Clinicopathological analysis of a type of "low grade" poorly cohesive gastric adenocarcinoma not otherwise specified with a good prognosis.

Poorly cohesive carcinoma not otherwise specified (PCGCA-NOS) is regarded in the most recent WHO classification as a high-grade malignancy; however, some cases may be associated with a relatively good prognosis. We have studied a series of 115 cases of PCGCA-NOS and were able to identify low-grade features in 14 cases based on three morphological manifestations. Immunohistochemical staining, EBER in situ hybridization, Feulgen staining and flow cytometry were employed. Among the 115 cases of PCGAC-NOS, 14 cases met the criteria of "low grade", accounting for 12.2 %. The "low grade" cases exhibited more shallow invasion and less lymph node metastasis (both P < 0.05); showed less frequent expression of MUC5AC, E-cadherin and p53 (all P < 0.05). Moreover, "low grade" PCGAC-NOS had a lower proliferative index(P < 0.001). We also found that the DNA content was lower in the "low grade" group, and aneuploidy was not detected in the "low grade" group, which was sharply different from the control group (50 %). Last, "low grade" PCGAC-NOS had a more favorable prognosis. A small subset of PCGAC-NOS cases have a low grade nature, and the clinicopathological features, immunophenotypes, and cytogenetics of these "low grade" cases differ from those of traditional PCGAC-NOS.

Periostin: A Potential Therapeutic Target For Pulmonary Hypertension?

RATIONALE: POSTN (Periostin) is an ECM (extracellular matrix) protein involved in tissue remodeling in response to injury and a contributing factor in tumorigenesis, suggesting that POSTN plays a role in the pathogenesis of pulmonary hypertension (PH). OBJECTIVE: We aimed to gain insight into the mechanistic contribution of POSTN in experimental mouse models of PH and correlate these findings with PH in humans. METHODS AND RESULTS: We used genetic epistasis approaches in human pulmonary artery endothelial cells (hPAECs), human pulmonary artery smooth muscle cells, and experimental mouse models of PH (Sugen 5416/hypoxia or chronic hypoxia) to discern the role of POSTN and its relationship to HIF (hypoxia-inducible factor)-1α signaling. We found that POSTN expression was correlated with the extent of PH in mouse models and in humans. Decreasing POSTN improved hemodynamic and cardiac responses in PH mice, blunted the release of growth factors and HIF-1α, and reversed the downregulated BMPR (bone morphogenetic protein receptor)-2 expression in hPAECs from patients with PH, whereas increasing POSTIN had the opposite effects and induced a hyperproliferative and promigratory phenotype in both hPAECs and human pulmonary artery smooth muscle cells. Overexpression of POSTN-induced activation of HIFs and increased the production of ET (endothelin)-1 and VEGF (vascular endothelial growth factor) in hPAECs. SiRNA-mediated knockdown of HIF-1α abolished the proangiogenic effect of POSTN. Blockade of TrkB (tyrosine kinase receptor B) attenuated the effect of POSTN on HIF-1α expression, while inhibition of HIF-1α reduced the expression of POSTN and TrkB. These results suggest that hPAECs produce POSTN via a HIF-1α-dependent mechanism. CONCLUSIONS: Our study reveals that POSTN expression is increased in human and animal models of PH and fosters PH development via a positive feedback loop between HIF-1α and POSTN during hypoxia. We propose that manipulating POSTIN expression may be an efficacious therapeutic target in the treatment of PH. Our results also suggest that POSTN may serve as a biomarker to estimate the severity of PH.

TGR5 receptor activation attenuates diabetic retinopathy through suppression of RhoA/ROCK signaling.

