Therapy-induced senescent tumor cell-derived extracellular vesicles promote colorectal cancer progression through SERPINE1-mediated NF-κB p65 nuclear translocation.

BACKGROUND: Cellular senescence frequently occurs during anti-cancer treatment, and persistent senescent tumor cells (STCs) unfavorably promote tumor progression through paracrine secretion of the senescence-associated secretory phenotype (SASP). Extracellular vesicles (EVs) have recently emerged as a novel component of the SASP and primarily mediate the tumor-promoting effect of the SASP. Of note, the potential effect of EVs released from STCs on tumor progression remains largely unknown. METHODS: We collected tumor tissues from two cohorts of colorectal cancer (CRC) patients to examine the expression of p16, p21, and SERPINE1 before and after anti-cancer treatment. Cohort 1 included 22 patients with locally advanced rectal cancer (LARC) who received neoadjuvant therapy before surgical resection. Cohort 2 included 30 patients with metastatic CRC (mCRC) who received first-line irinotecan-contained treatment. CCK-8, transwell, wound-healing assay, and tumor xenograft experiments were carried out to determine the impacts of EVs released from STCs on CRC progression in vitro and in vivo. Quantitative proteomic analysis was applied to identify protein cargo inside EVs secreted from STCs. Immunoprecipitation and mass spectrometer identification were utilized to explore the binding partners of SERPINE1. The interaction of SERPINE1 with p65 was verified by co-immunoprecipitation, and their co-localization was confirmed by immunofluorescence. RESULTS: Chemotherapeutic agents and irradiation could potently induce senescence in CRC cells in vitro and in human CRC tissues. The more significant elevation of p16 and p21 expression in patients after anti-cancer treatment displayed shorter disease-free survival (DFS) for LARC or progression-free survival (PFS) for mCRC. We observed that compared to non-STCs, STCs released an increased number of EVs enriched in SERPINE1, which further promoted the progression of recipient cancer cells. Targeting SERPINE1 with a specific inhibitor, tiplaxtinin, markedly attenuated the tumor-promoting effect of STCs-derived EVs. Additionally, the patients with greater increment of SERPINE1 expression after anti-cancer treatment had shorter DFS for LARC or PFS for mCRC. Mechanistically, SERPINE1 bound to p65, promoting its nuclear translocation and subsequently activating the NF-κB signaling pathway. CONCLUSIONS: We provide the in vivo evidence of the clinical prognostic implications of therapy-induced senescence. Our results revealed that STCs were responsible for CRC progression by producing large amounts of EVs enriched in SERPINE1. These findings further confirm the crucial role of therapy-induced senescence in tumor progression and offer a potential therapeutic strategy for CRC treatment.

miR-30d-5p inhibits proliferation, invasion and migration of breast cancer cells by targeting SERPINE1 and promoting fatty acid ß-oxidation.

Breast cancer (BC) is among the top three most prevalent cancers across the world, especially in women, and its pathogenesis is still unknown. Fatty acid ß-oxidation is highly associated with breast cancer. Serpin family E member 1 (SERPINE1)-induced down-regulation of fatty acid ß-oxidation can facilitate BC cell proliferation, invasion, and metastasis. In this paper, the difference of miR-30d-5p expressions in both cancerous tissues and para-carcinoma tissues was first detected. Next, the expressions of SERPINE1, long-chain acyl-CoA dehydrogenase (LCAD) and medium-chain acyl-CoA dehydrogenase (MCAD) in the aforementioned tissues were analyzed. Finally, miR-30d-5p mimics were supplemented to breast cancer cells to observe the miR-30d-5p effect upon breast cancer cells. Via immunofluorescence assay and Western blotting, it was found that cancerous tissues had lower expressions of miR-30d-5p, MCAD and LCAD and a higher expression of SERPINE1 than para-carcinoma tissues. The miR-30d-5p mimic group had a decreased SERPINE1 expression and increased MCAD and LCAD expressions compared with the NC group, thus inhibiting BC cell proliferation, invasion, and metastasis. To sum up, miR-30d-5p blocks the cell proliferation, invasion and metastasis by targeting SERPINE1 and promoting fatty acid ß-oxidation. Preclinical studies are further required to establish a fatty acid ß-oxidation-targeting therapy for breast cancer.

