Exploring the mechanism of Pim-1 upregulation of tissue factor to initiate hypercoagulable state in sepsis

doi:10.21203/rs.3.rs-4961936/v1

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Exploring the mechanism of Pim-1 upregulation of tissue factor to initiate hypercoagulable state in sepsis

https://doi.org/10.21203/rs.3.rs-4961936/v1

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Background

During sepsis-induced coagulopathy (SIC), the balance of coagulation, anticoagulation, and fibrinolysis is disrupted, and endothelial dysfunction plays a key role in the disease progression. Current studies have indicated that the Proviral integration site for Moloney murine leukemia virus 1 (Pim-1) can promote thrombosis and activate an autoimmune response. This study aimed to assess the relevance of inhibiting Pim-1 as a potential therapeutic target for SIC.

Methods

Wild-type, Pim-1-KO, and TLR4-KO mice were categorized into the sham and cecal ligation and puncture (CLP) groups. Human umbilical vein endothelial cells were classified into the control, lipopolysaccharide (LPS) stimulation, and intervention groups. Enzyme-linked immunosorbent assay was used to detect plasma coagulation index in mice. Western blotting and immunofluorescence were employed to examine protein expression in tissues or cells. Additionally, immunohistochemistry and hematoxylin and eosin staining were conducted to detect liver/lung tissue damage. Tissue factor (TF) promoter activity was detected using a dual-luciferase reporter assay. Moreover, the correlation between variables was determined using Pearson correlation analysis.

Results

Pim-1 inhibition can decrease the coagulation response of sepsis mice and improve the survival rate. Pim-1 administration activated LPS-induced endothelial injury via mTOR/Sp1/TF signaling pathways, and Pim-1 acts in endothelial cells via the TLR4 pathway.

Conclusions

These findings indicated that Pim-1 promotes TF upregulation, leading to the initiation of a hypercoagulable state in sepsis. Therefore, inhibiting Pim-1 activity may be a therapeutic approach for SIC.

Pim-1

tissue factor

sepsis-induced coagulopathy

hypercoagulation

Sepsis is a leading cause of mortality among critically ill patients (Williams et al. 2024). In 2017, Iba proposed sepsis-induced coagulopathy (SIC) for the first time and formulated SIC diagnostic criteria, which is the first scoring system designed specifically for sepsis coagulation disorders (Iba et al. 2017). The criteria include the international normalized ratio, platelet count, and sequential organ failure assessment (SOFA) score. Additionally, a total score of ≥ 4 points can be diagnosed as SIC (Iba et al. 2017). According to data published in the Lancet in 2020, approximately 48.9 million patients worldwide experience sepsis each year (Rudd et al. 2020). Among these, 11 million (22.5%) patients die of sepsis, accounting for 19.7% of global deaths (Rudd et al. 2020). In 2023, statistics from China showed that the incidence of sepsis in intensive care units (ICUs) was 28.4%, and the mortality rate was 30.0% (Weng et al. 2023). Between 50% and 70% of patients with sepsis experience coagulation dysfunction, characterized by systemic activation of the coagulation system, consumption of anticoagulant factor, fibrinolytic dysfunction, microcirculation failure, and eventual development of multiple organ dysfunction (Rudd et al. 2020). SIC is considered the early stage of disseminated intravascular coagulation, with a 28-day mortality rate of 34%, significantly higher than the 25% observed in patients without SIC (Iba et al. 2023). Therefore, exploring new targets for preventing and treating SIC is crucial for improving the prognosis and reducing the mortality rate of SIC.

