LPA2 Alleviates Septic Acute Lung Injury via Protective Endothelial Barrier Function Through Activation of PLC-PKC-FAK.

Background:Increased endothelial permeability of pulmonary vessels is a primary pathological characteristic of septic acute lung injury (ALI). Previously, elevated lysophosphatidic acid (LPA) levels and LPA2 (an LPA receptor) expression have been found in the peripheral blood and lungs of septic mice, respectively. However, the specific role of LPA2 in septic ALI remains unclear.Methods:A lipopolysaccharide (LPS)-induced model of sepsis was established in wild-type (WT) and global LPA2 knockout (Lpar2-/-) mice. We examined mortality, lung injury, assessed endothelial permeability through Evans blue dye (EBD) assay in vivo, and transendothelial electrical resistance (TEER) of mouse lung microvascular endothelial cells (MLMECs) in vitro. Enzyme-linked immunosorbent assay (ELISA), histopathological, immunofluorescence, immunohistochemistry, and Western blot were employed to investigate the role of LPA2 in septic ALI.Results:Lpar2 deficiency increased vascular endothelial permeability, impaired lung injury, and increased mortality. Histological examination revealed aggravated inflammation, edema, hemorrhage and alveolar septal thickening in the lungs of septic Lpar2-/- mice. In vitro, loss of Lpar2 resulted in increased permeability of MLMECs. Pharmacological activation of LPA2 by the agonist DBIBB led to significantly reduced inflammation, edema and hemorrhage, as well as increased expression of the vascular endothelial tight junction (TJ) protein zonula occludens-1 (ZO-1) and claudin-5, as well as the adheren junction (AJ) protein VE-cadherin. Moreover, DBIBB treatment was found to alleviate mortality by protecting against vascular endothelial permeability. Mechanistically, we demonstrated that vascular endothelial permeability was alleviated through LPA-LPA2 signaling via the PLC-PKC-FAK pathway.Conclusion:These data provide a novel mechanism of endothelial barrier protection via PLC-PKC-FAK pathway and suggest that LPA2 may contribute to the therapeutic effects of septic ALI.

[Research update on the roles and potential clinical application value of lysophosphatidic acid signaling in cardiovascular diseases].

Neutrophil levels upon admission for the assessment of acute pulmonary embolism with intermediate- and high-risk: an indicator of thrombosis and inflammation.

BACKGROUND:Risk prediction rules are important to establish appropriate treatment and management strategy for patients with different risk classification of pulmonary embolism (PE). Neutrophils are considered to be related to PE as an essential marker of inflammation. However, few studies have reported the association between neutrophil levels and risk classification of acute PE (APE). The aim of this study was to investigate the role of neutrophil levels upon admission in the assessment of risk classification of APE.METHODS:A total of 299 consecutive APE patients and 90 patients without APE confirmed by computed tomographic pulmonary angiography were retrospectively screened. APE patients were stratified into two subgroups according to clinical guidelines: low- (n = 233) and intermediate- and high-risk (n = 60) APE.RESULTS:The neutrophil levels in intermediate- and high-risk APE patients were significantly higher compared to low-risk APE or non-APE patients (P < 0.001). In multivariable logistic regression analysis, neutrophil levels were significantly and independently associated with intermediate- and high-risk APE (odds ratio = 1.239, 95% confidence interval [CI] 1.055-1.455, P = 0.009). Neutrophil levels were positively correlated with the pulmonary embolism severity index score (r = 0.357, P < 0.001), high sensitive C-reactive protein, D-dimer and pulmonary artery obstruction index (PAOI), in the overall population of APE patients. Receiver-operating characteristic curve analysis revealed that neutrophils had a better diagnostic value for intermediate- and high-risk APE (area under the curve [AUC] = 0.760, 95% CI 0.695-0.826; P < 0.001) compared to PAOI (AUC = 0.719) and D-dimer (AUC = 0.645).CONCLUSIONS:High neutrophil levels upon admission were significantly and independently associated with intermediate- and high-risk APE, which could be regarded as an indicator of inflammation and thrombosis in APE simultaneously. The potent diagnostic role of neutrophil levels and their competitive advantage over PAOI and D-dimer for the assessment of APE risk classification are suggested.

LPA2 Contributes to Vascular Endothelium Homeostasis and Cardiac Remodeling After Myocardial Infarction.