Exosomal miR-361-3p promotes the viability of breast cancer cells by targeting ETV7 and BATF2 to upregulate the PAI-1/ERK pathway.

BACKGROUND: Malignant progression is the major cause of poor prognosis in breast cancer (BC) patients. Plasma exosomal miRNAs have been reported to be involved in tumor progression, but their roles in BC remain unclear. METHODS: We performed plasma exosomal miRNA sequencing on 45 individuals, including healthy controls and nonmetastatic and metastatic BC patients. We examined the correlation between miRNA expression in tumor tissues and plasma exosomes in BC patients by qRT‒PCR. The effects of exosomal miR-361-3p on BC cells were determined by CellTiter-Glo, migration and wound healing assays. The target genes of miR-361-3p and downstream pathways were explored by dual-luciferase reporter assay, RNA knockdown, rescue experiments, and western blotting. We utilized murine xenograft model to further assess the impact of plasma exosomal miR-361-3p on the malignant progression of BC. RESULTS: We found that the expression level of plasma exosomal miR-361-3p gradually increased with malignant progression in BC patients, and the expression of miR-361-3p in plasma exosomes and BC tissues was positively correlated. Consistently, exosomal miR-361-3p enhanced the migration and proliferation of two BC cell lines, MDA-MB-231 and SK-BR-3. Furthermore, our data showed that miR-361-3p inhibited two novel target genes, ETV7 and BATF2, to activate the PAI-1/ERK pathway, leading to increased BC cell viability. Finally, the consistency of the in vivo experimental results supported that elevated plasma exosomal miR-361-3p promote the malignant progression of BC. CONCLUSIONS: We found for the first time that plasma exosomal miR-361-3p was associated with malignant progression in BC patients. Mechanistically, exosomal miR-361-3p can enhance the migration and proliferation of BC cells by targeting the ETV7 and BATF2/PAI-1/ERK pathways. Our data suggest that plasma exosomal miR-361-3p has the potential to serve as a biomarker for predicting malignant progression in BC patients.

Endothelial H2S-AMPK dysfunction upregulates the angiocrine factor PAI-1 and contributes to lung fibrosis.

Dysfunction of the vascular angiocrine system is critically involved in regenerative defects and fibrosis of injured organs. Previous studies have identified various angiocrine factors and found that risk factors such as aging and metabolic disorders can disturb the vascular angiocrine system in fibrotic organs. One existing key gap is what sense the fibrotic risk to modulate the vascular angiocrine system in organ fibrosis. Here, using human and mouse data, we discovered that the metabolic pathway hydrogen sulfide (H2S)-AMP-activated protein kinase (AMPK) is a sensor of fibrotic stress and serves as a key mechanism upregulating the angiocrine factor plasminogen activator inhibitor-1 (PAI-1) in endothelial cells to participate in lung fibrosis. Activation of the metabolic sensor AMPK was inhibited in endothelial cells of fibrotic lungs, and AMPK inactivation was correlated with enriched fibrotic signature and reduced lung functions in humans. The inactivation of endothelial AMPK accelerated lung fibrosis in mice, while the activation of endothelial AMPK with metformin alleviated lung fibrosis. In fibrotic lungs, endothelial AMPK inactivation led to YAP activation and overexpression of the angiocrine factor PAI-1, which was positively correlated with the fibrotic signature in human fibrotic lungs and inhibition of PAI-1 with Tiplaxtinin mitigated lung fibrosis. Further study identified that the deficiency of the antioxidative gas metabolite H2S accounted for the inactivation of AMPK and activation of YAP-PAI-1 signaling in endothelial cells of fibrotic lungs. H2S deficiency was involved in human lung fibrosis and H2S supplement reversed mouse lung fibrosis in an endothelial AMPK-dependent manner. These findings provide new insight into the mechanism underlying the deregulation of the vascular angiocrine system in fibrotic organs.

Relationship between Platelet Index and Lipid Metabolism, Coagulation Function, and Inflammation in Diabetic Patients.