In SIC, the balance of coagulation, anticoagulation, and fibrinolysis is disrupted, and endothelial dysfunction plays a key role in the disease progression (Reinhart et al. 2017). Endothelial cells line the inner surface of blood vessels and play an essential role in maintaining barrier permeability and regulating coagulation (Rohlenova et al. 2018). Tissue factor (TF) is a potent procoagulation factor in vivo and serves as a key initiator of the exogenous coagulation pathway (Tsantes et al. 2023). The extrinsic coagulation pathway is rapidly triggered by early sepsis, the blood coagulation effects of endothelial cells, the cells' TF mRNA in rapid synthesis, which peak within 3 h, and the activation of factor VII (FVIIa) (Franco et al. 2000). These factors combine to create complexes, rapid activation factors IX and X in 5 min (Franco et al. 2000). Thrombin exponentially increases after 4 min, and platelets are heavily consumed within 0–4 days (Brummel et al. 2002). Simultaneously, the anticoagulant mechanism of endothelial cells is activated, leading to the gradual increase in anticoagulant substances, such as TF pathway inhibitor, antithrombin Ⅲ, and activated protein C (Kinasewitz et al. 2004; Basavaraj et al. 2010). Sepsis can also activate the fibrinolytic system and increase plasminogen activator inhibitor-1 (PAI-1) level in the plasma of patients (Ito et al. 2019). Endothelial cells have been confirmed as the main source of PAI-1 in plasma (Chen et al. 2024). They exert physiological fibrinolysis by secreting tissue plasminogen activators and inhibitors of fibrinogen (FIB) activation, thereby limiting the hypercoagulable state to the damaged area of vascular endothelium and forming micro-thrombi (Chen et al. 2024).

Proviral integration site for Moloney murine leukemia virus 1 (Pim-1) is a member of the serine/threonine protein kinase Pim family, with its coding gene located on human chromosome 6p21, which is widely expressed in various tissues and organs of the whole body (Nawijn et al. 2011). Additionally, Pim-1 is highly expressed during the embryonic period of animals, and its expression level gradually decreases postnatally (Le et al. 2015). However, pathological injury can increase its expression level again. Previous studies on Pim-1 mainly focused on tumor and inflammation-related diseases (Weirauch et al. 2013; Bellon et al. 2016; Shah et al. 2008). Nawijn et al. and Zhao et al. both reported that Pim-1 can serve as a potential therapeutic target in diseases with high Pim-1 expression (Nawijn et al. 2011; Zhao et al. 2022). Current studies indicate that Pim-1 can promote coagulation reaction and thrombosis and activate an autoimmune response (Fu et al. 2019). In 2020, Unsworth et al. reported that inhibiting Pim-1 expression in platelets could reduce thrombosis without affecting normal hemostatic function (Unsworth et al. 2020). Additionally, in 2021, Cao et al. highlighted that the overexpression of Pim-1 in microvascular endothelial cells can reduce vascular endothelial cadherin expression (Cao et al. 2021). It also increases the expression of intercellular cell adhesion molecule-1, leading to endothelial cell injury (Cao et al. 2021).

Previous studies found that Pim-1 and protein kinase B (AKT) recognize similar substrates and regulate overlapping signaling pathways (Le et al. 2021). Pim-1 can phosphorylate the AKT Ser473 loci (Warfel et al. 2015). In combination with this AKT phosphorylation, they phosphorylated the the protein proline-rich Akt substrate of 40 kilodaltons (PRAS40, a protein that inhibit mTORC1 signaling) at the Thr246 site jointly, which ultimately causes mTOR activation and the activation of AKT-mTOR signaling pathways (Warfel et al. 2015). These results indicate that Pim-1 functions by activating the AKT-mTOR signaling pathway. Additionally, blocking the AKT-mTOR signaling pathways can inhibit the TF promoter activity, inhibit TF expression, reduce coagulant activity, and decrease fibrin deposition (Eisenreich et al. 2009; Hu et al. 2012; Cong et al. 2020; Lewis et al. 2019). These results suggest that Pim-1 promotes TF expression by activating the AKT-mTOR signaling pathway. However, whether the Pim-1 signaling pathways regulate the production of TF and subsequently participate in the pathological process of sepsis remains unclear. Three regulatory sites of transcription factor specificity protein 1 (Sp1) exist within the TF promoter sequence (Koizume et al. 2015). Bioinformatics analysis of transcription factors associated with the mTOR signaling pathway revealed that Sp1 appears with the highest frequency and is closely associated with mTOR-mediated protein synthesis (Cong et al. 2020). However, whether Pim-l initiates TF expression through the mTOR/Sp1 pathway is an imperfect mechanism that needs to be verified.