RATIONALE:Myocardial infarction (MI) is one of the most dangerous adverse cardiovascular events. Our previous study found that lysophosphatidic acid (LPA) is increased in human peripheral blood after MI, and LPA has a protective effect on the survival and proliferation of various cell types. However, the role of LPA and its receptors in MI is less understood.OBJECTIVES:To study the unknown role of LPA and its receptors in heart during MI.METHODS AND RESULTS:In this study, we found that mice also had elevated LPA level in peripheral blood, as well as increased cardiac expression of its receptor LPA2 in the early stages after MI. With adult and neonate MI models in global Lpar2 knockout (Lpar2-KO) mice, we found Lpar2 deficiency increased vascular leak leading to disruption of its homeostasis, so as to impaired heart function and increased early mortality. Histological examination revealed larger scar size, increased fibrosis, and reduced vascular density in the heart of Lpar2-KO mice. Furthermore, Lpar2-KO also attenuated blood flow recovery after femoral artery ligation with decreased vascular density in gastrocnemius. Our study revealed that Lpar2 was mainly expressed and altered in cardiac endothelial cells during MI, and use of endothelial-specific Lpar2 knockout mice phenocopied the global knockout mice. Additionally, adenovirus-Lpar2 and pharmacologically activated LPA2 significantly improved heart function, reduced scar size, increased vascular formation, and alleviated early mortality by maintaining vascular homeostasis owing to protecting vessels from leakage. Mechanistic studies demonstrated that LPA-LPA2 signaling could promote endothelial cell proliferation through PI3K-Akt/PLC-Raf1-Erk pathway and enhanced endothelial cell tube formation via PKD1-CD36 signaling.CONCLUSIONS:Our results indicate that endothelial LPA-LPA2 signaling promotes angiogenesis and maintains vascular homeostasis, which is vital for restoring blood flow and repairing tissue function in ischemic injuries. Targeting LPA-LPA2 signal might have clinical therapeutic potential to protect the heart from ischemic injury.

Transgelin exacerbates pulmonary artery smooth muscle cell dysfunction in shunt-related pulmonary arterial hypertension.

AIMS:Orchestrating the transition from reversible medial hypertrophy to irreversible plexiform lesions is crucial for pulmonary arterial hypertension related to congenital heart disease (CHD-PAH). Transgelin is an actin-binding protein that modulates pulmonary arterial smooth muscle cell (PASMC) dysfunction. In this study, we aimed to probe the molecular mechanism and biological function of transgelin in the pathogenesis of CHD-PAH.METHODS AND RESULTS:Transgelin expression was detected in lung tissues from both CHD-PAH patients and monocrotaline (MCT)-plus aortocaval (AV)-induced PAH rats by immunohistochemistry. In vitro, the effects of transgelin on the proliferation, migration, and apoptosis of human PASMCs (HPASMCs) were evaluated by the cell count and EdU assays, transwell migration assay, and TUNEL assay, respectively. And the effect of transgelin on the expression of HPASMC phenotype markers was assessed by the immunoblotting assay. (i) Compared with the normal control group (n = 12), transgelin expression was significantly overexpressed in the pulmonary arterioles of the reversible (n = 15) and irreversible CHD-PAH group (n = 4) (reversible group vs. control group: 18.2 ± 5.1 vs. 13.6 ± 2.6%, P < 0.05; irreversible group vs. control group: 29.9 ± 4.7 vs. 13.6 ± 2.6%, P < 0.001; irreversible group vs. reversible group: 29.9 ± 4.7 vs. 18.2 ± 5.1, P < 0.001). This result was further confirmed in MCT-AV-induced PAH rats. Besides, the transgelin expression level was positively correlated with the pathological grading of pulmonary arteries in CHD-PAH patients (r = 0.48, P = 0.03, n = 19). (ii) Compared with the normal control group (n = 12), TGF-β1 expression was notably overexpressed in the pulmonary arterioles of the reversible (n = 15) and irreversible CHD-PAH group (n = 4) (reversible group vs. control group: 14.8 ± 4.4 vs. 6.0 ± 2.5%, P < 0.001; irreversible group vs. control group: 20.1 ± 4.4 vs. 6.0 ± 2.5%, P < 0.001; irreversible group vs. reversible group: 20.1 ± 4.4 vs. 14.8 ± 4.4, P < 0.01). The progression-dependent correlation between TGF-β1 and transgelin was demonstrated in CHD-PAH patients (r = 0.48, P = 0.04, n = 19) and MCT-AV-induced PAH rats, which was further confirmed at sub-cellular levels. (iii) Knockdown of transgelin diminished proliferation, migration, apoptosis resistance, and phenotypic transformation of HPASMCs through repressing the TGF-β1 signalling pathway. On the contrary, transgelin overexpression resulted in the opposite effects.CONCLUSIONS:These results indicate that transgelin may be an indicator of CHD-PAH development via boosting HPASMC dysfunction through positive regulation of the TGF-β1 signalling pathway, as well as a potential therapeutic target for the treatment of CHD-PAH.

Lysophosphatidic Acid Receptor 3 Suppress Neutrophil Extracellular Traps Production and Thrombosis During Sepsis.