BACKGROUND: Platelets are overactive in type 2 diabetes mellitus (T2DM). This study analyzed the relationship between platelet index and lipid metabolism, coagulation function, and inflammation in T2DM patients. METHODS: This study enrolled 60 T2DM patients and 60 healthy subjects (age- and gender-matched). Mean platelet volume (MPV) and platelet distribution width (PDW) were evaluated, and their associations with lipid metabolism (TG and HDL-C), coagulation function (vWF and PAI-1), and inflammation (IL-6 and TNF-α) were analyzed. RESULTS: T2DM patients had increased MPV and PDW. Furthermore, the platelet index was correlated with the levels of TG, HDL-C, vWF, PAI-1, IL-6, and TNF-α. CONCLUSIONS: MPV and PDW are increased in T2DM patients. Moreover, platelet index was associated with lipid metabolism disorder, coagulation dysfunction, and inflammatory response in T2DM patients.

Nrf2 knockout attenuates the astragaloside IV therapeutic effect on kidney fibrosis from liver cancer by regulating pSmad3C/3L pathways.

Fibrotic kidney injury from hepatocarcinogenesis seriously impacts treatment effect. Astragaloside IV (AS-IV), an extract of Astragalus membranaceus, has several pharmacological activities, which are useful in the treatment of edema and fibrosis. Nrf2/HO-1 is a key antioxidant stress pathway and help treatment of kidney injury. Smad3 phosphorylation is implicated in hepatocarcinogenesis. Our previous study clarified that Smad3 is differentially regulated by different phosphorylated forms of Smad3 on hepatocarcinogenesis. Therefore, we investigated the contribution of AS-IV on the therapy of kidney fibrosis from hepatocarcinogenesis. And the focus was on whether the phosphorylation of Smad3 and the regulation of Nrf2/HO-1 pathway were involved during AS-IV therapy and whether there is an effect of Nrf2 knockout on the phosphorylation of Smad3. We performed TGF-ß1 stimulation on HK-2 cells and intervened with AS-IV. Furtherly, we investigated renal injury of AS-IV on Nrf2 knockout mice during hepatocarcinogenesis and its mechanism of action. On the one hand, in vitro results showed that AS-IV reduced the ROS and α-SMA expression of HK-2 by promoting the expression pSmad3C/p21 of and Nrf2/HO-1 and suppressed the expression of pSmad3L/PAI-1. On the other hand, the in vivo results of histopathological features, serological biomarkers, and oxidative damage indicators showed that Nrf2 knockout aggravated renal injury. Besides, Nrf2 deletion decreased the nephroprotective effect of AS-IV by suppressing the pSmad3C/p21 pathway and promoting the pSmad3L/PAI-1 pathway. The experimental results were as we suspected. And we identify for the first time that Nrf2 deficiency increases renal fibrosis from hepatocarcinogenesis and attenuates the therapeutic effects of AS-IV via regulating pSmad3C/3L signal pathway.

Plasminogen Activator Inhibitor 1 Is a Novel Faecal Biomarker for Monitoring Disease Activity and Therapeutic Response in Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: Crohn's disease [CD] and ulcerative colitis [UC] require lifelong treatment and patient monitoring. Current biomarkers have several limitations; therefore, there is an unmet need to identify novel biomarkers in inflammatory bowel disease [IBD]. Previously, the role of plasminogen activator inhibitor 1 [PAI-1] was established in the pathogenesis of IBD and suggested as a potential biomarker. Therefore, we aimed to comprehensively analyse the selectivity of PAI-1 in IBD, its correlation with disease activity, and its potential to predict therapeutic response. METHODS: Blood, colon biopsy, organoid cultures [OC], and faecal samples were used from active and inactive IBD patients and control subjects. Serpin E1 gene expressions and PAI-1 protein levels and localisation in serum, biopsy, and faecal samples were evaluated by qRT-PCR, ELISA, and immunostaining, respectively. RESULTS: The study population comprised 132 IBD patients [56 CD and 76 UC] and 40 non-IBD patients. We demonstrated that the serum, mucosal, and faecal PAI-1 concentrations are elevated in IBD patients, showing clinical and endoscopic activity. In responders [decrease of eMayo ≥3 in UC; or SES-CD  50% in CD], the initial PAI-1 level decreased significantly upon successful therapy. OCs derived from active IBD patients produced higher concentrations of PAI-1 than the controls, suggesting that epithelial cells could be a source of PAI-1. Moreover, faecal PAI-1 selectively increases in active IBD but not in other organic gastrointestinal diseases. CONCLUSIONS: The serum, mucosal, and faecal PAI-1 concentration correlates with disease activity and therapeutic response in IBD, suggesting that PAI-1 could be used as a novel, non-invasive, disease-specific, faecal biomarker in patient follow-up.

