Hypoxia and Foxn1 alter the proteomic signature of dermal fibroblasts to redirect scarless wound healing to scar-forming skin wound healing in Foxn1-/- mice.

BACKGROUND: Foxn1-/- deficient mice are a rare model of regenerative skin wound healing among mammals. In wounded skin, the transcription factor Foxn1 interacting with hypoxia-regulated factors affects re-epithelialization, epithelial-mesenchymal transition (EMT) and dermal white adipose tissue (dWAT) reestablishment and is thus a factor regulating scar-forming/reparative healing. Here, we hypothesized that transcriptional crosstalk between Foxn1 and Hif-1α controls the switch from scarless (regenerative) to scar-present (reparative) skin wound healing. To verify this hypothesis, we examined (i) the effect of hypoxia/normoxia and Foxn1 signalling on the proteomic signature of Foxn1-/- (regenerative) dermal fibroblasts (DFs) and then (ii) explored the effect of Hif-1α or Foxn1/Hif-1α introduced by a lentiviral (LV) delivery vector to injured skin of regenerative Foxn1-/- mice with particular attention to the remodelling phase of healing. RESULTS: We showed that hypoxic conditions and Foxn1 stimulation modified the proteome of Foxn1-/- DFs. Hypoxic conditions upregulated DF protein profiles, particularly those related to extracellular matrix (ECM) composition: plasminogen activator inhibitor-1 (Pai-1), Sdc4, Plod2, Plod1, Lox, Loxl2, Itga2, Vldlr, Ftl1, Vegfa, Hmox1, Fth1, and F3. We found that Pai-1 was stimulated by hypoxic conditions in regenerative Foxn1-/- DFs but was released by DFs to the culture media exclusively upon hypoxia and Foxn1 stimulation. We also found higher levels of Pai-1 protein in DFs isolated from Foxn1+/+ mice (reparative/scar-forming) than in DFs isolated from Foxn1-/- (regenerative/scarless) mice and triggered by injury increase in Foxn1 and Pai-1 protein in the skin of mice with active Foxn1 (Foxn1+/+ mice). Then, we demonstrated that the introduction of Foxn1 and Hif-1α via lentiviral injection into the wounded skin of regenerative Foxn1-/- mice activates reparative/scar-forming healing by increasing the wounded skin area and decreasing hyaluronic acid deposition and the collagen type III to I ratio. We also identified a stimulatory effect of LV-Foxn1 + LV-Hif-1α injection in the wounded skin of Foxn1-/- mice on Pai-1 protein levels. CONCLUSIONS: The present data highlight the effect of hypoxia and Foxn1 on the protein profile and functionality of regenerative Foxn1-/- DFs and demonstrate that the introduction of Foxn1 and Hif-1α into the wounded skin of regenerative Foxn1-/- mice activates reparative/scar-forming healing.

PAI-1 deficiency drives pulmonary vascular smooth muscle remodeling and pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PAs). Central to the remodeling process is a switch of pulmonary vascular cells to a proliferative, apoptosis-resistant phenotype. Plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2) are the primary physiological inhibitors of urokinase-type and tissue-type plasminogen activators (uPA and tPA), but their roles in PAH are unsettled. Here, we report that: 1) PAI-1, but not PAI-2, is deficient in remodeled small PAs and in early-passage PA smooth muscle and endothelial cells (PASMCs and PAECs) from subjects with PAH compared with controls; 2) PAI-1-/- mice spontaneously develop pulmonary vascular remodeling associated with upregulation of mTORC1 signaling, pulmonary hypertension (PH), and right ventricle (RV) hypertrophy; and 3) pharmacological inhibition of uPA in human PAH PASMCs suppresses proproliferative mTORC1 and SMAD3 signaling, restores PAI-1 levels, reduces proliferation, and induces apoptosis in vitro, and prevents the development of SU5416/hypoxia-induced PH and RV hypertrophy in vivo in mice. These data strongly suggest that downregulation of PAI-1 in small PAs promotes vascular remodeling and PH due to unopposed activation of uPA and consequent upregulation of mTOR and transforming growth factor-ß (TGF-ß) signaling in PASMCs, and call for further studies to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate and/or reverse pulmonary vascular remodeling and PH.NEW & NOTEWORTHY This study identifies a novel role for the deficiency of plasminogen activator inhibitor (PAI)-1 and resultant unrestricted uPA activity in PASMC remodeling and PH in vitro and in vivo, provides novel mechanistic link from PAI-1 loss through uPA-induced Akt/mTOR and TGFß-Smad3 upregulation to pulmonary vascular remodeling in PH, and suggests that inhibition of uPA to rebalance the uPA-PAI-1 tandem might provide a novel approach to complement current therapies used to mitigate this pulmonary vascular disease.