Sepsis consists of life-threatening organ dysfunction resulting from a dysregulated response to infection. Recent studies have found that excessive neutrophil extracellular traps (NETs) contribute to the pathogenesis of sepsis, thereby increasing morbidity and mortality. Lysophosphatidic acid (LPA) is a small glycerophospholipid molecule that exerts multiple functions by binding to its receptors. Although LPA has been functionally identified to induce NETs, whether and how LPA receptors, especially lysophosphatidic acid receptor 3 (LPA3), play a role in the development of sepsis has never been explored. A comprehensive understanding of the impact of LPA3 on sepsis is essential for the development of medical therapy. After intraperitoneal injection of lipopolysaccharide (LPS), Lpar3 -/-mice showed a substantially higher mortality, more severe injury, and more fibrinogen content in the lungs than wild-type (WT) mice. The values of blood coagulation markers, plasma prothrombin time (PT) and fibrinogen (FIB), indicated that the Lpar3 -/- mice underwent a severe coagulation process, which resulted in increased thrombosis. The levels of NETs in Lpar3 -/- mice were higher than those in WT mice after LPS injection. The mortality rate and degree of lung damage in Lpar3 -/- mice with sepsis were significantly reduced after the destruction of NETs by DNaseI treatment. Furthermore, in vitro experiments with co-cultured monocytes and neutrophils demonstrated that monocytes from Lpar3 -/- mice promoted the formation of NETs, suggesting that LPA3 acting on monocytes inhibits the formation of NETs and plays a protective role in sepsis. Mechanistically, we found that the amount of CD14, an LPS co-receptor, expressed by monocytes in Lpar3 -/-mice was significantly elevated after LPS administration, and the MyD88-p65-NFκB signaling axis, downstream of toll-like receptor 4 signaling, in monocytes was overactivated. Finally, after an injection of the LPA3 agonist (2S)-1-oleoyl-2-methylglycero-3-phosphothionate (OMPT), the survival rate of mice with sepsis was improved, organ damage was reduced, and the production of NETs was decreased. This suggested the possible translational value and application prospects of (2S)-OMPT in the treatment of sepsis. Our study confirms an important protective role of LPA3 in curbing the development of sepsis by suppressing NETs production and thrombosis and provides new ideas for sepsis treatment strategies.

Clopidogrel versus ticagrelor in the treatment of Chinese patients undergoing percutaneous coronary intervention: effects on platelet function assessed by platelet function tests and mean platelet volume.

BACKGROUND:Knowledge on the pharmacodynamic effects of antiplatelet drugs including clopidogrel and ticagrelor on Asian patients is scarce. We aim to evaluate the effects of the two drugs on platelet reactivity in the treatment of Chinese patients who underwent percutaneous coronary intervention (PCI), using two platelet function tests (PFT). Meanwhile, the relationship between mean platelet volume (MPV), a routine index of platelet size, and high on-treatment platelet reactivity (HPR) is also investigated.METHODS:Patients receiving dual antiplatelet therapy (DAPT) were scheduled for the assessment of platelet reactivity at 2-3 days after PCI. Two PFTs, light transmission aggregometry (LTA) and vasodilator-stimulated phosphoprotein (VASP)-FCM assay, were applied in the evaluation of platelet reactivity. The MPV was measured simultaneously with EDTA plasma using a Sysmex XN 2000 automated hematology analyzer.RESULTS:The final study population included the aspirin + clopidogrel group (n = 46) and the aspirin + ticagrelor group (n = 66). In the aspirin + ticagrelor group, the maximal light transmittance (LT) changes in response to 5 μM ADP assessed by LTA was obviously lower than that in the aspirin + clopidogrel group (P <  0.001). The platelet reactivity index (PRI) level in the VASP test was also markedly lower in the group given aspirin and ticagrelor (P <  0.001). There was a significant difference in HPR between the two groups. MPV showed a potent ability to predict the presence of HPR at VASP assay (AUC = 0.788, 95% CI: 0.701-0.875, P <  0.001) in receiver-operating characteristic curve analysis.CONCLUSIONS:Compared with clopidogrel, ticagrelor has dramatically greater antiplatelet effect, with a superiority in suppressing platelet function and a lower HPR rate. In addition, there existed a significant independent association between MPV and high prevalence of HPR in the VASP assay.

LPA3-mediated lysophosphatidic acid signaling promotes postnatal heart regeneration in mice.