Pim-1 activates the body’s autoimmune response, and its inhibition can significantly enhance the rabbit epidemic inhibitory activity of cells (Fu et al. 2019). Based on this, we hypothesized that Pim-1 kinase is involved in immune activation in SIC. mTOR is also involved in the immune regulation of the body and can recognize and bind toll-like receptors (TLRs) on the surface of antigen-presenting cells when the host is infected by pathogenic microorganisms such as bacteria (Powell et al. 2012; Panwar et al. 2023). Anomalies in TLR signaling pathways can lead to the occurrence of sepsis and autoimmune diseases (Wang et al. 2020). In sepsis, toll-like receptor 4 (TLR4) can identify bacterial cell wall components such as lipopolysaccharides (LPSs), activate similar or different downstream signaling pathways, and simultaneously use myeloid differentiation primary response protein 88 (MyD88) and toll/interleukin-1R domain-containing adaptor-inducing interferon-β (TRIF) signals, which are involved in inflammatory response associated with the induction of mononuclear cells, regulation of the body’s natural immune, and adaptive immune response (Ye et al. 2021). After TLR4 is successfully activated, mTOR within the TLR4/MyD88 or TLR4/TRIF pathway on associated inflammatory cytokine expression prior to regulation can cause endothelial cells to express TF (Rink et al. 2014). Pim-1 is also involved in the body’s immune response (Asati et al. 2019) and initiates the coagulation response in sepsis (Zheng et al. 2016). However, whether the activation of the TLR4 signaling pathway regulates Pim-1 expression or causes TF expression and release is yet to be verified.

Therefore, we hypothesized that Pim-1 activates the mTOR signaling pathway and induces TF expression in SIC via phosphorylated Sp1 (p-Sp1), which initiates the coagulation cascade and leads to extensive microthrombosis and other coagulation system imbalance. Furthermore, the TLR4 signaling pathway activates TF, causing Pim-1 to initiate the coagulation cascade and autoimmune reactions, resulting in a high coagulation state and immune disorders. This study aimed to explore the mechanism of Pim-1 upregulation of tissue factor to initiate hypercoagulable state in sepsis using (include the model used).

Reagents and antibodies

LPS (Escherichia coli O111:B4) were purchased from Sigma Aldrich Company. Evans Blue (0109A14) was bought from Leagene, China. Wright-Giemsa (G1020), 4% paraformaldehyde (P1110), 5% BSA (SW3015) and Triton X-100 (T8200) were procured from Solarbio Life Sciences (Beijing, China). Anti-β-actin (4970), anti-GAPDH (5174), anti-mTOR (2983), anti-p-mTOR (5536) and anti-Sp1 (9389) were acquired from Cell Signaling Technology, USA. Anti-Pim-1 (PA5-15123) and anti-p-Sp1 (PA5-104770) were acquired from Invitrogen, USA. Anti-TF (P13726) for WB and IF/ICC from Affinity, USA. Anti-TF (ZRB1811) for IHC from Sigma-Aldrich, Germany. Anti-mouse HRP-conjugated polyclonal antibody (170–6516) and anti-rabbit HRP-conjugated polyclonal antibody (170–6515) were garnered from Bio-Rad, USA. Phenylmethanesulfonyl fuoride (PMSF) were acquired from Beyotime, China. BCA kit and Radio-Immunoprecipitation Assay (RIPA) were received from Termo Scientifc, USA. Protein phosphatase inhibitor mixture (P1260) was gained from Applygen (Beijing, China). The following ELISA kits (human or mouse) were from Abcam, UK or Invitrogen, USA: Thrombin-Antithrombin Complex (TAT, ab108907 or ab137994), D-Dimer (D-D, ab315310 or EEL094), Fibrinogen (FIB, ab241383 or ab108843) and Thrombomodulin (TM, ab214029 or ab209880).

Animals

We contracted Cyagen Biosciences Inc. to construct a mouse model of specific endothelial cell Pim-1-KO. TLR4 mutant male C57BL/10ScNJ mice (TLR4 mut) were purchased from the Jackson Laboratory. Male C57BL/6 mice (6–8 weeks old, 20–25 g/body weight) were purchased from Shanghai SLAC Laboratory, Animal Limited Liability Company (Shanghai, China). All animals were raised in a local laboratory animal center free of specific pathogens, maintained at an ambient temperature of 23 ± 3°C, relative humidity of 55 ± 10%, and 12 h light/dark cycles according to the guidelines of the Agency’s Animal Care and Use Committee. Additionally, all animal care and experimental procedures were approved by the Institutional Animal Care and Use Committees of Wenzhou Medical University (WYYY-AEC-YS-2022-0169).