Endothelial extracellular vesicles induce acute lung injury via follistatin-like protein 1.

Cardiopulmonary bypass has been speculated to elicit systemic inflammation to initiate acute lung injury (ALI), including acute respiratory distress syndrome (ARDS), in patients after cardiac surgery. We previously found that post-operative patients showed an increase in endothelial cell-derived extracellular vesicles (eEVs) with components of coagulation and acute inflammatory responses. However, the mechanism underlying the onset of ALI owing to the release of eEVs after cardiopulmonary bypass, remains unclear. Plasma plasminogen-activated inhibitor-1 (PAI-1) and eEV levels were measured in patients with cardiopulmonary bypass. Endothelial cells and mice (C57BL/6, Toll-like receptor 4 knockout (TLR4-/-) and inducible nitric oxide synthase knockout (iNOS-/-)) were challenged with eEVs isolated from PAI-1-stimulated endothelial cells. Plasma PAI-1 and eEVs were remarkably enhanced after cardiopulmonary bypass. Plasma PAI-1 elevation was positively correlated with the increase in eEVs. The increase in plasma PAI-1 and eEV levels was associated with post-operative ARDS. The eEVs derived from PAI-1-stimulated endothelial cells could recognize TLR4 to stimulate a downstream signaling cascade identified as the Janus kinase 2/3 (JAK2/3)-signal transducer and activator of transcription 3 (STAT3)-interferon regulatory factor 1 (IRF-1) pathway, along with iNOS induction, and cytokine/chemokine production in vascular endothelial cells and C57BL/6 mice, ultimately contributing to ALI. ALI could be attenuated by JAK2/3 or STAT3 inhibitors (AG490 or S3I-201, respectively), and was relieved in TLR4-/- and iNOS-/- mice. eEVs activate the TLR4/JAK3/STAT3/IRF-1 signaling pathway to induce ALI/ARDS by delivering follistatin-like protein 1 (FSTL1), and FSTL1 knockdown in eEVs alleviates eEV-induced ALI/ARDS. Our data thus demonstrate that cardiopulmonary bypass may increase plasma PAI-1 levels to induce FSTL1-enriched eEVs, which target the TLR4-mediated JAK2/3/STAT3/IRF-1 signaling cascade and form a positive feedback loop, leading to ALI/ARDS after cardiac surgery. Our findings provide new insight into the molecular mechanisms and therapeutic targets for ALI/ARDS after cardiac surgery.

Transcriptomics reveal a molecular signature in the progression of nonalcoholic steatohepatitis and identifies PAI­1 and MMP­9 as biomarkers in in vivo and in vitro studies.

Nonalcoholic steatohepatitis (NASH) is emerging as the primary driver of liver disease­induced fibrosis. The imperative need for noninvasive biomarkers to ascertain disease progression stage is evident. The present study elucidated the biological roles of hub genes that could potentially serve as diagnostic markers for NASH. Using an in vivo approach, C57BL/6J mice were subjected to a high­fat and fructose diet (HFFD) for 6, 10, 14, 18 or 22 weeks. Serological biochemical indices were assessed and liver specimens were obtained to identify potential markers linked to the NASH process, employing a comprehensive strategy that combined transcriptomic and histopathological analyses. The HFFD regimen induced hyperlipidemia, obesity and insulin resistance, progressively culminating in NASH with fibrosis over time. The transcriptomic analyses indicated temporal patterns of pivotal gene sets intricately connected to NASH progression, which encompassed processes such as glucose homeostasis, inflammatory responses, reactive oxygen species­mediated damage, lipid metabolism disruptions and the formation of fibrotic tissue. Among these genes, Serpine1 and Mmp9 demonstrated promising diagnostic potential for NASH, with their intrahepatic mRNA expression levels serving as robust indicators. Moreover, the levels of PAI­1 (encoded by the Serpine1 gene) and MMP­9 in the serum of mice demonstrated a parallel increase with the duration of HFFD intervention. In vitro experiments utilizing HepG2 cells further validated these findings, demonstrating a significant elevation in the protein expression levels of both PAI­1 and MMP­9 upon exposure to free fatty acids, in agreement with the results of the animal study. Consequently, PAI­1 and MMP­9 are promising noninvasive biomarkers for assessing the progression of NASH.