Background: Lysophosphatidic acid (LPA) is a small glycerophospholipid that acts as a potent extracellular signal in various biological processes and diseases. Our previous work demonstrated that the expression of the LPA receptors LPA1 and LPA3 is elevated in the early postnatal heart. However, the role of this stage-specific expression of LPA1 and LPA3 in the heart is unknown. Methods and Results: By using LPA3 and LPA1 knockout mice, and neonatal SD rats treated with Ki16425 (LPA1/LPA3 inhibitor), we found that the number of proliferating cardiomyocytes, detected by coimmunostaining pH3, Ki67 or BrdU with cardiac troponin T, was significantly decreased in the LPA3 knockout mice and the Ki16425-treated rats but not in the LPA1 knockout mice during the first week of postnatal life. Using a myocardial infarction (MI) model, we found that cardiac function and the number of proliferating cardiomyocytes were decreased in the neonatal LPA3 KO mice and increased in the AAV9-mediated cardiac-specific LPA3 overexpression mice. By using lineage tracing and AAV9-LPA3, we further found that LPA3 overexpression in adult mice enhances cardiac function and heart regeneration as assessed by pH3-, Ki67-, and Aurora B-positive cardiomyocytes and clonal cardiomyocytes after MI. Genome-wide transcriptional profiling and additional mechanistic studies showed that LPA induces cardiomyocyte proliferation through the PI3K/AKT, BMP-Smad1/5, Hippo/YAP and MAPK/ERK pathways in vitro, whereas only ERK was confirmed to be activated by LPA-LPA3 signaling in vivo. Conclusion: Our study reports that LPA3-mediated LPA signaling is a crucial factor for cardiomyocyte proliferation in the early postnatal heart. Cardiac-specific LPA3 overexpression improved cardiac function and promoted cardiac regeneration after myocardial injury induced by MI. This finding suggested that activation of LPA3 potentially through AAV-mediated gene therapy might be a therapeutic strategy to improve the outcome after MI.

Hydrogen Sulfide Promotes Cardiomyocyte Proliferation and Heart Regeneration via ROS Scavenging.

Neonatal mouse hearts can regenerate completely in 21 days after cardiac injury, providing an ideal model to exploring heart regenerative therapeutic targets. The oxidative damage by Reactive Oxygen Species (ROS) is one of the critical reasons for the cell cycle arrest of cardiomyocytes (CMs), which cause mouse hearts losing the capacity to regenerate in 7 days or shorter after birth. As an antioxidant, hydrogen sulfide (H2S) plays a protective role in a variety of diseases by scavenging ROS produced during the pathological processes. In this study, we found that blocking H2S synthesis by PAG (H2S synthase inhibitor) suspended heart regeneration and CM proliferation with ROS deposition increase after cardiac injury (myocardial infarction or apex resection) in 2-day-old mice. NaHS (a H2S donor) administration improved heart regeneration with CM proliferation and ROS elimination after myocardial infarction in 7-day-old mice. NaHS protected primary neonatal mouse CMs from H2O2-induced apoptosis and promoted CM proliferation via SOD2-dependent ROS scavenging. The oxidative DNA damage in CMs was reduced with the elimination of ROS by H2S. Our results demonstrated for the first time that H2S promotes heart regeneration and identified NaHS as a potent modulator for cardiac repair.

Lysophosphatidic acid promotes thrombus stability by inducing rapid formation of neutrophil extracellular traps: A new mechanism of thrombosis.

BACKGROUND:Lysophosphatidic acid (LPA), a bioactive phospholipid released by activated platelets, can induce platelet shape changes and aggregation, which may play an important role in thrombosis. In contrast, the interaction of LPA with neutrophils in thrombosis has not been studied. Recently, neutrophil extracellular traps (NETs) have been shown to bind plasma proteins and activate platelets, which promotes thrombosis.OBJECTIVES:To investigate whether LPA could activate neutrophils to release NETs, predisposing to thrombosis and promoting thrombus stability.METHODS:Levels of neutrophils, NETs, and LPA were detected in 56 participants. Immunofluorescence of NETs and autotaxin, the LPA-producing ectoenzyme, were performed. Induction of NETs and signaling pathways were explored in vitro.RESULTS:Patients with acute pulmonary embolism showed elevated levels of neutrophils, NETs (dsDNA, MPO-DNA, citrullinated histone H3, and nucleosomes), LPA18:1, and LPA20:4. NETs were present in human intrapulmonary thrombi and were surrounded by autotaxin. LPA18:1 induced rapid release of NETs from human neutrophils via a peptidylarginine deiminase 4-dependent pathway. LPA-induced NETs provided a scaffolding for plasma protein binding and generated a tissue plasminogen activator (tPA)-resistant blood clot. Addition of deoxyribonuclease I to tPA significantly accelerated the lysis of clots and human intrapulmonary thrombi. Furthermore, LPA-induced NETs could activate platelets to release LPA.CONCLUSION:This is the first study to implicate LPA in regulating the stability of thrombi by inducing rapid release of NETs in vitro and ex vivo, which could be a new mechanism of thrombosis. These findings provide new insight into the prevention and therapy of venous thromboembolic disease by targeting the LPA-NET signaling pathway.