Efficacy and safety of TM5614 in combination with paclitaxel in the treatment of paclitaxel-resistant cutaneous angiosarcoma: Phase II study protocol.

Cutaneous angiosarcoma (CAS) is an endothelial cell-derived, highly aggressive type of vascular tumour. Although chemoradiotherapy with paclitaxel (PTX) is recognized as a first-line therapy for CAS, second-line therapy for CAS remains controversial, and there is no standard therapy for taxane-resistant CAS. Plasminogen activator inhibitor-1 (PAI-1) is associated with poor clinical outcomes, and elevated levels of PAI-1 in both tissue and serum are correlated with poor response to therapy in various cancers, including skin cancers. Since PAI-1 protects endothelial cells from Fas ligand-mediated apoptosis, PAI-1 inhibition might induce apoptosis of endothelial cell-derived tumours such as CAS. This is a single-arm, open-label, multi-institutional, Phase 2 clinical trial to assess the efficacy and safety of PTX in combination with TM5614 (PAI-1 inhibitor) in patients with PTX-resistant CAS. PTX will be administered for 28 weeks, with oral administration of TM5614. The primary endpoint of this study will be the overall response rate (ORR) at 28 weeks after starting treatment (central image evaluation). The secondary endpoint will include the ORR at 28 weeks after starting treatment (investigator evaluation), ORR at 8 weeks and 16 weeks after initiation of treatment (central and investigator evaluation), progression-free survival, overall survival, disease control rate and safety profiles. Assuming the null hypothesis of a response rate of 13.6% and an alternative hypothesis of 45%, a minimum of 15 patients are required to achieve a two-sided, Type I error of 5% and power of 70% based on the exact binomial distribution. Data quality control will be conducted by a combination of centralized (remote) and on-site monitoring. This study will contribute to the development of novel combination therapy for PTX-resistant CAS patients, which remains an unmet clinical need.

Possible effects of plasminogen activator inhibitor-1 on promoting angiogenesis through matrix metalloproteinase 9 in advanced mycosis fungoides.

Mycosis fungoides (MF) progresses slowly before advancing to skin tumors followed by lymph node and visceral involvement. Among MF progression, stage IIB is an initial time point of tumor formation in MF. Since MF in tumor stage possess abundant blood vessels, it is important to evaluate the pro-angiogenic factors before and after MF in stage IIB. In this report, we investigated pro-angiogenic soluble factors in MF patients, as well as its pro-angiogenetic effects on tumor cells and stroma cells. We first evaluated the serum levels of pro-angiogenic factors in 9 MF patients without tumor formation and 8 MF patients with tumor formation. Among them, the serum MMP-9 and plasminogen activator inhibitors 1 (PAI-1) was significantly increased in MF with tumor formation compared in MF without tumor formation, leading to favorable formation of human dermal microvascular endothelial cells tube networks. Moreover, PAI-1 stimulation significantly increased the mRNA expression and protein production MMP-9 on monocytes derived M2 macrophages and HUT-78. Furthermore, since MMP-9 production from tumor cells as well as stromal cells is suppressed by bexarotene, we evaluate the baseline serum pro-angiogenic factors including MMP-9 in 16 patients with advanced cutaneous T cell lymphoma treated with bexarotene. The serum levels of MMP-2 and MMP-9 was significantly increased in bexarotene non-responded patients compared to responded patients. Our present study suggested the significance of MMP-9 and PAI-1 for the progression of MF stage toward to the tumor stage, and could be a therapeutic target in future.