CLP and animal tests

Wild-type (WT) and knockout mice were randomly assigned to the sham operation and cecal ligation and puncture (CLP) groups. Mice were anesthetized, and the CLP model was prepared as described previously (Dejager et al. 2011). Say simply, an abdominal incision was made in mice, and the cecum was ligated at two points with a 21-gauge needle, followed by gentle expression of some contents. Next, the cecum was returned to the abdominal cavity, and the incision was carefully sutured. The CLP model was not established in the sham group. Pim-1 inhibitor (15 and 30 mg/kg SMI-4a) was injected in the intervention group after CLP. The untreated group was given equal amounts of phosphate buffered saline (PBS). All mice received 1 mL of warm saline solution immediately after injection. The mice were evaluated every 12 h over the next 4 days and in the last stage of euthanasia. Finally, the experimental specimens were collected 24 h after CLP.

Cell culture and experiments

Human umbilical vein endothelial cells (HUVECs, 8000, ScienCell, USA) were cultured in endothelial cell medium (ECM, 1001, ScienCell, USA), supplemented with 500 mL base medium, 25 mL fetal bovine serum, 5 mL endothelial growth factor, and 5 mL penicillin/streptomycin solution in a humidified incubator under 5% CO₂ at 37°C. The HUVECs were cultured with 1 µg/ml LPS with or without reagent for 1 h or 6 h. A reduced serum medium was used to transfect cells with overexpressed plasmids, and Lipofectamine 3000 was used for all transfection reagents (L3000001, Invitrogen, USA).

ELISA test

Plasma (human and mouse) was quantified using enzyme-linked immunosorbent assay kits for FIB, D-D, TAT and TM. The experimental procedures were performed in accordance with the manufacturer’s instructions. Briefly, each reagent was left on the well plate for some time and washed several times. Color changes were assessed in the colored liquid rather than the reaction liquid. The results were examined using a microplate reader (Molecular Devices, Hercules, CA, USA).

Histology

Four groups of mice were sacrificed, and the lung and liver were harvested. Formalin-fixed tissues were processed for 4 h, embedded in paraffin, and cut into 3 mm sections. Subsequently, the sections were stained with hematoxylin and eosin (H&E, Sigma). The staining procedures and subsequent analyses followed the protocols described in a previous study (Ryu et al. 2022). Briefly, tissues were fixed in 4% paraformaldehyde and embedded in paraffin. After sectioning and deparaffinization, tissues were stained, dehydrated in ascending alcohol grades, cleared in xylene, and mounted. The stained tissues were photographed with the Olympus BX61 Microscope (Japan).

Immunofuorescence

Fresh lung tissues were embedded in optimal cutting temperature compound and frozen. The tissue was cut into 5 µm sections, placed in an oven at 65°C for 1 h, and fixed at room temperature for 20 min with 4% paraformaldehyde and sodium citrate buffer for 6 min. Next, 0.2% Triton was added and incubated at room temperature for 20 min, followed by blocking with 10% goat serum for 1 h. The primary antibody was incubated overnight at 4°C and after 30 min at room temperature the next day. Secondary antibodies were added dropfold to the corresponding tissues and incubated in the dark at room temperature for 1 h. Subsequently, 4',6-diamidino-2-phenylindole containing an anti-fluorescence quench agent was added dropfold and incubated in the dark for 15 min. After sealing, the slices were dried and stored at 37°C. Confocal microscopy (Leica, Germany) was used for observation and image acquisition.

Western blotting

Proteins from liver, lung, or HUVEC cells were extracted using radioimmunoprecipitation assay buffer supplemented with 1% phenylmethylsulfonyl fluoride and 1% protein phosphatase inhibitor. Protein concentration was subsequently determined using a Bicinchoninic Acid kit. Proteins were subjected to electrophoresis, transmembrane, blocked, and incubated with the first antibody overnight at 4°C. Polyvinylidene difluoride membranes were incubated with homologous secondary antibodies. After incubation, the membrane was examined using an enhanced chemiluminescence reagent (Advansta, USA).

Luciferase report

The TF promoter was inserted into the luciferase reporter gene expression vector (pGL3-basic). Next, the reporter plasmid and phRL-TK (internal reference) were co-transfected into cells (with Lipofectamine 3000 as the transfection reagent), HUVEC was collected 24 h after transfection, and lysed buffer was added. The activity of Firefly luciferase was measured first, followed by the addition of Renilla luciferase substrate to determine the activity of Renilla luciferase. Finally, the ratio of Firefly luciferase/Renilla luciferase for each tube was calculated.

