Spleen Tyrosine Kinase phosphorylates VE-cadherin to cause endothelial barrier disruption in acute lung injury.

Increased endothelial cell (EC) permeability is a cardinal feature of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Tyrosine phosphorylation of VE-cadherin is a key determinant of EC barrier disruption. However, the identity and role of tyrosine kinases in this context are incompletely understood. Here we report that Spleen Tyrosine Kinase (Syk) is a key mediator of EC barrier disruption and lung vascular leak in sepsis. Inhibition of Syk by pharmacological or genetic approaches, each reduced thrombin-induced EC permeability. Mechanistically, Syk associates with and phosphorylates VE-cadherin to cause EC permeability. To study the causal role of endothelial Syk in sepsis-induced ALI, we used a remarkably efficient and cost-effective approach based on gene transfer to generate EC-ablated Syk mice. These mice were protected against sepsis-induced loss of VE-cadherin and inflammatory lung injury. Notably, the administration of Syk inhibitor R788 (fostamatinib); currently in phase II clinical trial for the treatment of COVID-19, mitigated lung injury and mortality in mice with sepsis. These data identify Syk as a novel kinase for VE-cadherin and a druggable target against ALI in sepsis.

The novel coronary artery disease risk gene JCAD/KIAA1462 promotes endothelial dysfunction and atherosclerosis.

AIMS: Recent genome-wide association studies (GWAS) have identified that the JCAD locus is associated with risk of coronary artery disease (CAD) and myocardial infarction (MI). However, the mechanisms whereby candidate gene JCAD confers disease risk remain unclear. We addressed whether and how JCAD affects the development of atherosclerosis, the common cause of CAD. METHODS AND RESULTS: By mining data in the Genotype-Tissue Expression (GTEx) database, we found that CAD-associated risk variants at the JCAD locus are linked to increased JCAD gene expression in human arteries, implicating JCAD as a candidate causal CAD gene. We therefore generated global and endothelial cell (EC) specific-JCAD knockout mice, and observed that JCAD deficiency attenuated high fat diet-induced atherosclerosis in ApoE-deficient mice. JCAD-deficiency in mice also improved endothelium-dependent relaxation. Genome-wide transcriptional profiling of JCAD-depleted human coronary artery ECs showed that JCAD depletion inhibited the activation of YAP/TAZ pathway, and the expression of downstream pro-atherogenic genes, including CTGF and Cyr61. As a result, JCAD-deficient ECs attracted fewer monocytes in response to lipopolysaccharide (LPS) stimulation. Moreover, JCAD expression in ECs was decreased under unidirectional laminar flow in vitro and in vivo. Proteomics studies suggest that JCAD regulates YAP/TAZ activation by interacting with actin-binding protein TRIOBP, thereby stabilizing stress fiber formation. Finally, we observed that endothelial JCAD expression was increased in mouse and human atherosclerotic plaques. CONCLUSION: The present study demonstrates that the GWAS-identified CAD risk gene JCAD promotes endothelial dysfunction and atherosclerosis, thus highlighting the possibility of new therapeutic strategies for CAD by targeting JCAD.

Autophagy inhibitor 3-methyladenine protects against endothelial cell barrier dysfunction in acute lung injury.