Growth differentiation factor myostatin regulates epithelial-mesenchymal transition genes and enhances invasion by increasing serine protease inhibitors E1 and E2 in human trophoblast cells.

Placental insufficiency disorders, including preeclampsia and intrauterine growth restriction, are major obstetric complications that can have devastating effects on both the mother and the fetus. These syndromes have underlying poor placental trophoblast cell invasion into uterine tissues. Placental invasion is controlled by many hormones and growth factors. Myostatin (MSTN) is a transforming growth factor-ß superfamily member recognized for its important role in muscle growth control. MSTN has also been shown to be secreted and functioning in the placenta, and its serum and/or placental levels were found to be upregulated in preeclampsia and intrauterine growth restriction. Considering that the mechanistic role of MSTN in placentation remains poorly understood, we hypothesized that MSTN uses ALK4/5-SMAD2/3/4 signaling to increase human trophoblast invasion through a group of epithelial-mesenchymal transition genes including SERPINE2, PAI-1, and SOX4. mRNA sequencing of control and MSTN-treated primary human trophoblast cells (n = 5) yielded a total of 610 differentially expressed genes (false discovery rate <0.05) of which 380 genes were upregulated and 230 were downregulated. These differentially expressed genes were highly enriched in epithelial-mesenchymal transition genes, and a subset including SERPINE2, PAI-1, and SOX4 was investigated for its role in MSTN-induced trophoblast cell invasion. We found that MSTN induced upregulation of SERPINE2 via ALK4/5-SMAD2/3/4 signaling; however, SMAD2 was not involved in MSTN-induced PAI-1 upregulation. SOX4 was involved in MSTN-induced upregulation of SERPINE2, but not PAI-1. Collectively, this study discovers novel molecular mechanisms of MSTN-induced human trophoblast cell invasion and provides insight into the functional consequences of its dysregulation in placental insufficiency disorders.

PAI-1 transfected-conditioned media promotes osteogenic differentiation of hBMSCs.

Reconstruction of injured bone remains challenging in the clinic owing to the lack of suitable bone grafts. The utilization of PAI-1 transfected-conditioned media (P-CM) has demonstrated its ability to facilitate the differentiation process of mesenchymal stem cells (MSCs), potentially serving as a crucial mediator in tissue regeneration. This research endeavored to explore the therapeutic potential of P-CM concerning the differentiation of human bone marrow mesenchymal stem cells (hBMSCs). To assess new bone formation, a rat calvaria critical defect model was employed, while in vitro experiments involved the use of the alizarin Red-S mineral induction test. In the rat calvaria critical defect model, P-CM treatment resulted in significan new bone formation. In vitro, P-CM treated hBMSCs displayed robust osteogenesis compared to the control group, as demonstrated by the mineral induction test using alizarin Red-S. P-CM with hydroxyapatite/ß-tricalcium phosphate/fibrin gel treatment significantly exhibited new bone formation, and the expression of osteogenic associated markers was enhanced in the P-CM-treated group. In conclusion, results demonstrate that P-CM treatment significantly enhanced the osteogenic differantiation efficiency and new bone formation, thus could be used as an ideal therapeutic biomolecule for constructing bone-specific implants, especially for orthopedic and dental applications.

Clinical value of echocardiography combined with serum Cav-1, NFATc1, and PAI-1 in the diagnosis of Kawasaki disease complicated with coronary artery lesions.