Diabetic retinopathy (DR) is a common microvascular complication of diabetes mellitus. Abnormal energy metabolism in microvascular endothelium is involved in the progression of diabetic retinopathy. Bile Acid G-Protein-Coupled Membrane Receptor (TGR5) has emerged as a novel regulator of metabolic disorders. However, the role of TGR5 in diabetes mellitus-induced microvascular dysfunction in retinas is largely unknown. Herein, enzyme-linked immunosorbent assay was used for analyzing bile acid (BA) profiles in diabetic rat retinas and retinal microvascular endothelial cells (RMECs) cultured in high glucose medium. The effects of TGR5 agonist on streptozotocin (STZ)-induced diabetic retinopathy were evaluated by HE staining, TUNEL staining, retinal trypsin digestion, and vascular permeability assay. A pharmacological inhibitor of RhoA was used to study the role of TGR5 on the regulation of Rho/Rho-associated coiled-coil containing protein kinase (ROCK) and western blot, immunofluorescence and siRNA silencing were performed to study the related signaling pathways. Here we show that bile acids were downregulated during DR progression in the diabetic rat retinas and RMECs cultured in high glucose medium. The TGR5 agonist obviously ameliorated diabetes-induced retinal microvascular dysfunction in vivo, and inhibited the effect of TNF-α on endothelial cell proliferation, migration, and permeability in vitro. In contrast, knockdown of TGR5 by siRNA aggravated TNF-α-induced actin polymerization and endothelial permeability. Mechanistically, the effects of TGR5 on the improvement of endothelial function was due to its regulatory role on the ROCK signaling pathway. An inhibitor of RhoA significantly reversed the loss of tight junction protein under TNF-α stimulation. Taken together, our findings suggest that insufficient BA signaling plays an important pathogenic role in the development of DR. Upregulation or activation of TGR5 may inhibit RhoA/ROCK-dependent actin remodeling and represent an important therapeutic intervention for DR.

Prostaglandin E2/EP2 receptor signalling pathway promotes diabetic retinopathy in a rat model of diabetes.

AIMS/HYPOTHESIS: Diabetic retinopathy is a common microvascular complication of diabetes mellitus and is initiated by inflammation and apoptosis-associated retinal endothelial cell damage. Prostaglandin E2 (PGE2) has emerged as a critical regulator of these biological processes. We hypothesised that modulating PGE2 and its E-prostanoid receptor (EP2R) would prevent diabetes mellitus-induced inflammation and microvascular dysfunction. METHODS: In a streptozotocin (STZ)-induced rat model of diabetes, rats received intravitreal injection of PGE2, butaprost (a PGE2/EP2R agonist) or AH6809 (an EP2R antagonist). Retinal histology, optical coherence tomography, ultrastructure of the retinal vascular and biochemical markers were assessed. RESULTS: Intravitreal injection of PGE2 and butaprost significantly accelerated retinal vascular leakage, leucostasis and endothelial cell apoptosis in STZ-induced diabetic rats. This response was ameliorated in diabetic rats pre-treated with AH6809. In addition, pre-treatment of human retinal microvascular endothelial cells with AH6809 attenuated PGE2- and butaprost-induced activation of caspase 1, activation of the complex containing nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) and apoptosis-associated speck-like protein containing a C-terminal caspase-activation and recruitment domain (ASC), and activation of the EP2R-coupled cAMP/protein kinase A/cAMP response element-binding protein signalling pathway. CONCLUSIONS/INTERPRETATION: The PGE2/EP2R signalling pathway is involved in STZ-induced diabetic retinopathy and could be considered as a potential target for diabetic retinopathy prevention and treatment.

CCL5 deficiency rescues pulmonary vascular dysfunction, and reverses pulmonary hypertension via caveolin-1-dependent BMPR2 activation.