Autophagy is an evolutionarily conserved cellular process that facilitates the continuous recycling of intracellular components (organelles and proteins) and provides an alternative source of energy when nutrients are scarce. Recent studies have implicated autophagy in many disorders, including pulmonary diseases. However, the role of autophagy in endothelial cell (EC) barrier dysfunction and its relevance in the context of acute lung injury (ALI) remain uncertain. Here, we provide evidence that autophagy is a critical component of EC barrier disruption in ALI. Using an aerosolized bacterial lipopolysaccharide (LPS) inhalation mouse model of ALI, we found that administration of the autophagy inhibitor 3-methyladenine (3-MA), either prophylactically or therapeutically, markedly reduced lung vascular leakage and tissue edema. 3-MA was also effective in reducing the levels of proinflammatory mediators and lung neutrophil sequestration induced by LPS. To test the possibility that autophagy in EC could contribute to lung vascular injury, we addressed its role in the mechanism of EC barrier disruption. Knockdown of ATG5, an essential regulator of autophagy, attenuated thrombin-induced EC barrier disruption, confirming the involvement of autophagy in the response. Similarly, exposure of cells to 3-MA, either before or after thrombin, protected against EC barrier dysfunction by inhibiting the cleavage and loss of vascular endothelial cadherin at adherens junctions, as well as formation of actin stress fibers. 3-MA also reversed LPS-induced EC barrier disruption. Together, these data imply a role of autophagy in lung vascular injury and reveal the protective and therapeutic utility of 3-MA against ALI.

Phospholipase C-ε signaling mediates endothelial cell inflammation and barrier disruption in acute lung injury.

Phospholipase C-ε (PLC-ε) is a unique PLC isoform that can be regulated by multiple signaling inputs from both Ras family GTPases and heterotrimeric G proteins and has primary sites of expression in the heart and lung. Whereas the role of PLC-ε in cardiac function and pathology has been documented, its relevance in acute lung injury (ALI) is unclear. We used PLC-ε(-/-) mice to address the role of PLC-ε in regulating lung vascular inflammation and injury in an aerosolized bacterial LPS inhalation mouse model of ALI. PLC-ε(-/-) mice showed a marked decrease in LPS-induced proinflammatory mediators (ICAM-1, VCAM-1, TNF-α, IL-1ß, IL-6, macrophage inflammatory protein 2, keratinocyte-derived cytokine, monocyte chemoattractant protein 1, and granulocyte-macrophage colony-stimulating factor), lung neutrophil infiltration and microvascular leakage, and loss of VE-cadherin compared with PLC-ε(+/+) mice. These data identify PLC-ε as a critical determinant of proinflammatory and leaky phenotype of the lung. To test the possibility that PLC-ε activity in endothelial cells (EC) could contribute to ALI, we determined its role in EC inflammation and barrier disruption. RNAi knockdown of PLC-ε inhibited NF-κB activity in response to diverse proinflammatory stimuli, thrombin, LPS, TNF-α, and the nonreceptor agonist phorbol 13-myristate 12-acetate (phorbol esters) in EC. Depletion of PLC-ε also inhibited thrombin-induced expression of NF-κB target gene, VCAM-1. Importantly, PLC-ε knockdown also protected against thrombin-induced EC barrier disruption by inhibiting the loss of VE-cadherin at adherens junctions and formation of actin stress fibers. These data identify PLC-ε as a novel regulator of EC inflammation and permeability and show a hitherto unknown role of PLC-ε in the pathogenesis of ALI.

Estrogen receptor α in cancer associated fibroblasts suppresses prostate cancer invasion via reducing CCL5, IL6 and macrophage infiltration in the tumor microenvironment.

BACKGROUND: Cancer associated fibroblasts (CAF) play important roles in tumor growth that involves inflammation and epithelial cell differentiation. Early studies suggested that estrogen receptor alpha (ERα) was expressed in stromal cells in normal prostates and prostate cancer (PCa), but the detailed functions of stromal ERα in the PCa remain to be further elucidated. METHODS: Migration and invasion assays demonstrated the presence of high levels of ERα in CAF cells (CAF.ERα(+)) suppressed PCa invasion via influencing the infiltration of tumor associated macrophages. ERα decreased CAF CCL5 secretion via suppressing the CCL5 promoter activity was examined by luciferase assay. ERα decreased CCL5 and IL-6 expression in conditioned media that was collected from CAF cell only or CAF cell co-cultured with macrophages as measured by ELISA assay. RESULTS: Both in vitro and in vivo studies demonstrated CAF.ERα(+) led to a reduced macrophage migration toward PCa via inhibiting CAF cells secreted chemokine CCL5. This CAF.ERα(+) suppressed macrophage infiltration affected the neighboring PCa cells invasion and the reduced invasiveness of PCa cells are at least partly due to reduced IL6 expression in the macrophages and CAF. CONCLUSION: Our data suggest that CAF ERα could be applied as a prognostic marker to predict cancer progression, and targeting CCL5 and IL6 may be applied as an alternative therapeutic approach to reduce M2 type macrophages and PCa invasion in PCa patients with low or little ERα expression in CAF cells.