LPA2 Alleviates Septic Acute Lung Injury via Protective Endothelial Barrier Function Through Activation of PLC-PKC-FAK.

Background:Increased endothelial permeability of pulmonary vessels is a primary pathological characteristic of septic acute lung injury (ALI). Previously, elevated lysophosphatidic acid (LPA) levels and LPA2 (an LPA receptor) expression have been found in the peripheral blood and lungs of septic mice, respectively. However, the specific role of LPA2 in septic ALI remains unclear.Methods:A lipopolysaccharide (LPS)-induced model of sepsis was established in wild-type (WT) and global LPA2 knockout (Lpar2-/-) mice. We examined mortality, lung injury, assessed endothelial permeability through Evans blue dye (EBD) assay in vivo, and transendothelial electrical resistance (TEER) of mouse lung microvascular endothelial cells (MLMECs) in vitro. Enzyme-linked immunosorbent assay (ELISA), histopathological, immunofluorescence, immunohistochemistry, and Western blot were employed to investigate the role of LPA2 in septic ALI.Results:Lpar2 deficiency increased vascular endothelial permeability, impaired lung injury, and increased mortality. Histological examination revealed aggravated inflammation, edema, hemorrhage and alveolar septal thickening in the lungs of septic Lpar2-/- mice. In vitro, loss of Lpar2 resulted in increased permeability of MLMECs. Pharmacological activation of LPA2 by the agonist DBIBB led to significantly reduced inflammation, edema and hemorrhage, as well as increased expression of the vascular endothelial tight junction (TJ) protein zonula occludens-1 (ZO-1) and claudin-5, as well as the adheren junction (AJ) protein VE-cadherin. Moreover, DBIBB treatment was found to alleviate mortality by protecting against vascular endothelial permeability. Mechanistically, we demonstrated that vascular endothelial permeability was alleviated through LPA-LPA2 signaling via the PLC-PKC-FAK pathway.Conclusion:These data provide a novel mechanism of endothelial barrier protection via PLC-PKC-FAK pathway and suggest that LPA2 may contribute to the therapeutic effects of septic ALI.

[Research update on the roles and potential clinical application value of lysophosphatidic acid signaling in cardiovascular diseases].

Neutrophil levels upon admission for the assessment of acute pulmonary embolism with intermediate- and high-risk: an indicator of thrombosis and inflammation.

BACKGROUND:Risk prediction rules are important to establish appropriate treatment and management strategy for patients with different risk classification of pulmonary embolism (PE). Neutrophils are considered to be related to PE as an essential marker of inflammation. However, few studies have reported the association between neutrophil levels and risk classification of acute PE (APE). The aim of this study was to investigate the role of neutrophil levels upon admission in the assessment of risk classification of APE.METHODS:A total of 299 consecutive APE patients and 90 patients without APE confirmed by computed tomographic pulmonary angiography were retrospectively screened. APE patients were stratified into two subgroups according to clinical guidelines: low- (n = 233) and intermediate- and high-risk (n = 60) APE.RESULTS:The neutrophil levels in intermediate- and high-risk APE patients were significantly higher compared to low-risk APE or non-APE patients (P < 0.001). In multivariable logistic regression analysis, neutrophil levels were significantly and independently associated with intermediate- and high-risk APE (odds ratio = 1.239, 95% confidence interval [CI] 1.055-1.455, P = 0.009). Neutrophil levels were positively correlated with the pulmonary embolism severity index score (r = 0.357, P < 0.001), high sensitive C-reactive protein, D-dimer and pulmonary artery obstruction index (PAOI), in the overall population of APE patients. Receiver-operating characteristic curve analysis revealed that neutrophils had a better diagnostic value for intermediate- and high-risk APE (area under the curve [AUC] = 0.760, 95% CI 0.695-0.826; P < 0.001) compared to PAOI (AUC = 0.719) and D-dimer (AUC = 0.645).CONCLUSIONS:High neutrophil levels upon admission were significantly and independently associated with intermediate- and high-risk APE, which could be regarded as an indicator of inflammation and thrombosis in APE simultaneously. The potent diagnostic role of neutrophil levels and their competitive advantage over PAOI and D-dimer for the assessment of APE risk classification are suggested.

LPA2 Contributes to Vascular Endothelium Homeostasis and Cardiac Remodeling After Myocardial Infarction.