To analyze the clinical value of echocardiography combined with serum lacuna protein-1 (Cav-1), activated T cell nuclear factor C1 (NFATc1), and plasminogen activator inhibitor-1 (PAI-1) in the diagnosis of Kawasaki disease (KD) complicated with coronary artery lesions (CAL). A total of 200 children with KD treated in our hospital from January 2019 to October 2021 were grouped as the KD alone group (n = 56) and the KD complicated with CAL group (n = 144) according to the results of coronary angiography. The levels of Cav-1, NFATc1, and PAI-1 were detected by enzyme-linked immunosorbent assay. Echocardiography was performed and the internal diameters of left and right coronary arteries were compared between the two groups. The area under the curve (AUC), sensitivity, and specificity of echocardiography combined with serum Cav-1, NFATc1, and PAI-1 in the diagnosis of KD complicated with CAL were analyzed with receiver operating characteristic (ROC) curve. Coronary angiography, as the gold standard, showed that the sensitivity of echocardiography in diagnosing KD with CAL was 88.19% (127/144), the specificity was 66.07% (37/56), and the accuracy was 82.00% (164/200). ROC curve analysis revealed that the AUC of KD complicated with CAL diagnosed by echocardiography, Cav-1, NFATc1, and PAI-1 was 0.819, 0.715, 0.688, and 0.663, respectively, and the AUC of combined diagnosis of the four was 0.896. The combination of echocardiography, Cav-1, NFATc1, and PAI-1 has high value in diagnosing KD complicated with CAL, which can be widely used in clinical practice.

JCAD promotes arterial thrombosis through PI3K/Akt modulation: a translational study.

AIMS: Variants of the junctional cadherin 5 associated (JCAD) locus associate with acute coronary syndromes. JCAD promotes experimental atherosclerosis through the large tumor suppressor kinase 2 (LATS2)/Hippo pathway. This study investigates the role of JCAD in arterial thrombosis. METHODS AND RESULTS: JCAD knockout (Jcad-/-) mice underwent photochemically induced endothelial injury to trigger arterial thrombosis. Primary human aortic endothelial cells (HAECs) treated with JCAD small interfering RNA (siJCAD), LATS2 small interfering RNA (siLATS2) or control siRNA (siSCR) were employed for in vitro assays. Plasma JCAD was measured in patients with chronic coronary syndrome or ST-elevation myocardial infarction (STEMI). Jcad-/- mice displayed reduced thrombogenicity as reflected by delayed time to carotid occlusion. Mechanisms include reduced activation of the coagulation cascade [reduced tissue factor (TF) expression and activity] and increased fibrinolysis [higher thrombus embolization episodes and D-dimer levels, reduced vascular plasminogen activator inhibitor (PAI)-1 expression]. In vitro, JCAD silencing inhibited TF and PAI-1 expression in HAECs. JCAD-silenced HAECs (siJCAD) displayed increased levels of LATS2 kinase. Yet, double JCAD and LATS2 silencing did not restore the control phenotype. si-JCAD HAECs showed increased levels of phosphoinositide 3-kinases (PI3K)/ proteinkinase B (Akt) activation, known to downregulate procoagulant expression. The PI3K/Akt pathway inhibitor-wortmannin-prevented the effect of JCAD silencing on TF and PAI-1, indicating a causative role. Also, co-immunoprecipitation unveiled a direct interaction between JCAD and Akt. Confirming in vitro findings, PI3K/Akt and P-yes-associated protein levels were higher in Jcad-/- animals. Lastly, as compared with chronic coronary syndrome, STEMI patients showed higher plasma JCAD, which notably correlated positively with both TF and PAI-1 levels. CONCLUSIONS: JCAD promotes arterial thrombosis by modulating coagulation and fibrinolysis. Herein, reported translational data suggest JCAD as a potential therapeutic target for atherothrombosis.

Involvement of Matricellular Proteins in Cellular Senescence: Potential Therapeutic Targets for Age-Related Diseases.

Senescence is a physiological and pathological cellular program triggered by various types of cellular stress. Senescent cells exhibit multiple characteristic changes. Among them, the characteristic flattened and enlarged morphology exhibited in senescent cells is observed regardless of the stimuli causing the senescence. Several studies have provided important insights into pro-adhesive properties of cellular senescence, suggesting that cell adhesion to the extracellular matrix (ECM), which is involved in characteristic morphological changes, may play pivotal roles in cellular senescence. Matricellular proteins, a group of structurally unrelated ECM molecules that are secreted into the extracellular environment, have the unique ability to control cell adhesion to the ECM by binding to cell adhesion receptors, including integrins. Recent reports have certified that matricellular proteins are closely involved in cellular senescence. Through this biological function, matricellular proteins are thought to play important roles in the pathogenesis of age-related diseases, including fibrosis, osteoarthritis, intervertebral disc degeneration, atherosclerosis, and cancer. This review outlines recent studies on the role of matricellular proteins in inducing cellular senescence. We highlight the role of integrin-mediated signaling in inducing cellular senescence and provide new therapeutic options for age-related diseases targeting matricellular proteins and integrins.