Estrogen receptor α in cancer-associated fibroblasts suppresses prostate cancer invasion via modulation of thrombospondin 2 and matrix metalloproteinase 3.

The prostate cancer (PCa) microenvironment contains active stromal cells known as cancer-associated fibroblasts (CAF) that may play important roles in influencing tumor progression. Here we studied the role of CAF estrogen receptor alpha (ERα) and found that it could protect against PCa invasion. Immunohistochemistry on prostatectomy specimens showed that PCa patients with ERα-positive stroma had a significantly lower risk for biochemical recurrence. In vitro invasion assays further confirmed that the stromal ERα was able to reduce PCa cell invasion. Dissection of the molecular mechanism revealed that the CAF ERα could function through a CAF-epithelial interaction via selectively upregulating thrombospondin 2 (Thbs2) and downregulating matrix metalloproteinase 3 (MMP3) at the protein and messenger RNA levels. Chromatin immunoprecipitation assays further showed that ERα could bind to an estrogen response element on the promoter of Thbs2. Importantly, knockdown of Thbs2 led to increased MMP3 expression and interruption of the ERα mediated invasion suppression, providing further evidence of an ERα-Thbs2-MMP3 axis in CAF. In vivo studies using athymic nude mice injected with CWR22Rv1 (22Rv1) PCa epithelial cells and CAF cells ± ERα also confirmed that mice coimplanted with PCa cells and CAF ERα+ cells had less tumor foci in the pelvic lymph nodes, less metastases, and tumors showed less angiogenesis, MMP3, and MMP9 (an MMP3 downstream target) positive staining. Together, these data suggest that CAF ERα could play protective roles in suppressing PCa metastasis. Our results may lead to developing new and alternative therapeutic approaches to battle PCa via controlling ERα signaling in CAF.

Suppression of ERß signaling via ERß knockout or antagonist protects against bladder cancer development.

Epidemiological studies showed that women have a lower bladder cancer (BCa) incidence, yet higher muscle-invasive rates than men, suggesting that estrogen and the estrogen receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERß), may play critical roles in BCa progression. Using in vitro cell lines and an in vivo carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced mouse BCa model, we found that ERß plays a positive role in promoting BCa progression. Knockdown of ERß with ERß-shRNA in ERß-positive human BCa J82, 647v and T24 cell lines led to suppressed cell growth and invasion. Mice lacking ERß have less cancer incidence with reduced expression of the proliferation marker Ki67 in BBN-induced BCa. Consistently, our results show that non-malignant urothelial cells with ERß knockdown are more resistant to carcinogen-induced malignant transformation. Mechanism dissection found that targeting ERß suppressed the expression of minichromosome maintenance complex component 5 (MCM5), a DNA replication licensing factor that is involved in tumor cell growth. Restoring MCM5 expression can partially reverse ERß knockdown-mediated growth reduction. Supportively, treating cells with the ERß-specific antagonist, 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP), reduced BCa cell growth and invasion, as well as MCM5 expression. Furthermore, we provide the first evidence that BCa burden and mortality can be controlled by PHTPP treatment in the carcinogen-induced BCa model. Together, these results demonstrate that ERß could play positive roles in promoting BCa progression via MCM5 regulation. Targeting ERß through ERß-shRNA, PHTPP or via downstream targets, such as MCM5, could serve as potential therapeutic approaches to battle BCa.