Pulmonary arterial hypertension (PAH) is a devastating cardiopulmonary disorder characterized by pulmonary arterial remodeling mainly due to excess cellular proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Reduced bone morphogenetic protein receptor 2 (BMPR2) expression in patients with PAH impairs pulmonary arterial endothelial cells (PAECs) function. This can adversely affect PAEC survival and promote PASMCs proliferation. We hypothesized that interventions to normalize the expression of genes that are targets of the BMPR2 signaling could restore PAECs function and prevent or reverse PAH. Here we characterized for the first time, in human PAECs, chemokine (C-C motif) ligand 5 (CCL5/RANTES) deficiency restore BMP-mediated PAECs function. In the cell culture experiments, we found that CCL5 deficiency increased apoptosis and tube formation of PAECs, but suppressed proliferation and migration of PASMCs. Silencing CCL5 expression in PAH PAECs restored bone morphogenetic protein (BMP) signaling responses and promoted phosphorylation of SMADs and transcription of ID genes. Moreover, CCL5 deficiency inhibited angiogenesis by increasing pSMAD-dependent and-independent BMPR2 signaling. This was linked mechanistically to enhanced interaction of BMPR2 with caveolin-1 via CCL5 deficiency-mediated stabilization of endothelial surface caveolin-1. Consistent with these functions, deletion of CCL5 significantly attenuated development of Sugen5416/hypoxia-induced PAH by restoring BMPR2 signaling in mice. Taken together, our findings suggest that CCL5 deficiency could reverse obliterative changes in pulmonary arteries via caveolin-1-dependent amplification of BMPR2 signaling. Our results shed light on better understanding of the disease pathobiology and provide a possible novel target for the treatment of PAH.

Andrographolide Attenuates Established Pulmonary Hypertension via Rescue of Vascular Remodeling.

Pulmonary hypertension (PH) is characterized by vascular remodeling caused by marked proliferation of pulmonary artery smooth muscle cells (PASMCs). Andrographolide (ANDRO) is a potent anti-inflammatory agent which possesses antioxidant, and has anticarcinogenic activity. The present study examined potential therapeutic effects of ANDRO on PH in both chronic hypoxia and Sugen5416/hypoxia mouse PH models. Effects of ANDRO were also studied in cultured human PASMCs isolated from either healthy donors or PH patients. In vivo, ANDRO decreased distal pulmonary arteries (PAs) remodeling, mean PA pressure and right ventricular hypertrophy in chronic hypoxia- and Sugen/hypoxia-induced PH in mice. ANDRO reduced cell viability, proliferation and migration, but increased cell apoptosis in the PASMCs isolated from PH patients. ANDRO also reversed the dysfunctional bone morphogenetic protein receptor type-2 (BMPR2) signaling, suppressed [Ca2+]i elevation, reactive oxygen species (ROS) generation, and the upregulated expression of IL-6 and IL-8, ET-1 and VEGF in PASMCs from PH patients. Moreover, ANDRO significantly attenuated the activation of TLR4/NF-κB, ERK- and JNK-MAPK signaling pathways and reversed the inhibition of p38-MAPK in PASMCs of PH patients. Further, ANDRO blocked hypoxia-triggered ROS generation by suppressing NADPH oxidase (NOX) activation and augmenting nuclear factor erythroid 2-related factor 2 (Nrf2) expression both in vitro and in vivo. Conventional pulmonary vasodilators have limited efficacy for the treatment of severe PH. We demonstrated that ANDRO may reverse pulmonary vascular remodeling through modulation of NOX/Nrf2-mediated oxidative stress and NF-κB-mediated inflammation. Our findings suggest that ANDRO may have therapeutic value in the treatment of PH.

SB203580 protects against inflammatory response and lung injury in a mouse model of lipopolysaccharide­induced acute lung injury.