RATIONALE:Myocardial infarction (MI) is one of the most dangerous adverse cardiovascular events. Our previous study found that lysophosphatidic acid (LPA) is increased in human peripheral blood after MI, and LPA has a protective effect on the survival and proliferation of various cell types. However, the role of LPA and its receptors in MI is less understood.OBJECTIVES:To study the unknown role of LPA and its receptors in heart during MI.METHODS AND RESULTS:In this study, we found that mice also had elevated LPA level in peripheral blood, as well as increased cardiac expression of its receptor LPA2 in the early stages after MI. With adult and neonate MI models in global Lpar2 knockout (Lpar2-KO) mice, we found Lpar2 deficiency increased vascular leak leading to disruption of its homeostasis, so as to impaired heart function and increased early mortality. Histological examination revealed larger scar size, increased fibrosis, and reduced vascular density in the heart of Lpar2-KO mice. Furthermore, Lpar2-KO also attenuated blood flow recovery after femoral artery ligation with decreased vascular density in gastrocnemius. Our study revealed that Lpar2 was mainly expressed and altered in cardiac endothelial cells during MI, and use of endothelial-specific Lpar2 knockout mice phenocopied the global knockout mice. Additionally, adenovirus-Lpar2 and pharmacologically activated LPA2 significantly improved heart function, reduced scar size, increased vascular formation, and alleviated early mortality by maintaining vascular homeostasis owing to protecting vessels from leakage. Mechanistic studies demonstrated that LPA-LPA2 signaling could promote endothelial cell proliferation through PI3K-Akt/PLC-Raf1-Erk pathway and enhanced endothelial cell tube formation via PKD1-CD36 signaling.CONCLUSIONS:Our results indicate that endothelial LPA-LPA2 signaling promotes angiogenesis and maintains vascular homeostasis, which is vital for restoring blood flow and repairing tissue function in ischemic injuries. Targeting LPA-LPA2 signal might have clinical therapeutic potential to protect the heart from ischemic injury.

Transgelin exacerbates pulmonary artery smooth muscle cell dysfunction in shunt-related pulmonary arterial hypertension.

AIMS:Orchestrating the transition from reversible medial hypertrophy to irreversible plexiform lesions is crucial for pulmonary arterial hypertension related to congenital heart disease (CHD-PAH). Transgelin is an actin-binding protein that modulates pulmonary arterial smooth muscle cell (PASMC) dysfunction. In this study, we aimed to probe the molecular mechanism and biological function of transgelin in the pathogenesis of CHD-PAH.METHODS AND RESULTS:Transgelin expression was detected in lung tissues from both CHD-PAH patients and monocrotaline (MCT)-plus aortocaval (AV)-induced PAH rats by immunohistochemistry. In vitro, the effects of transgelin on the proliferation, migration, and apoptosis of human PASMCs (HPASMCs) were evaluated by the cell count and EdU assays, transwell migration assay, and TUNEL assay, respectively. And the effect of transgelin on the expression of HPASMC phenotype markers was assessed by the immunoblotting assay. (i) Compared with the normal control group (n = 12), transgelin expression was significantly overexpressed in the pulmonary arterioles of the reversible (n = 15) and irreversible CHD-PAH group (n = 4) (reversible group vs. control group: 18.2 ± 5.1 vs. 13.6 ± 2.6%, P < 0.05; irreversible group vs. control group: 29.9 ± 4.7 vs. 13.6 ± 2.6%, P < 0.001; irreversible group vs. reversible group: 29.9 ± 4.7 vs. 18.2 ± 5.1, P < 0.001). This result was further confirmed in MCT-AV-induced PAH rats. Besides, the transgelin expression level was positively correlated with the pathological grading of pulmonary arteries in CHD-PAH patients (r = 0.48, P = 0.03, n = 19). (ii) Compared with the normal control group (n = 12), TGF-β1 expression was notably overexpressed in the pulmonary arterioles of the reversible (n = 15) and irreversible CHD-PAH group (n = 4) (reversible group vs. control group: 14.8 ± 4.4 vs. 6.0 ± 2.5%, P < 0.001; irreversible group vs. control group: 20.1 ± 4.4 vs. 6.0 ± 2.5%, P < 0.001; irreversible group vs. reversible group: 20.1 ± 4.4 vs. 14.8 ± 4.4, P < 0.01). The progression-dependent correlation between TGF-β1 and transgelin was demonstrated in CHD-PAH patients (r = 0.48, P = 0.04, n = 19) and MCT-AV-induced PAH rats, which was further confirmed at sub-cellular levels. (iii) Knockdown of transgelin diminished proliferation, migration, apoptosis resistance, and phenotypic transformation of HPASMCs through repressing the TGF-β1 signalling pathway. On the contrary, transgelin overexpression resulted in the opposite effects.CONCLUSIONS:These results indicate that transgelin may be an indicator of CHD-PAH development via boosting HPASMC dysfunction through positive regulation of the TGF-β1 signalling pathway, as well as a potential therapeutic target for the treatment of CHD-PAH.

Lysophosphatidic Acid Receptor 3 Suppress Neutrophil Extracellular Traps Production and Thrombosis During Sepsis.