Essential Role of Rho-Associated Kinase in ABO Immune Complex-Mediated Endothelial Barrier Disruption.

ABO immune complexes (ABO-IC) formed by ABO-incompatible antigen-antibody interaction are associated with hemolysis and platelet destruction in patients transfused with ABO-nonidentical blood products. However, the effects of ABO-IC on endothelial cells (EC) are unclear. ABO-IC were formed in vitro from normal donor-derived plasma and serum. Human pulmonary artery EC (HPAEC) were cultured and treated with media, ABO-identical and -non-identical plasma, and ABO-IC. EC barrier integrity was evaluated using transendothelial electrical resistance (TEER), scanning electron microscopy (SEM), vascular endothelial (VE)-cadherin and phalloidin staining, and Rho-associated Kinase (ROCK) inhibitor treatment. TEER revealed significant/irreversible barrier disruption within 1-2 h of exposure to ABO non-identical plasma and ABO-IC; this occurred independently of EC ABO type. Treatment with ABO-IC resulted in decreased VE-cadherin staining and increased phalloidin staining in a time-dependent manner, suggesting that the resultant increased EC barrier permeability is secondary to actin stress fiber formation and loss of cell surface VE-cadherin. Inhibition of ROCK was effective in protecting against IC-induced barrier disruption even two hours after ABO-IC exposure. ABO-IC causes increased EC barrier permeability by decreasing cell surface VE-cadherin and promoting stress fiber formation, which is preventable by inhibiting ROCK activation to protect against EC contraction and gap formation.

Autophagy protein ATG7 is a critical regulator of endothelial cell inflammation and permeability.

Endothelial cell (EC) inflammation and permeability are critical pathogenic mechanisms in many inflammatory conditions including acute lung injury. In this study, we investigated the role of ATG7, an essential autophagy regulator with no autophagy-unrelated functions, in the mechanism of EC inflammation and permeability. Knockdown of ATG7 using si-RNA significantly attenuated thrombin-induced expression of proinflammatory molecules such as IL-6, MCP-1, ICAM-1 and VCAM-1. Mechanistic study implicated reduced NF-κB activity in the inhibition of EC inflammation in ATG7-silenced cells. Moreover, depletion of ATG7 markedly reduced the binding of RelA/p65 to DNA in the nucleus. Surprisingly, the thrombin-induced degradation of IκBα in the cytosol was not affected in ATG7-depleted cells, suggesting a defect in the translocation of released RelA/p65 to the nucleus in these cells. This is likely due to suppression of thrombin-induced phosphorylation and thereby inactivation of Cofilin1, an actin-depolymerizing protein, in ATG7-depleted cells. Actin stress fiber dynamics are required for thrombin-induced translocation of RelA/p65 to the nucleus, and indeed our results showed that ATG7 silencing inhibited this response via inactivation of Cofilin1. ATG7 silencing also reduced thrombin-mediated EC permeability by inhibiting the disassembly of VE-cadherin at adherens junctions. Together, these data uncover a novel function of ATG7 in mediating EC inflammation and permeability, and provide a mechanistic basis for the linkage between autophagy and EC dysfunction.

Critical role of autophagy regulator Beclin1 in endothelial cell inflammation and barrier disruption.