Intrapleural Fibrinolytic Interventions for Retained Hemothoraces in Rabbits.

Bleeding within the pleural space may result in persistent clot formation called retained hemothorax (RH). RH is prone to organization, which compromises effective drainage, leading to lung restriction and dyspnea. Intrapleural fibrinolytic therapy is used to clear the persistent organizing clot in lieu of surgery, but fibrinolysin selection, delivery strategies, and dosing have yet to be identified. We used a recently established rabbit model of RH to test whether intrapleural delivery of single-chain urokinase (scuPA) can most effectively clear RH. scuPA, or single-chain tissue plasminogen activator (sctPA), was delivered via thoracostomy tube on day 7 as either one or two doses 8 h apart. Pleural clot dissolution was assessed using transthoracic ultrasonography, chest computed tomography, two-dimensional and clot displacement measurements, and gross analysis. Two doses of scuPA (1 mg/kg) were more effective than a bolus dose of 2 mg/kg in resolving RH and facilitating drainage of pleural fluids (PF). Red blood cell counts in the PF of scuPA, or sctPA-treated rabbits were comparable, and no gross intrapleural hemorrhage was observed. Both fibrinolysins were equally effective in clearing clots and promoting pleural drainage. Biomarkers of inflammation and organization were likewise comparable in PF from both groups. The findings suggest that single-agent therapy may be effective in clearing RH; however, the clinical advantage of intrapleural scuPA remains to be established by future clinical trials.

Serum Levels of Plasminogen Activator Inhibitor-1 in Patients with Parkinson's Disease.

OBJECTIVES: The aim of the study was to investigate serum plasminogen activator inhibitor-1 (PAI-1) levels of patients with Parkinson's disease (PD) and their relationship with clinical findings and treatment of disease. METHODS: The study included 125 PD patients and 48 healthy controls. Patients have been taking effective dopaminergic treatment regularly. The clinical severity of parkinsonism was assessed using the Hoehn and Yahr (HY) staging scale and the Unified PD Rating Scale (UPDRS). PAI-1 level analysis was performed by enzyme-linked immunosorbent assay. RESULTS: Patients with PD had significantly lower serum PAI-1 levels than healthy controls (p < 0.001). Correlations with clinical findings showed only a marginally positive correlation between serum PAI-1 and HY score (r = 0.170, p = 0.05). In contrast, no significant correlation was demonstrated with the UPDRS score or other clinical parameters. CONCLUSION: This is the first comprehensive analysis of serum PAI-1 levels in patients with PD. The distribution of PAI-1 in PD appears to be complex. The study results implicate that the paradoxical effects of tissue plasminogen activator on the brain parenchyma can be important in the pathophysiology of PD. Future studies are needed to elucidate the role of fibrinolytic system components in PD.

An Inhibitory Function of TRPA1 Channels in TGF-ß1-driven Fibroblast-to-Myofibroblast Differentiation.

TRPA1 (transient receptor potential ankyrin 1) is a nonselective Ca2+-permeable cation channel, which was originally cloned from human lung fibroblasts (HLFs). TRPA1-mediated Ca2+ entry is evoked by exposure to several chemicals, including allyl isothiocyanate (AITC), and a protective effect of TRPA1 activation in the development of cardiac fibrosis has been proposed. Yet the function of TRPA1 in TGF-ß1 (transforming growth factor-ß1)-driven fibroblast-to-myofibroblast differentiation and the development of pulmonary fibrosis remains elusive. TRPA1 expression and function were analyzed in cultured primary HLFs, and mRNA concentrations were significantly reduced after adding TGF-ß1. Expression of genes encoding fibrosis markers (e.g., ACTA2, SERPINE1 [plasminogen activator inhibitor 1], FN1 [fibronectin], COL1A1 [type I collagen]) was increased after siRNA-mediated downregulation of TRPA1 mRNA in HLFs. Moreover, AITC-induced Ca2+ entry in HLFs was decreased after TGF-ß1 treatment and by application of TRPA1 siRNAs, while AITC treatment alone did not reduce cell viability or enhance apoptosis. Most interestingly, AITC-induced TRPA1 activation augmented ERK1/2 (extracellular signal-regulated kinase 1/2) and SMAD2 linker phosphorylation, which might inhibit TGF-ß-receptor signaling. Our results suggest an inhibitory function of TRPA1 channels in TGF-ß1-driven fibroblast-to-myofibroblast differentiation. Therefore, activation of TRPA1 channels might be protective during the development of pulmonary fibrosis in patients.