Sepsis consists of life-threatening organ dysfunction resulting from a dysregulated response to infection. Recent studies have found that excessive neutrophil extracellular traps (NETs) contribute to the pathogenesis of sepsis, thereby increasing morbidity and mortality. Lysophosphatidic acid (LPA) is a small glycerophospholipid molecule that exerts multiple functions by binding to its receptors. Although LPA has been functionally identified to induce NETs, whether and how LPA receptors, especially lysophosphatidic acid receptor 3 (LPA3), play a role in the development of sepsis has never been explored. A comprehensive understanding of the impact of LPA3 on sepsis is essential for the development of medical therapy. After intraperitoneal injection of lipopolysaccharide (LPS), Lpar3 -/-mice showed a substantially higher mortality, more severe injury, and more fibrinogen content in the lungs than wild-type (WT) mice. The values of blood coagulation markers, plasma prothrombin time (PT) and fibrinogen (FIB), indicated that the Lpar3 -/- mice underwent a severe coagulation process, which resulted in increased thrombosis. The levels of NETs in Lpar3 -/- mice were higher than those in WT mice after LPS injection. The mortality rate and degree of lung damage in Lpar3 -/- mice with sepsis were significantly reduced after the destruction of NETs by DNaseI treatment. Furthermore, in vitro experiments with co-cultured monocytes and neutrophils demonstrated that monocytes from Lpar3 -/- mice promoted the formation of NETs, suggesting that LPA3 acting on monocytes inhibits the formation of NETs and plays a protective role in sepsis. Mechanistically, we found that the amount of CD14, an LPS co-receptor, expressed by monocytes in Lpar3 -/-mice was significantly elevated after LPS administration, and the MyD88-p65-NFκB signaling axis, downstream of toll-like receptor 4 signaling, in monocytes was overactivated. Finally, after an injection of the LPA3 agonist (2S)-1-oleoyl-2-methylglycero-3-phosphothionate (OMPT), the survival rate of mice with sepsis was improved, organ damage was reduced, and the production of NETs was decreased. This suggested the possible translational value and application prospects of (2S)-OMPT in the treatment of sepsis. Our study confirms an important protective role of LPA3 in curbing the development of sepsis by suppressing NETs production and thrombosis and provides new ideas for sepsis treatment strategies.

Clopidogrel versus ticagrelor in the treatment of Chinese patients undergoing percutaneous coronary intervention: effects on platelet function assessed by platelet function tests and mean platelet volume.

BACKGROUND:Knowledge on the pharmacodynamic effects of antiplatelet drugs including clopidogrel and ticagrelor on Asian patients is scarce. We aim to evaluate the effects of the two drugs on platelet reactivity in the treatment of Chinese patients who underwent percutaneous coronary intervention (PCI), using two platelet function tests (PFT). Meanwhile, the relationship between mean platelet volume (MPV), a routine index of platelet size, and high on-treatment platelet reactivity (HPR) is also investigated.METHODS:Patients receiving dual antiplatelet therapy (DAPT) were scheduled for the assessment of platelet reactivity at 2-3 days after PCI. Two PFTs, light transmission aggregometry (LTA) and vasodilator-stimulated phosphoprotein (VASP)-FCM assay, were applied in the evaluation of platelet reactivity. The MPV was measured simultaneously with EDTA plasma using a Sysmex XN 2000 automated hematology analyzer.RESULTS:The final study population included the aspirin + clopidogrel group (n = 46) and the aspirin + ticagrelor group (n = 66). In the aspirin + ticagrelor group, the maximal light transmittance (LT) changes in response to 5 μM ADP assessed by LTA was obviously lower than that in the aspirin + clopidogrel group (P <  0.001). The platelet reactivity index (PRI) level in the VASP test was also markedly lower in the group given aspirin and ticagrelor (P <  0.001). There was a significant difference in HPR between the two groups. MPV showed a potent ability to predict the presence of HPR at VASP assay (AUC = 0.788, 95% CI: 0.701-0.875, P <  0.001) in receiver-operating characteristic curve analysis.CONCLUSIONS:Compared with clopidogrel, ticagrelor has dramatically greater antiplatelet effect, with a superiority in suppressing platelet function and a lower HPR rate. In addition, there existed a significant independent association between MPV and high prevalence of HPR in the VASP assay.

LPA3-mediated lysophosphatidic acid signaling promotes postnatal heart regeneration in mice.

