, p< 0.001), anti-β1(-23± 5%, p< 0.05), and anti-α1+β1(- 35± 5%, p< 0.001). The mRNA expression of TIMP-1 was inhibited by anti-α1(- 59 ± 9%, p< 0.01) and anti-β1(- 63± 7%, p< 0.001) while PAI-1 mRNA expression was increased by anti-α1 + β1(+ 285 ± 70%, p< 0.0001). In the conditioned medium, anti-α1 reduced MMP-2(-28± 1%, p< 0.001), MMP-9(-27± 8%, p< 0.01), and sFlt-1(-27± 5%, p< 0.001) production. Anti-β1 reduced MMP-2(- 15± 2%, p< 0.05) production. There were no changes in PlGF. Appropriate integrins α1β1 modulate trophoblast cell integration into endothelial cellular networks in vitro through invasive pathways including galectin-1, TIMP-1, PAI-1, MMP-2, and MMP-9 production.