An antibody that targets cell-surface glucose-regulated protein-78 inhibits expression of inflammatory cytokines and plasminogen activator inhibitors by macrophages.

Glucose-regulated protein-78 (Grp78) is an endoplasmic reticulum chaperone, which is secreted by cells and associates with cell surfaces, where it functions as a receptor for activated α2 -macroglobulin (α2 M) and tissue-type plasminogen activator (tPA). In macrophages, α2 M and tPA also bind to the transmembrane receptor, LDL receptor-related protein-1 (LRP1), activating a cell-signaling receptor assembly that includes the NMDA receptor (NMDA-R) to suppress innate immunity. Herein, we demonstrate that an antibody targeting Grp78 (N88) inhibits NFκB activation and expression of proinflammatory cytokines in bone marrow-derived macrophages (BMDMs) treated with the toll-like receptor-4 (TLR4) ligand, lipopolysaccharide, or with agonists that activate TLR2, TLR7, or TLR9. Pharmacologic inhibition of the NMDA-R or deletion of the gene encoding LRP1 (Lrp1) in BMDMs neutralizes the activity of N88. The fibrinolysis protease inhibitor, plasminogen activator inhibitor-1 (PAI1), has been implicated in diverse diseases including metabolic syndrome, cardiovascular disease, and type 2 diabetes. Deletion of Lrp1 independently increased expression of PAI1 and PAI2 in BMDMs, as did treatment of wild-type BMDMs with TLR agonists. tPA, α2 M, and N88 inhibited expression of PAI1 and PAI2 in BMDMs treated with TLR-activating agents. Inhibiting Src family kinases blocked the ability of both N88 and tPA to function as anti-inflammatory agents, suggesting that the cell-signaling pathway activated by tPA and N88, downstream of LRP1 and the NMDA-R, may be equivalent. We conclude that targeting cell-surface Grp78 may be effective in suppressing innate immunity by a mechanism that requires LRP1 and the NMDA-R.

Myofibroblast depletion reduces kidney cyst growth and fibrosis in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) involves the development and persistent growth of fluid filled kidney cysts. In a recent study, we showed that ADPKD kidney cyst epithelial cells can stimulate the proliferation and differentiation of peri-cystic myofibroblasts. Although dense myofibroblast populations are often found surrounding kidney cysts, their role in cyst enlargement or fibrosis in ADPKD is unclear. To clarify this, we examined the effect of myofibroblast depletion in the Pkd1RC/RC (RC/RC) mouse model of ADPKD. RC/RC;αSMAtk mice that use the ganciclovir-thymidine kinase system to selectively deplete α-smooth muscle actin expressing myofibroblasts were generated. Ganciclovir treatment for four weeks depleted myofibroblasts, reduced kidney fibrosis and preserved kidney function in these mice. Importantly, myofibroblast depletion significantly reduced cyst growth and cyst epithelial cell proliferation in RC/RC;αSMAtk mouse kidneys. Similar ganciclovir treatment did not alter cyst growth or fibrosis in wild-type or RC/RC littermates. In vitro, co-culture with myofibroblasts from the kidneys of patients with ADPKD increased 3D microcyst growth of human ADPKD cyst epithelial cells. Treatment with conditioned culture media from ADPKD kidney myofibroblasts increased microcyst growth and cell proliferation of ADPKD cyst epithelial cells. Further examination of ADPKD myofibroblast conditioned media showed high levels of protease inhibitors including PAI1, TIMP1 and 2, NGAL and TFPI-2, and treatment with recombinant PAI1 and TIMP1 increased ADPKD cyst epithelial cell proliferation in vitro. Thus, our findings show that myofibroblasts directly promote cyst epithelial cell proliferation, cyst growth and fibrosis in ADPKD kidneys, and their targeting could be a novel therapeutic strategy to treat PKD.