SET8 is a novel negative regulator of TGF-ß signaling in a methylation-independent manner.

Transforming growth factor ß (TGF-ß) is a multifunctional cytokine that induces a diverse set of cellular processes principally through Smad-dependent transcription. Transcriptional responses induced by Smads are tightly regulated by Smad cofactors and histone modifications; however, the underlying mechanisms have not yet been elucidated in detail. We herein report lysine methyltransferase SET8 as a negative regulator of TGF-ß signaling. SET8 physically associates with Smad2/3 and negatively affects transcriptional activation by TGF-ß in a catalytic activity-independent manner. The depletion of SET8 results in an increase in TGF-ß-induced plasminogen activator inhibitor-1 (PAI-1) and p21 expression and enhances the antiproliferative effects of TGF-ß. Mechanistically, SET8 occupies the PAI-1 and p21 promoters, and a treatment with TGF-ß triggers the replacement of the suppressive binding of SET8 with p300 on these promoters, possibly to promote gene transcription. Collectively, the present results reveal a novel role for SET8 in the negative regulation of TGF-ß signaling.

Site-specific PEGylation of recombinant tissue-type plasminogen activator.

Tissue-type plasminogen activator (tPA) is the gold standard for emergency treatment of ischemic stroke, which is the third leading cause of death worldwide. Major challenges of tPA therapy are its rapid elimination by plasminogen activator inhibitor-1 (PAI-1) and hepatic clearance, leading to the use of high doses and consequent serious side effects, including internal bleeding, swelling and low blood pressure. In this regard, we developed three polyethylene glycol (PEG)ylated tPA bioconjugates based on the recombinant human tPA drug Alteplase using site-specific conjugation strategies. The first bioconjugate with PEGylation at the N-terminus of tPA performed by reductive alkylation showed a reduced proteolytic activity of 68 % compared to wild type tPA. PEGylation at the single-free cysteine of tPA with linear and branched PEG revealed similar proteolytic activities as the wild-type protein. Moreover, both bioconjugates with PEG-cysteine-modification showed 2-fold slower inhibition kinetics by PAI-1. All bioconjugates increased in hydrodynamic size as a critical requirement for half-life extension.

ADAMTS12 is a stromal modulator in chronic liver disease.

Adamalysins, a family of metalloproteinases containing a disintegrin and metalloproteinases (ADAMs) and ADAM with thrombospondin motifs (ADAMTSs), belong to the matrisome and play important roles in various biological and pathological processes, such as development, immunity and cancer. Using a liver cancer dataset from the International Cancer Genome Consortium, we developed an extensive in silico screening that identified a cluster of adamalysins co-expressed in livers from patients with hepatocellular carcinoma (HCC). Within this cluster, ADAMTS12 expression was highly associated with recurrence risk and poorly differentiated HCC signatures. We showed that ADAMTS12 was expressed in the stromal cells of the tumor and adjacent fibrotic tissues of HCC patients, and more specifically in activated stellate cells. Using a mouse model of carbon tetrachloride-induced liver injury, we showed that Adamts12 was strongly and transiently expressed after a 24 h acute treatment, and that fibrosis was exacerbated in Adamts12-null mice submitted to carbon tetrachloride-induced chronic liver injury. Using the HSC-derived LX-2 cell line, we showed that silencing of ADAMTS12 resulted in profound changes of the gene expression program. In particular, genes previously reported to be induced upon HSC activation, such as PAI-1, were mostly down-regulated following ADAMTS12 knock-down. The phenotype of these cells was changed to a less differentiated state, showing an altered actin network and decreased nuclear spreading. These phenotypic changes, together with the down-regulation of PAI-1, were offset by TGF-ß treatment. The present study thus identifies ADAMTS12 as a modulator of HSC differentiation, and a new player in chronic liver disease.

A dual role for ERK-1/2 in the regulation of plasmin activity and cell migration in metastatic NSCLC-H1299 cells.