Clinical data

Convenience sampling was used to select 55 patients and 50 healthy check-ups from October 2022 to October 2023 in the intensive care unit (ICU) of the First Affiliated Hospital of Wenzhou Medical University into the exclusion standard patients with sepsis-induced coagulopathy (SIC). The inclusion criteria were as follows: (1) within the first 24 h of ICU admission; (2) Meet SIC diagnostic criteria; (3) aged ≤ 18 and ≤ 80 years; and (4) Availablity of complete clinical data. The exclusion criteria were as follows: (1) pregnancy or lactation; (2) a state of near-death; (3) Blood history; (4) A history of malignant tumor; (5) A history of chronic inflammatory disease; and (6) Refusal to participate in the research. The collection of ICU admission was conducted within 24 h of various laboratory tests, including assessments of platelets (PLT), plasma prothrombin time, activated partial thromboplastin time (APTT), FIB, D-D, TAT, TM, sequential organ failure assessment (SOFA) score, and SIC score. The clinical study was approved by the Ethics Committee of Wenzhou Medical University (YS2022-108).

Statistical analyses

Values are expressed as the mean ± standard deviation. Statistical analyses were performed using IBM SPSS Statistics for Windows, version 26.0 (IBM Corp., Armonk, N.Y., USA). The chart was plotted using GraphPad Prism 8.4.2 (GraphPad Software, USA). The two groups were compared using an unpaired t-test, while differences among three or more sets of samples were analyzed using a one-way analysis of variance. Each set of data was obtained after at least three independent experiments. Survival analysis was conducted using the Kaplan–Meier survival curve. Spearman’s correlation coefficients were used to evaluate associations between the main variables. Statistical significance was set at P < 0.05.

Pim-1 plays an important role in CLP-induced sepsis

CLP after 24 h, compared with the control group (Sham), lung tissue of mice and liver tissue in the Pim-1 + cells in average optical density (AOD) significantly elevated (Fig. 1a). Simultaneously, protein expression of Pim-1 was detected in the extracted lung/liver tissue, revealing increased Pim-1 protein expression in the lung/liver tissue of the CLP group (Fig. 1b). In vitro studies using 5 µg/mL of LPS stimulated HUVECs for 24 h, and the LPS Pim-1 set of expression was significantly higher than the control group (Fig. 1c). Pim-1 inhibitor (SMI-4a, at 15 and 30 mg/kg) was used for 72 h. The results showed that the survival rate of CLP mice increased with increasing SMI-4a concentration, and the difference was statistically significant (Fig. 1d). Additionally, the low-dose and high-dose SMI-4a improved the CLP mice clotting index (Fig. 1e). The TM, TAT, and D-D levels in the blood of CLP mice in the high-dose SMI-4a group were significantly lower than those in the low-dose SMI-4a group (Fig. 1e).

Pim-1 relies on the transcription factor Sp1 to regulate TF

Western blot results showed that the expressions of p-Sp1 and TF were significantly increased after LPS stimulation, followed inhibited by SMI-4a (Pim-1 inhibitor) at a concentration of 80 µM (Fig. 2a). The cells in the p-Sp1 protein expression significantly reduced gradually with increasing concentrations of SMI-4a (30, 60, 90, and 120 µM), with statistically significant differences (Fig. 2b). Protein expressions of p-Sp1 and TF were increased after transfection of Pim-1 overexpression plasmid into HUVECs followed by LPS stimulation (Fig. 2c). Furthermore, the luciferase reporter gene vectors of TF promoter [PGL3-Basic-TF (-12/-189) and PGL3-Basic-TF (-1073/-1598)], overexpression plasmid (PCDNA3.1-Sp1), and internal reference plasmid (thymidine kinase pr(Fig. 2d)omoter-Renilla luciferase reporter plasmid) were co-transfected into HUVECs. The results showed that the overexpression of Sp1 significantly increased the activity of the TF promoter region regardless of LPS stimulation, and TF promoter activity increased with higher levels of Sp1 overexpression. Conversely, this effect was validated in the experimental group where Mithramycin A (an Sp1 inhibitor) was added (Fig. 2d).