Fibroblast growth factor 2 (FGF2) ameliorates the coagulation abnormalities in sepsis.

BACKGROUND: Sepsis is defined as a life-threatening organ dysfunction caused by dysregulation of the host response to infection. There is still a lack of specific treatment for sepsis. Here, we report that Fibroblast growth factor-2 (FGF2) can reduce the mortality of sepsis by ameliorating the coagulation abnormalities. METHODS: FGF2 was intraperitoneally injected into septic mice induced by lipopolysaccharide (LPS) and then assessed for coagulation response, organ damage and survival. RAW264.7 cells with or without FGF2 pretreating were exposed to LPS, and then changes in coagulation related factors expression and signaling were tested. RESULTS: The findings showed that intraperitoneal injection of FGF2 inhibited coagulation activity, reduced lung and liver damage, and increased survival in septic mice. In RAW264.7 cells, LPS upregulated the expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1); however, pretreatment with FGF2 prevented this upregulation, while FGF2 knockdown exacerbated TF upregulation. Moreover, FGF2 suppressing the AKT/mTOR/S6K1 signaling pathway in septic mice and RAW264.7 cells stimulated by LPS. CONCLUSIONS: This study revealed a therapeutic role of FGF2 in ameliorating the coagulation abnormalities during sepsis.

Plasminogen activating inhibitor-1 promotes angiogenesis in cutaneous angiosarcomas.

Plasminogen activating inhibitor-1 (PAI-1) is associated with poor clinical outcomes, and elevated levels of PAI-1 in both tissue and serum are correlated with poor response to therapy in various cancers, including skin cancer. Cutaneous angiosarcoma (CAS) is a vascular tumor histologically characterized by detachment of endothelial cell-derived tumor cells. Since CAS expresses multiple angiogenic growth factors and has increased expressions of angiogenic receptor tyrosine kinase transcripts including VEGFR1/2/3, angiogenesis-promoting factors are potential drug targets in CAS. In this study, the expression of PAI-1 was examined in 31 cases of CAS, and the immunomodulatory effects of PAI-1 on a human CAS cell line, ISO-HAS-B, were evaluated. We found that, of the angiogenesis-promoting factors, PAI-1 was expressed in almost all cases of CAS, and PAI-1 increased the mRNA expressions of IL-23p19, VEGF-C, CXCL5 and CCL20 on ISO-HAS-B. Moreover, PAI-1 stimulated ISO-HAS-B culture supernatant promoted favourable tube networks, suggesting that these tumor-derived factors promote the pro-angiogenic effect on tumor development. In addition, IL-23p19 was expressed in 61.3% of cases, whereas VEGF-C was expressed in 41% of cases. The results of the present study suggest that PAI-1 promotes angiogenesis that results in tumor progression in CAS.

Damage-associated molecular patterns and fibrinolysis perturbation are associated with lethal outcomes in traumatic injury.