Occupational and environmental exposure of various toxins or cigarette smoke causes non-small cell lung carcinoma (NSCLC); a devastating disease with a very low survival rate after metastasis. Increased activity of plasmin is a hallmark in NSCLC metastasis. It is accepted that metastatic cells exhibit higher plasmin activity than cells from primary tumors. Mechanisms behind this elevation, however, are barely understood. We compared plasmin activity and cell migration of A549 cells derived from a primary lung tumor with metastatic H1299 lung cells isolated from lymph nodes. Surprisingly, we found higher plasmin activity and migration for A549 cells. mRNA levels of the plasminogen activator inhibitor-1 (PAI-1) were higher in H1299 cells and activity of extracellular-regulated kinases-1/2 (ERK-1/2) was increased. An inhibitor of ERK-1/2 decreased PAI-1 mRNA levels and increased plasmin activity or cell migration in H1299 cells. Transforming growth factor-ß (TGF-ß) decreased plasmin activity and migration in A549 cells but enhanced both in H1299 cells. The cytokine massively increased PAI-1 and decreased urokinase plasminogen activator (uPA) levels in A549 cells but strongly induced uPA and only weakly PAI- 1 expression in H1299 cells. Consequently, TGF-ß enhanced plasmin activity and cell migration in H1299. Additionally, TGF-ß activated ERK-1/2 stronger in H1299 than in A549 cells. Accordingly, an ERK-1/2 inhibitor completely reversed the effects of TGF-ß on uPA expression, plasmin activity and migration in H1299 cells. Hence, we provide first data indicating TGF-ß-promoted increased plasmin activity and suggest that blocking TGF-ß-promoted ERK-1/2 activity might be a straightforward approach to inhibit NSCLC metastasis.

Pharmacological inhibition of plasminogen activator inhibitor-1 prevents memory deficits and reduces neuropathology in APP/PS1 mice.

RATIONALE: Extracellular proteolytic activity plays an important role in memory formation and the preservation of cognitive function. Previous studies have shown increased levels of plasminogen activator inhibitor-1 (PAI-1) in the brain of mouse models of Alzheimer's disease (AD) and plasma of AD patients, associated with memory and cognitive decline; however, the exact function of PAI-1 in AD onset and progression is largely unclear. OBJECTIVE: In this study, we evaluated a novel PAI-1 inhibitor, TM5A15, on its ability to prevent or reverse memory deficits and decrease Aß levels and plaque deposition in APP/PS1 mice. METHODS: We administered TM5A15 mixed in a chow diet to 3-month and 9-month-old APP/PS1 mice before and after neuropathological changes were distinguishable. We then evaluated the effects of TM5A15 on memory function and neuropathology at 9 months and 18 months of age. RESULTS: In the younger mice, 6 months of TM5A15 treatment protected against recognition and short-term working memory impairment. TM5A15 also decreased oligomer levels and amyloid plaques, and increased mBDNF expression in APP/PS1 mice at 9 months of age. In aged mice, 9 months of TM5A15 treatment did not significantly improve memory function nor decrease amyloid plaques. However, TM5A15 treatment showed a trend in decreasing oligomer levels in APP/PS1 mice at 18 months of age. CONCLUSION: Our results suggest that PAI-1 inhibition could improve memory function and reduce the accumulation of amyloid levels in APP/PS1 mice. Such effects are more prominent when TM5A15 is administered before advanced AD pathology and memory deficits occur.

Intracellular tPA-PAI-1 interaction determines VLDL assembly in hepatocytes.

Apolipoprotein B (apoB)-lipoproteins initiate and promote atherosclerotic cardiovascular disease. Plasma tissue plasminogen activator (tPA) activity is negatively associated with atherogenic apoB-lipoprotein cholesterol levels in humans, but the mechanisms are unknown. We found that tPA, partially through the lysine-binding site on its Kringle 2 domain, binds to the N terminus of apoB, blocking the interaction between apoB and microsomal triglyceride transfer protein (MTP) in hepatocytes, thereby reducing very-low-density lipoprotein (VLDL) assembly and plasma apoB-lipoprotein cholesterol levels. Plasminogen activator inhibitor 1 (PAI-1) sequesters tPA away from apoB and increases VLDL assembly. Humans with PAI-1 deficiency have smaller VLDL particles and lower plasma levels of apoB-lipoprotein cholesterol. These results suggest a mechanism that fine-tunes VLDL assembly by intracellular interactions among tPA, PAI-1, and apoB in hepatocytes.

Caveolin-1-derived peptide attenuates cigarette smoke-induced airway and alveolar epithelial injury.

Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease with no effective treatment that can reduce mortality or slow the disease progression. COPD is the third leading cause of global death and is characterized by airflow limitations due to chronic bronchitis and alveolar damage/emphysema. Chronic cigarette smoke (CS) exposure damages airway and alveolar epithelium and remains a major risk factor for the pathogenesis of COPD. We found that the expression of caveolin-1, a tumor suppressor protein; p53; and plasminogen activator inhibitor-1 (PAI-1), one of the downstream targets of p53, was markedly increased in airway epithelial cells (AECs) as well as in type II alveolar epithelial (AT2) cells from the lungs of patients with COPD or wild-type mice with CS-induced lung injury (CS-LI). Moreover, p53- and PAI-1-deficient mice resisted CS-LI. Furthermore, treatment of AECs, AT2 cells, or lung tissue slices from patients with COPD or mice with CS-LI with a seven amino acid caveolin-1 scaffolding domain peptide (CSP7) reduced mucus hypersecretion in AECs and improved AT2 cell viability. Notably, induction of PAI-1 expression via increased caveolin-1 and p53 contributed to mucous cell metaplasia and mucus hypersecretion in AECs, and reduced AT2 viability, due to increased senescence and apoptosis, which was abrogated by CSP7. In addition, treatment of wild-type mice having CS-LI with CSP7 by intraperitoneal injection or nebulization via airways attenuated mucus hypersecretion, alveolar injury, and significantly improved lung function. This study validates the potential therapeutic role of CSP7 for treating CS-LI and COPD. NEW & NOTEWORTHY Chronic cigarette smoke (CS) exposure remains a major risk factor for the pathogenesis of COPD, a debilitating disease with no effective treatment. Increased caveolin-1 mediated induction of p53 and downstream plasminogen activator inhibitor-1 (PAI-1) expression contributes to CS-induced airway mucus hypersecretion and alveolar wall damage. This is reversed by caveolin-1 scaffolding domain peptide (CSP7) in preclinical models, suggesting the therapeutic potential of CSP7 for treating CS-induced lung injury (CS-LI) and COPD.

PAI1 inhibits the pathogenesis of primary focal hyperhidrosis by targeting CHRNA1.

BACKGROUND: Primary focal hyperhidrosis (PFH) may be attributed to the up-regulation of the cholinergic receptor nicotinic alpha 1 subunit (CHRNA1) in eccrine glands. Plasminogen activator inhibitor-1 (PAI1, encoded by SERPINE1) is reported to inhibit the expression of CHRNA1, while the role of PAI1 in hyperhidrosis is unknown. METHODS: Serpine1 KO mice, Serpine1-Tg mice, and wild type BALB/c mice were intraperitoneally injected with pilocarpine hydrochloride to induce PFH. Cisatracurium (CIS, antagonist of CHRNA1) or PAI-039 (small-molecule inhibitor of PAI1) was pre-administrated before the induction of hyperhidrosis. On the other hand, Chrna1-expressing AAV was constructed and administered to Serpine1-Tg mice with hydrochloride stimulation. Hydrochloride-related biomarkers, such as acetylcholine (ACH) in the serum, calcium voltage-gated channel subunit alpha1 C (CACNA1C), and aquaporin 5 (AQP5) in sweat glands of mice were assayed with ELISA, RT-PCR, and Western blot. RESULTS: The administration of PAI-039 or Pai1 knock-out increased Chrna1 expression, sweat secretion, and hydrochloride-related biomarkers (ACH, CACNA1C, and AQP5) expression. On the other hand, CIS administration diminished the strengthened hyperhidrosis phenotype induced by Pai1 knock-out with decreased sweat gland secretion. CONCLUSION: PAI1 inhibits CHRNA1-mediated hydrochloride-induced hyperhidrosis, with decreased sweat gland secretion and diminished ACH, AQP5, and CACNA1C expression. These results indicate the potential to utilize PAI1 to alleviate PFH.

PAI-1 Regulation of p53 Expression and Senescence in Type II Alveolar Epithelial Cells.