Pim-1 activates the mTOR/Sp1/TF axis to initiate SIC

After transfecting the HUVECs with Pim-1 overexpression plasmid, the expression of phosphorylated mTOR (p-mTOR) was significantly increased by LPS stimulation (Fig. 3a). Rapamycin (5 µM) inhibited mTOR, and p-Sp1 and TF expressions were significantly decreased after LPS stimulation (Fig. 3b). In contrast, p-Sp1 and TF expressions were significantly increased by LPS stimulation after the HUVECs were transfected with mTOR overexpression plasmid (Fig. 3c). More importantly, when mTOR cDNA was transfected into Pim-1-inhibited human pulmonary microvascular endothelial cells upon LPS stimulation, the increase in p-Sp1 and TF protein expressions due to mTOR was reversed (Fig. 3d). These results suggest that mTOR mediates TF expression through p-Sp1 when activated by Pim-1 in HUVECs.

Role of Pim-1 endothelial cell-specific knockout in mice with SIC

H&E staining showed that the Pim-1^−/−CLP group had reduced pathological manifestations of lung injury and disappearance of microthrombi in the central hepatic lobular vein compared with the WT CLP group (Fig. 4a). The results of fibrin immunohistochemistry showed that the Pim-1^−/−CLP group had reduced fibrin deposition in both the lungs and liver of septic mice (Fig. 4a). Additionally, plasma TM, TAT, and D-D levels were markedly decreased, while FIB levels were significantly increased in the WT CLP group compared with the Pim-1^−/−Sham group (Fig. 4b). Immunofluorescence results revealed that the protein fluorescence intensity of TF in the lung tissue of mice was significantly lower in the Pim-1^−/−CLP group than in the WT CLP group (Fig. 4c). Furthermore, western blot results showed that p-mTOR in the lung tissue of mice, p-Sp1, and TF protein content were significantly lower in the WT CLP group than in the Pim-1^−/−CLP group (Fig. 4d).

Effect of TLR4 on Pim-1 in SIC

Immunofluorescence results showed that the protein fluorescence intensities of Pim-1 and TF in the lung tissue of mice were significantly decreased in the TLR4^−/−CLP group compared with the WT CLP group (Fig. 5a). Western blot results revealed that the Pim-1 in the lung tissue of mice and TF protein content were significantly lower in the TLR4^−/−CLP group than in the WT CLP group (Fig. 5b). Resatorvid (TAK-242) is a selective inhibitor of TLR4 (Zandi et al. 2020). Western blot results showed that the TF expression was significantly decreased in the LPS + TAK-242 group compared with the LPS group (Fig. 5c). LPS is an activator of TLR4 (Nijland et al. 2014). Furthermore, Pim-1 and TF expressions gradually increased with increasing LPS concentration, and the difference was statistically significant (Fig. 5d).

Pim-1 may predict the occurrence of SIC

Pim-1 was not associated with the 28-day mortality (Fig. 6a) but positively correlated with SOFA and SIC scores (Fig. 6b). We analyzed the plasma concentration of Pim-1 and its association with clinical parameters related to coagulation in patients with sepsis. The serum levels of Pim-1 were positively correlated with APTT, FIB, D-D, TAT, and TM, and negatively correlated with PLT (Fig. 6c). Based on these clinical data, Pim-1 may be a biomarker for predicting the development of SIC.

We initially investigated the expression level of Pim-1 in the tissues and endothelial cells of WT septic mice and examined its effect on survival rate and coagulation parameters in these mice. We found a significant increase in Pim-1 level, and CLP mice exhibited a significant reduction in Pim-1-induced mortality, as well as an improvement in blood coagulation indexes (TM, TAT, FIB and D-D). Considering previous studies on the procoagulant ability of Pim-1, we hypothesized that its inhibition might improve the outcome of CLP-induced TF-initiated hypercoagulable state in sepsis. In line with our expectations, we found Pim-1 upstream (TLR4) and downstream (mTOR/Sp1/TF) molecules in the body induced by the high coagulation state of CLP or LPS in the mechanism.