Background: Lysophosphatidic acid (LPA) is a small glycerophospholipid that acts as a potent extracellular signal in various biological processes and diseases. Our previous work demonstrated that the expression of the LPA receptors LPA1 and LPA3 is elevated in the early postnatal heart. However, the role of this stage-specific expression of LPA1 and LPA3 in the heart is unknown. Methods and Results: By using LPA3 and LPA1 knockout mice, and neonatal SD rats treated with Ki16425 (LPA1/LPA3 inhibitor), we found that the number of proliferating cardiomyocytes, detected by coimmunostaining pH3, Ki67 or BrdU with cardiac troponin T, was significantly decreased in the LPA3 knockout mice and the Ki16425-treated rats but not in the LPA1 knockout mice during the first week of postnatal life. Using a myocardial infarction (MI) model, we found that cardiac function and the number of proliferating cardiomyocytes were decreased in the neonatal LPA3 KO mice and increased in the AAV9-mediated cardiac-specific LPA3 overexpression mice. By using lineage tracing and AAV9-LPA3, we further found that LPA3 overexpression in adult mice enhances cardiac function and heart regeneration as assessed by pH3-, Ki67-, and Aurora B-positive cardiomyocytes and clonal cardiomyocytes after MI. Genome-wide transcriptional profiling and additional mechanistic studies showed that LPA induces cardiomyocyte proliferation through the PI3K/AKT, BMP-Smad1/5, Hippo/YAP and MAPK/ERK pathways in vitro, whereas only ERK was confirmed to be activated by LPA-LPA3 signaling in vivo. Conclusion: Our study reports that LPA3-mediated LPA signaling is a crucial factor for cardiomyocyte proliferation in the early postnatal heart. Cardiac-specific LPA3 overexpression improved cardiac function and promoted cardiac regeneration after myocardial injury induced by MI. This finding suggested that activation of LPA3 potentially through AAV-mediated gene therapy might be a therapeutic strategy to improve the outcome after MI.

Hydrogen Sulfide Promotes Cardiomyocyte Proliferation and Heart Regeneration via ROS Scavenging.

Neonatal mouse hearts can regenerate completely in 21 days after cardiac injury, providing an ideal model to exploring heart regenerative therapeutic targets. The oxidative damage by Reactive Oxygen Species (ROS) is one of the critical reasons for the cell cycle arrest of cardiomyocytes (CMs), which cause mouse hearts losing the capacity to regenerate in 7 days or shorter after birth. As an antioxidant, hydrogen sulfide (H2S) plays a protective role in a variety of diseases by scavenging ROS produced during the pathological processes. In this study, we found that blocking H2S synthesis by PAG (H2S synthase inhibitor) suspended heart regeneration and CM proliferation with ROS deposition increase after cardiac injury (myocardial infarction or apex resection) in 2-day-old mice. NaHS (a H2S donor) administration improved heart regeneration with CM proliferation and ROS elimination after myocardial infarction in 7-day-old mice. NaHS protected primary neonatal mouse CMs from H2O2-induced apoptosis and promoted CM proliferation via SOD2-dependent ROS scavenging. The oxidative DNA damage in CMs was reduced with the elimination of ROS by H2S. Our results demonstrated for the first time that H2S promotes heart regeneration and identified NaHS as a potent modulator for cardiac repair.

Lysophosphatidic acid promotes thrombus stability by inducing rapid formation of neutrophil extracellular traps: A new mechanism of thrombosis.

BACKGROUND:Lysophosphatidic acid (LPA), a bioactive phospholipid released by activated platelets, can induce platelet shape changes and aggregation, which may play an important role in thrombosis. In contrast, the interaction of LPA with neutrophils in thrombosis has not been studied. Recently, neutrophil extracellular traps (NETs) have been shown to bind plasma proteins and activate platelets, which promotes thrombosis.OBJECTIVES:To investigate whether LPA could activate neutrophils to release NETs, predisposing to thrombosis and promoting thrombus stability.METHODS:Levels of neutrophils, NETs, and LPA were detected in 56 participants. Immunofluorescence of NETs and autotaxin, the LPA-producing ectoenzyme, were performed. Induction of NETs and signaling pathways were explored in vitro.RESULTS:Patients with acute pulmonary embolism showed elevated levels of neutrophils, NETs (dsDNA, MPO-DNA, citrullinated histone H3, and nucleosomes), LPA18:1, and LPA20:4. NETs were present in human intrapulmonary thrombi and were surrounded by autotaxin. LPA18:1 induced rapid release of NETs from human neutrophils via a peptidylarginine deiminase 4-dependent pathway. LPA-induced NETs provided a scaffolding for plasma protein binding and generated a tissue plasminogen activator (tPA)-resistant blood clot. Addition of deoxyribonuclease I to tPA significantly accelerated the lysis of clots and human intrapulmonary thrombi. Furthermore, LPA-induced NETs could activate platelets to release LPA.CONCLUSION:This is the first study to implicate LPA in regulating the stability of thrombi by inducing rapid release of NETs in vitro and ex vivo, which could be a new mechanism of thrombosis. These findings provide new insight into the prevention and therapy of venous thromboembolic disease by targeting the LPA-NET signaling pathway.