Acute lung injury (ALI) is characterized by acute hypoxic respiratory failure, pulmonary edema and inflammatory infiltration. ALI has a high mortality rate (~30%) in the clinical setting; therefore, focusing on the treatment of lung edema and inflammatory responses in ALI is of significance. The present study investigated the effect of the p38 mitogen­activated protein kinase (p38MAPK) inhibitor, SB203580, on lung edema and inflammatory responses in ALI in vivo. A mouse model of ALI was established to assess the effect of SB203580 on edema, proinflammatory cytokine production, and the expression of interferon regulatory factor 5 (IRF5) and inducible nitric oxide synthase (iNOS) in lung tissues using immunoblotting, immunohistochemistry, immunofluorescence, hematoxylin and eosin staining, and ELISA. SB203580 inhibited LPS­induced lung injury and proinflammatory cytokine expression, including tumor necrosis factor­α and interleukin­1ß. SB203580 also downregulated LPS­induced IRF5 and iNOS expression, which are widely used as markers of proinflammatory macrophages. Collectively, the present study demonstrated that SB203580 protected against inflammatory responses and lung injury by inhibiting lung edema and downregulating proinflammatory mediators in LPS­induced lung injury.

MicroRNA-221-3p promotes pulmonary artery smooth muscle cells proliferation by targeting AXIN2 during pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a pathological condition characterized by excessive cell proliferation and migration of pulmonary arterial smooth muscle cells (PASMC). PAH pathogenesis shares similarities with cancers such as excessive cell proliferation and apoptosis resistance. A previous study by our group revealed that decreased expression of a tumor suppressor-AXIN2 (Axis inhibition protein 2) was responsible for enhanced PASMC proliferation and suppressed apoptosis. Nevertheless, the mechanisms that regulate the downregulation of AXIN2 in PAH remain elusive. Data from the present study demonstrated that miR-221-3p acts as an upstream regulator of AXIN2 and functions to induce PASMC proliferation. We first showed that miR-221-3p expression was elevated in lung tissue and PASMC of PAH patients as well as in animal models of PAH. Human PASMC were transfected with a miR-221-3p mimic and miR-221-3p inhibitor, respectively, and their effects on the proliferation and migration was assessed using BrdU incorporation, PCNA staining and wound healing assays. In addition, we investigated the molecular mechanism through which miR-221-3p contributes to cell proliferation in PASMC and identified AXIN2 as a direct target gene of miR-221-3p by dual luciferase reporter gene assays, qRT-qPCR and western blotting. Furthermore, we found that ectopic expression of AXIN2 or pharmacological inhibition of ß-catenin by XAV-939 can attenuate the effect of miR-221-3p on cell proliferation in PASMC. Moreover, intravenous injection of miR-221-3p inhibitor attenuated the progression of SU5416-hypoxia-induced PAH in rats. The results of the present study identified a new regulatory axis in which miR-221-3p and AXIN2 regulate the proliferation of PASMC.

PKCγ promotes axonal remodeling in the cortico-spinal tract via GSK3ß/ß-catenin signaling after traumatic brain injury.

Traumatic brain injury (TBI) is a common cause of death and disability. Enhancing the midline-crossing of the contralateral corticospinal tract (CST) to the denervated side of spinal cord facilitates functional recovery after TBI. Activation of the gamma isoform of PKC (PKCγ) in contralateral CST implicates its roles in promoting CST remodeling after TBI. In this study, we deployed loss and gain of function strategies in N2a cells and primary cortical neurons in vitro, and demonstrated that PKCγ is not only important but necessary for neuronal differentiation, neurite outgrowth and axonal branching but not for axonal extension. Mechanically, through the phosphorylation of GSK3ß, PKCγ stabilizes the expression of cytosolic ß-catenin and increase GAP43 expression, thus promoting axonal outgrowth. Further, rAAV2/9-mediated delivery of constitutive PKCγ in the corticospinal tract after unilateral TBI in vivo additionally showed that specifically delivery of active PKCγ mutant to cortical neuron promotes midline crossing of corticospinal fibers from the uninjured side to the denervated cervical spinal cord. This PKCγ-mediated injury response promoted sensorimotor functional recovery. In conclusion, PKCγ mediates stability of ß-catenin through the phosphorylation of GSK3ß to facilitate neuronal differentiation, neurite outgrowth and axonal branching, and PKCγ maybe a novel therapeutic target for physiological and functional recovery after TBI.