Recent studies have implicated autophagy in several inflammatory diseases involving aberrant endothelial cell (EC) responses, such as acute lung injury (ALI). However, the mechanistic basis for a role of autophagy in EC inflammation and permeability remain poorly understood. In this study, we impaired autophagy by silencing the essential Beclin1 autophagy gene in human pulmonary artery EC. This resulted in reduced expression of proinflammatory genes in response to thrombin, a procoagulant and proinflammatory mediator whose concentration is elevated in many diseases including sepsis and ALI. These (Beclin1-depleted) cells also displayed a marked decrease in NF-κB activity secondary to impaired DNA binding of RelA/p65 in the nucleus, but exhibited normal IκBα degradation in the cytosol. Further analysis showed that Beclin1 knockdown was associated with impaired RelA/p65 translocation to the nucleus. Additionally, Beclin1 knockdown attenuated thrombin-induced phosphorylation of RelA/p65 at Ser536, a critical event necessary for the transcriptional activity of RelA/p65. Beclin1 silencing also protected against thrombin-induced EC barrier disruption by preventing the loss of VE-cadherin at adherens junctions. Moreover, Beclin1 knockdown reduced thrombin-induced phosphorylation/inactivation of actin depolymerizing protein Cofilin1 and thereby actin stress fiber formation required for EC permeability as well as RelA/p65 nuclear translocation. Together, these data identify Beclin1 as a novel mechanistic link between autophagy and EC dysfunction (inflammation and permeability).

Recruited T cells promote the bladder cancer metastasis via up-regulation of the estrogen receptor ß/IL-1/c-MET signals.

Clinical data indicates that T cells can be recruited to bladder cancer (BCa), yet the impact of T cells on BCa progression remains unclear. In the present study, we found that T cells were recruited more to BCa tissues than to the surrounding normal bladder tissues. Results from an in vitro co-culture system also found that BCa recruited more CD4+ T cells than did normal bladder cells. The recruiting of T cells to BCa tissues may increase the proliferation and invasion of BCa cells. Mechanistic studies revealed that infiltrating T cells stimulate BCa estrogen receptor beta (ERß) signaling and consequently increase the expression of MET proto-oncogene, receptor tyrosine kinase (c-MET), through either direct binding to its promoter or via modulation of IL-1 expression. Interruption of ERß/c-MET or ERß/IL-1/c-MET signaling via ERß-shRNA, IL-1 antagonist, or the c-MET inhibitor, SU11274, could partially reverse the T cell-enhanced BCa cell invasion and proliferation. Finally, the mouse BCa model with xenografted BCa 5637 cells with T (HH) cells confirmed the results of in vitro co-culture studies showing that infiltrating T cells could promote BCa metastasis via modulation of the ERß/c-MET or ERß/IL-1/c-MET signaling pathways. These findings may provide a new therapeutic approach to better combat BCa progression via targeting these newly identified signaling pathways.

Estrogen receptor alpha prevents bladder cancer via INPP4B inhibited akt pathway in vitro and in vivo.

Clinical reports show males have a higher bladder cancer (BCa) incidence than females. The sexual difference of BCa occurrence suggests that estrogen and its receptors may affect BCa development. Estrogen receptor alpha (ERα) is the classic receptor to convey estrogen signaling, however, the function of ERα in BCa development remains largely unknown. To understand the in vivo role of ERα in BCa development, we generated total and urothelial specific ERα knockout mice (ERαKO) and used the pre- carcinogen BBN to induce BCa. Earlier reports showed that ERα promotes breast and ovarian cancers in females. Surprisingly and of clinical importance, our results showed that ERα inhibits BCa development and loss of the ERα gene results in an earlier onset and higher incidence of BBN-induced in vivo mouse BCa. Supportively, carcinogen induced malignant transformation ability was reduced in ERα expressing urothelial cells as compared to ERα negative cells. Mechanism studies suggest that ERα could control the expression of INPP4B to reduce AKT activity and consequently reduce BCa cell growth. In addition, IHC staining of clinical sample analyses show that INPP4B expression, in correlation with reduced ERα, is significantly reduced in human BCa specimens. Together, this is the first report using the in vivo cre-loxP gene knockout mouse model to characterize ERα roles in BCa development. Our studies provide multiple in vitro cell studies and in vivo animal model data as well as human BCa tissue analyses to prove ERα plays a protective role in BCa initiation and growth at least partly via modulating the INPP4B/Akt pathway.