In the knockout mice, we found that Pim-1 knockout had a protective effect on the lung/liver tissues of septic mice. Consistent with previous findings, Cao et al. also found significant upregulation of Pim-1 mRNA and protein expression levels in mouse lung tissues during acute lung injury induced by intraperitoneal LPS administration in mice (Cao et al. 2021). Previous studies have found that the Pim-1 specific inhibitor, SMI-4a, reduced neutrophil infiltration, decreased the severity of pulmonary edema, improved histopathological changes, and increased survival in LPS-induced acute lung injury in PMVECs (Cao et al. 2021). Pim-1 is primarily associated with tumors, and a study suggests that high level of Pim-1 serves as biomarkers and therapeutic targets for some solid tumor types (Shah et al. 2008). This study found that Pim-1 in the sepsis model also has research value, speculating that it participates in the sepsis clotting response signal transduction, which induces a high coagulation state in the body. Pim-1 has been identified to have Pim-1L (44 kDa) and Pim-1S (33 kDa) subtypes due to their different location (Linn et al. 2012); therefore, the functional differences between the long and short subtypes need to be further discussed, particularly regarding their respective roles in sepsis blood coagulation responses.

Our previous study found that inhibition of Pim-1 effectively improved coagulation parameters and down-regulated p-mTOR and TF expressions in LPS-induced macrophages with sepsis. However, whether Pim-1 plays a role in endothelial cells and how Pim-1 regulates TF expression through mTOR remains unclear. Studies have reported that Sp1 is a type of inhibitory factor involved in endotoxin-induced sepsis (Liu et al. 2022). Therefore, we hypothesized that the transcription factor Sp1 has serine/threonine kinase sites that can be phosphorylated by Pim-1. In the LPS-induced endothelial cell sepsis model, Pim-1 inhibition downregulated p-mTOR/Sp1 and TF expression levels, mTOR inhibition downregulated p-Sp1 and TF expression levels, and Pim-1 inhibition reversed p-Sp1 and TF expressions after mTOR overexpression. In the in vivo study, we also found that Pim-1 knockdown downregulated mTOR/Sp1 and TF protein levels in septic mice. Therefore, these results suggest that the Pim-1/mTOR/Sp1 signaling pathway causes TF elevation and coagulation dysfunction in HUVECs induced by LPS. Previous studies found that Pim-1 and activated AKT co-phosphorylate mTOR to activate mTOR, and Pim-1 may directly regulate the AKT-mTOR signaling pathway (Jiang et al. 2019). In sepsis coagulation studies, the AKT-mTOR signaling pathway has been shown to inhibit TF expression and secretion, thereby reducing fibrin deposition and microthrombosis (Fang et al. 2022). Additionally, Pim-1 and signal transducer and activator of transcription (STAT) have been shown to be closely related, and previous studies have highlighted that the Janus kinase-STAT is the most important pathway to initiate Pim-1 up-regulation (Szydłowski et al. 2017). STAT3 mediates inflammatory response in sepsis; however, only a few reports exist on the coagulation system imbalance in sepsis (Ye et al. 2021). In contrast, inflammation and coagulation promote and affect each other in the pathological process of sepsis. Therefore, the role of Pim-1 in sepsis coagulation reactions and its participation in signal transduction mechanisms are of research significance and should be further investigated in future studies.

The regulation of TF gene expression in human endothelial cells occurs at the transcriptional level (Ott et al. 2004). This study found that the transcription factor Sp1 reduces TF-specific promoter luciferase activity. The study of TF promoter by Parry et al. determined that the regulation of TF gene transcription in human endothelial cells is mainly controlled by cis-element, which includes a 56 base pair enhancer region, with two AP-1 sites and one nuclear factor kappa B binding site (Parry et al. 1995). The TF promoter sequence − 111 to + 121 controls the basal transcription of the TF gene, and transcription starts at -227 to + 123 sites under the induction of LPS (Mechtcheriakova et al. 2001). However, the far side of the TF gene promoter region has not been widely studied, Terry etc. Research shows that the 5' untranslated region far side of the TF gene also plays a role in regulating TF expression (Terry et al. 2004). Therefore, future studies on TF causing hypercoagulable state in sepsis should not only involve protein and RNA levels but also aim to predict and verify the regulatory mechanism of TF promoter level by transcription factors.