BACKGROUND: Upon cellular injury, damage-associated molecular patterns (DAMPs) are released into the extracellular space and evoke proinflammatory and prothrombotic responses in animal models of sterile inflammation. However, in clinical settings, the dynamics of DAMP levels after trauma and links between DAMPs and trauma-associated coagulopathy remain largely undetermined. METHODS: Thirty-one patients with severe trauma, who were transferred to Kagoshima City Hospital between June 2018 and December 2019, were consecutively enrolled in this study. Blood samples were taken at the time of delivery, and 6 and 12 h after the injury, and once daily thereafter. The time-dependent changes of coagulation/fibrinolysis markers, including thrombin-antithrombin complex, α2-plasmin inhibitor (α2-PI), plasmin-α2-PI complex, and plasminogen activator inhibitor-1 (PAI-1), and DAMPs, including high mobility group box 1 and histone H3, were analyzed. The relationship between coagulation/fibrinolysis markers, DAMPs, Injury Severity Score, in-hospital death, and amount of blood transfusion were analyzed. RESULTS: The activation of coagulation/fibrinolysis pathways was evident at the time of delivery. In contrast, PAI-1 levels remained low at the time of delivery, and then were elevated at 6-12 h after traumatic injury. Histone H3 and high mobility group box 1 levels were elevated at admission, and gradually subsided over time. PAI-1 levels at 6 h were associated with serum histone H3 levels at admission. Increased histone H3 levels and plasmin-α2-PI complex levels were associated with in-hospital mortality. α2-PI levels at admission showed the strongest negative correlation with the amount of blood transfusion. CONCLUSION: The elevation of histone H3 levels and fibrinolysis perturbation are associated with fatal outcomes in patients with traumatic injury. Patients with low α2-PI levels at admission tend to require blood transfusion.

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.

PAI-1 production by reactive astrocytes drives tissue dysfibrinolysis in multiple sclerosis models.

BACKGROUND: In multiple sclerosis (MS), disturbance of the plasminogen activation system (PAS) and blood brain barrier (BBB) disruption are physiopathological processes that might lead to an abnormal fibrin(ogen) extravasation into the parenchyma. Fibrin(ogen) deposits, usually degraded by the PAS, promote an autoimmune response and subsequent demyelination. However, the PAS disruption is not well understood and not fully characterized in this disorder. METHODS: Here, we characterized the expression of PAS actors during different stages of two mouse models of MS (experimental autoimmune encephalomyelitis-EAE), in the central nervous system (CNS) by quantitative RT-PCR, immunohistofluorescence and fluorescent in situ hybridization (FISH). Thanks to constitutive PAI-1 knockout mice (PAI-1 KO) and an immunotherapy using a blocking PAI-1 antibody, we evaluated the role of PAI-1 in EAE models and its impact on physiopathological processes such as fibrin(ogen) deposits, lymphocyte infiltration and demyelination. RESULTS: We report a striking overexpression of PAI-1 in reactive astrocytes during symptomatic phases, in two EAE mouse models of MS. This increase is concomitant with lymphocyte infiltration and fibrin(ogen) deposits in CNS parenchyma. By genetic invalidation of PAI-1 in mice and immunotherapy using a blocking PAI-1 antibody, we demonstrate that abolition of PAI-1 reduces the severity of EAE and occurrence of relapses in two EAE models. These benefits are correlated with a decrease in fibrin(ogen) deposits, infiltration of T4 lymphocytes, reactive astrogliosis, demyelination and axonal damage. CONCLUSION: These results demonstrate that a deleterious overexpression of PAI-1 by reactive astrocytes leads to intra-parenchymal dysfibrinolysis in MS models and anti-PAI-1 strategies could be a new therapeutic perspective for MS.

SOX2-OT induced by PAI-1 promotes triple-negative breast cancer cells metastasis by sponging miR-942-5p and activating PI3K/Akt signaling.

Triple-negative breast cancer (TNBC) has an aggressive biological behavior and poor outcome. Our published study showed that PAI-1 could induce the migration and metastasis of TNBC cells. However, the underlying mechanism by which PAI-1 regulates TNBC metastasis has not been addressed. Here, we demonstrated that PAI-1 is high expressed in TNBC and promotes TNBC cells tumorigenesis. Using microarray analysis of lncRNA expression profiles, we identified a lncRNA SOX2-OT, which is induced by PAI-1 and could function as an oncogenic lncRNA in TNBC. Mechanistic analysis demonstrated that SOX2-OT acts as a molecular sponge for miR-942-5p to regulate the expression of PIK3CA, ultimately leading to activating PI3K/Akt signaling pathway and promoting TNBC metastasis. Taken together, our findings suggest that SOX2-OT regulates PAI-1-induced TNBC cell metastasis through miR-942-5p/PIK3CA signaling and illustrate the great potential of developing new SOX2-OT-targeting therapy for TNBC patients.