α-Solanine reverses pulmonary vascular remodeling and vascular angiogenesis in experimental pulmonary artery hypertension.

OBJECTIVE: Similar to cancer, pulmonary arterial hypertension (PAH) is characterized by vascular remodeling, which leads to obliteration of the small pulmonary arteriole, with marked proliferation of pulmonary artery smooth muscle cells (PASMC) and/or endothelial cells dysfunction. Aberrant expression of tumor suppressor genes is closely associated with susceptibility to PAH. We hypothesized that α-solanine, a glycoalkaloid found in members of the nightshade family known to have antitumor activity in different cancers, reverses experimental PAH by activating the tumor suppressor-axis inhibition protein 2 (AXIN2). METHODS AND RESULTS: We investigated the effects of α-solanine on PASMC proliferation and apoptosis by using 5-ethynyl-2'-deoxyuridine proliferation assay, proliferating cell nuclear antigen and Ki67 staining, TUNEL and Anexine V assays. Scratch wound healing and tube formation assays were also used to study migration of endothelial cells. In vitro, we demonstrated, using cultured human PASMC from PAH patients, that α-solanine reversed dysfunctional AXIN2, ß-catenin and bone morphogenetic protein receptor type-2 signaling, whereas restored [Ca]i, IL-6 and IL-8, contributing to the decrease of PAH-PASMC proliferation and resistance to apoptosis. Meanwhile, α-solanine inhibits proliferation, migration and tube formation of PAH-pulmonary artery endothelial cells by inhibiting Akt/GSK-3α activation. In vivo, α-solanine administration decreases distal pulmonary arteries remodeling, mean pulmonary arteries pressure and right ventricular hypertrophy in both monocrotaline-induced and Sugen/hypoxia-induced PAH in mice. CONCLUSION: This study demonstrates that AXIN2/ß-catenin axis and Akt pathway can be therapeutically targeted by α-solanine in PAH. α-Solanine could be used as a new therapeutic strategy for the treatment of PAH.

Nur77 downregulation triggers pulmonary artery smooth muscle cell proliferation and migration in mice with hypoxic pulmonary hypertension via the Axin2-ß-catenin signaling pathway.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by remodeling of the pulmonary vasculature, including marked proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs). Members of the nuclear receptor 4A (NR4A) subfamily are involved in a variety of biological events, such as cell apoptosis, proliferation, inflammation, and metabolism. Activation of Nur77 (an orphan nuclear receptor that belongs to NR4A subfamily) has recently been reported to be as a beneficial agent in the treatment of cardiovascular and metabolic diseases. In the present study, we investigated the effects of NR4A on human PASMCs function in vitro and determined the underlying mechanisms. We found a robust expression of NR4A receptors in lung tissues of PAH patients and hypoxic mice but a highly significant downregulation within pulmonary arteries (PAs) as assessed by quantitative polymerase chain reaction, immunoblotting, and immunohistochemistry. In vitro, NR4A receptors were found significantly decreased in PASMCs derived from PAH patients. To explore the pathological effects of decreased Nur77 in PASMCs, PASMCs were transduced with siRNA against Nur77. The siRNA-mediated knockdown of Nur77 significantly augmented PASMCs proliferation and migration. In contrast, Nur77 overexpression prevented PASMCs from proliferation and migration. Mechanistically, overexpression of Axis inhibition protein 2 (Axin2) or inhibition of ß-catenin signaling was shown to be responsible for Nur77 knockdown-induced proliferation of PASMCs. Following hypoxia-induced angiogenesis of the pulmonary artery in C57BL/6 mice, small-molecule Nur77 agonists-Octaketide Cytosporone B (Csn-B) can significantly decreased thickness of vascular wall and markedly attenuated the development of chronic hypoxia-induced PAH in vivo. Therefore, reconstitution of Nur77 levels represents a promising therapeutic option to prevent vascular remodeling processes.