The innate immune system is the first line of defense against pathogen invasion, and sepsis coagulation disorder is also an autoimmune response in nature (Martinez-Orengo et al. 2022). TLRs in mammals, such as TLR4, play a pivotal role in the innate immune system. In our study using a TLR4 knockout mouse model of sepsis, we observed reduced expression of Pim-1 and TF. Similar results were observed in endothelial cells, suggesting that Pim-1 regulate TF expression through TLR4 as its upstream mediator. Previous studies have shown that after the activation of TLR4, mTOR regulates the expression of inflammatory cytokines in the TLR4/phosphoinositide 3-kinase/Akt pathway, leading to TF expression in endothelial cells (Watts et al. 2013). Additionally, Pim-1 may be involved in the innate immune response of the body. Studies have shown that Pim-1 can enhance the survival and memory of immune cells (CD8⁺ T cells) and promote the proliferation of B cells (Volberding et al. 2021). Ko et al. also indicated that TLR4 agonist (LPS) can induce the expression of Pim-1 in macrophages (Ko et al. 2022). Therefore, these results may provide insights into potential target therapies for controlling endotoxin-induced sepsis. Research has highlighted that sepsis may be systemic lupus erythematosus (an autoimmune disease associated with elevated TLR family) in patients with severe complications (Yang et al. 2020). Therefore, our results may contribute additional evidence to the clinical paradox; however, further studies on immune-related molecular and regulatory mechanisms involving Pim-1 are needed in the future.

Our study had some limitations. In the study of gene knockout mice, we did not intervene in the gene knockout mice, only grouping and detecting the expression of related genes (TF, among others). In the cell study, we only performed experiments on HUVECs. In fact, macrophages also play an important role in sepsis coagulation, which should be further explored in the future. In clinical research, our study participants were only from one hospital, and the sample may not completely represent all patients with SIC. In the future, large sample and multi-center research should be conducted. Second, cross-sectional studies cannot infer causal relationships between variables; therefore, future longitudinal studies should be designed to explore causal relationships between variables.

Pim-1 likely plays an important role in mice with CLP-induced SIC, as well as in the clotting cascade in HUVECs with LPS-induced SIC via the TLR4 and mTOR/Sp1/TF pathways, which indicates that inhibiting Pim-1 is a new promising treatment for the hypercoagulable state in sepsis.

APTT: Activated partial thromboplastin time; CLP: Cecal ligation and puncture; FIB: Fibrinogen; H&E: Hematoxylin and eosin; HUVECs: Human umbilical vein endothelial cells; ICU: Intensive care unit; LPS: Lipopolysaccharide; mTOR: Mammalian target of rapamycin; MYD88: Myeloid Differentiation Primary Response Protein MyD88; p-mTOR: Phosphorylated mTOR; p-Sp1: Phosphorylated Specificity protein 1; TAT: Thrombin-antithrombin; Pim-1: proviral integration site for Moloney murine leukemia virus 1; PMVECs: Pulmonary microvascular endothelial cells; SOFA: Sequential organ failure assessment; SIC: Sepsis-induced coagulopathy; STAT: Signal transducer and activator of transcription; Sp1: Specificity protein 1; TLR4: Toll-like receptor 4; TLR: Toll-like receptor; TRIF: Toll/interleukin-1R domain-containing adaptor-inducing Interferon-β; TF: Tissue factor; TM: Thrombomodulin

Ethics approval and consent to participate

The study was approved by the Professional Ethics Committee of Clinical Research, (The First Afliated Hospital of Wenzhou Medical University).

Consent for publication

Not applicable.

Availability of data and materials

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the National Natural Science Foundation of China (8227081631 and 8217082338).

Authors’ contributions

QW was responsible for experimental design, acquisition of data, interpretation of data and drafting the manuscript. YW was responsible for formal analysis, interpretation of data, Statistical analysis. YH was responsible for conception and design and formal analysis, supervision. RZ and CZ were responsible for conception and design, data curation, supervision, review and editing. JP was responsible for project administration, Critical revision of the manuscript for important intellectual content and review and editing. All authors read and approved the final manuscript.

Acknowledgments

Not applicable.

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Exploring the mechanism of Pim-1 upregulation of tissue factor to initiate hypercoagulable state in sepsis

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Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Background

Materials and methods

Reagents and antibodies

Animals

CLP and animal tests

Cell culture and experiments

ELISA test

Histology

Immunofuorescence

Western blotting

Luciferase report

Clinical data

Statistical analyses

Results

Pim-1 plays an important role in CLP-induced sepsis

Pim-1 relies on the transcription factor Sp1 to regulate TF

Pim-1 activates the mTOR/Sp1/TF axis to initiate SIC

Role of Pim-1 endothelial cell-specific knockout in mice with SIC

Effect of TLR4 on Pim-1 in SIC

Pim-1 may predict the occurrence of SIC

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1