Cellular senescence contributes importantly to aging and aging-related diseases, including idiopathic pulmonary fibrosis (IPF). Alveolar epithelial type II (ATII) cells are progenitors of alveolar epithelium, and ATII cell senescence is evident in IPF. Previous studies from this lab have shown that increased expression of plasminogen activator inhibitor 1 (PAI-1), a serine protease inhibitor, promotes ATII cell senescence through inducing p53, a master cell cycle repressor, and activating p53-p21-pRb cell cycle repression pathway. In this study, we further show that PAI-1 binds to proteasome components and inhibits proteasome activity and p53 degradation in human lung epithelial A549 cells and primary mouse ATII cells. This is associated with a senescence phenotype of these cells, manifested as increased p53 and p21 expression, decreased phosphorylated retinoblastoma protein (pRb), and increased senescence-associated beta-galactose (SA-ß-gal) activity. Moreover, we find that, although overexpression of wild-type PAI-1 (wtPAI-1) or a secretion-deficient, mature form of PAI-1 (sdPAI-1) alone induces ATII cell senescence (increases SA-ß-gal activity), only wtPAI-1 induces p53, suggesting that the premature form of PAI-1 is required for the interaction with the proteasome. In summary, our data indicate that PAI-1 can bind to proteasome components and thus inhibit proteasome activity and p53 degradation in ATII cells. As p53 is a master cell cycle repressor and PAI-1 expression is increased in many senescent cells, the results from this study will have a significant impact not only on ATII cell senescence/lung fibrosis but also on the senescence of other types of cells in different diseases.

Association of pro-fibrinolytic receptor AnnexinA2 with tissue plasminogen activator/Inhibitor-1 in pre-eclampsia.

Background: The clinical manifestations of pre-eclampsia are related to placental anti-angiogenic factor alteration. These variations are mainly due to the alteration of plasminolytic components. The study aims to compare the expression of plasminolytic components in the placenta of women with and without pre-eclampsia. Material and Methods: The study included pregnant women with pre-eclampsia as PE group (n = 30) and without pre-eclampsia as a control group (n = 30). Placental bed biopsy tissues were collected. AnxA2, tPA, PAI-1 expression in the placental villous tissue was quantitatively evaluated using immunohistochemistry, western blot, and real time-PCR analysis. Results: The results of the study showed a significant decrease in the expression of ANXA2 and increased expression of tPA and PAI-1 in PE group compared to control group (p<0.005). AnxA2 expression showed positive correlation with tPA (r=+0.895, p=0.002) and negative correlation with PAI-1(r=-0.905, p=0.020) in control group whereas in the PE group AnxA2 expression was negatively correlated with tPA ((r=-0.801, p=0.016) and PAI-1 (R=-0.831, P=0.010). Conclusion: Decreased AnxA2 with increased expression of PAI-1 and tPA may be responsible for the altered fibrinolytic activity and play a significant role in pre-eclampsia pathogenesis.

Distinct inflammatory signatures of upper and lower body adipose tissue and adipocytes in women with normal weight or obesity.

Introduction: Upper and lower body fat accumulation poses an opposing obesity-related cardiometabolic disease risk. Depot-differences in subcutaneous adipose tissue (SAT) function may underlie these associations. We aimed to investigate the inflammatory signatures of abdominal (ABD) and femoral (FEM) SAT in postmenopausal women with normal weight or obesity. Methods: We included 23 postmenopausal women with normal weight (n = 13) or obesity (n = 10). In vivo secretion of adipokines from ABD and FEM SAT was measured using the arterio-venous balance technique. Adipokine gene expression and adipocyte morphology were examined in ABD and FEM SAT. Furthermore, adipokine expression and secretion were investigated in vitro using differentiated human primary ABD and FEM subcutaneous adipocytes derived from the study participants. Results: Plasma leptin and plasminogen activator inhibitor (PAI)-1 concentrations were higher, and ABD and FEM adipocytes were larger in women with obesity than normal weight. No differences in adipocyte size and blood flow were apparent between ABD and FEM SAT. We found significant release of leptin and monocyte chemoattractant protein (MCP)-1 from ABD and FEM SAT, with higher fractional release of MCP-1 from ABD than FEM SAT. Gene expression of leptin, PAI-1, and tumor necrosis factor-α was lower in ABD than FEM SAT and higher in women with obesity than normal weight. In ABD adipocytes, interleukin-6, PAI-1, and leptin gene expression were higher, while adiponectin and dipeptidyl-peptidase-4 gene expression were lower than in FEM adipocytes. Finally, ABD adipocytes secreted less MCP-1 compared to FEM adipocytes. Discussion: These findings demonstrate that upper and lower body SAT and adipocytes are characterized by distinct inflammatory signatures in postmenopausal women, which seem independent of adipocyte size.