Insulin promotes vascular smooth muscle cell proliferation and apoptosis via differential regulation of tumor necrosis factor‐related apoptosis‐inducing ligand

Background: Insulin regulates glucose homeostasis but can also promote vascular smooth muscle (VSMC) proliferation, important in atherogenesis. Recently, we showed that tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) stimulates intimal thickening via accelerated growth of VSMCs. The aim of the present study was to determine whether insulin‐induced effects on VSMCs occur via TRAIL. Methods: Expression of TRAIL and TRAIL receptor in response to insulin and glucose was determined by polymerase chain reaction. Transcriptional activity was assessed using wild‐type and site‐specific mutations of the TRAIL promoter. Chromatin immunoprecipitation studies were performed. VSMC proliferation and apoptosis was measured. Results: Insulin and glucose exposure to VSMC for 24 h stimulated TRAIL mRNA expression. This was also evident at the transcriptional level. Both insulin‐ and glucose‐inducible TRAIL transcriptional activity was blocked by dominant‐negative specificity protein‐1 (Sp1) overexpression. There are five functional Sp1‐binding elements (Sp1‐1, Sp1‐2, Sp‐5/6 and Sp1‐7) on the TRAIL promoter. Insulin required the Sp1‐1 and Sp1‐2 sites, but glucose needed all Sp1‐binding sites to induce transcription. Furthermore, insulin (but not glucose) was able to promote VSMC proliferation over time, associated with increased decoy receptor‐2 (DcR2) expression. In contrast, chronic 5‐day exposure of VSMC to 1 µg/mL insulin repressed TRAIL and DcR2 expression, and reduced Sp1 enrichment on the TRAIL promoter. This was associated with increased cell death. Conclusions: The findings of the present study provide a new mechanistic insight into how TRAIL is regulated by insulin. This may have significant implications at different stages of diabetes‐associated cardiovascular disease. Thus, TRAIL may offer a novel therapeutic solution to combat insulin‐induced vascular pathologies

Family functioning of adolescents with nonsuicidal self-injury

INTRODUCTION: The aim of this study was to investigate the features of nonsuicidal self-injury (NSSI) behaviors, family functioning and parental well-being in adolescents with NSSI referred to child and adolescent psychiatry department and also in order to detect if there is an association between the act of NSSI and the issues above mentioned. METHODS: 37 cases with NSSI in the last year and their 51 parents and 31 controls with any psychopathology and their 47 parents referred to child and adolescents psychiatry clinic were included. Psychiatric diagnostic interview (Schedule for Affective Disorders and Schizophrenia for School Age Children Lifetime Version), Mc Master Family Assessment Device were applied to all participants. Mc Master Family Assessment Device and General Health Questionnaire were also applied to all participants’ parents. RESULTS: Cutting was the most common self harm method. The adolescents with NSSI and their parents had greater scores in Mc Master Family Assessment Device subscales by pointing the limitations in functioning. The parents of adolescents with NSSI had greater ratio 2 and upper scores in GHQ-12. DISCUSSION AND CONCLUSION: The family functioning of adolescents with NSSI was poorer. There was a significant association between NSSI behaviors and poor family functioning. The parents of adolescents with NSSI had more risks in mental health disorders. It is important to interview with parents of adolescents with NSSI about family functioning problems and to support parental well-being

Tumor Necrosis Factor - Related Apoptosis-Inducing Ligand (TRAIL) promotes angiogenesis and ischemia- induced neovascularization via NADPH Oxidase 4 (NOX4) and Nitric Oxide - dependent mechanisms

Background — Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has the ability to inhibit angiogenesis by inducing endothelial cell death, as well as being able to promote pro- angiogenic activity in vitro. These seemingly opposite effects make its role in ischemic disease unclear. Using Trail_/_ and wildtype mice, we sought to determine the role of TRAIL in angiogenesis and neovascularization following hindlimb ischemia. Methods and Results — Reduced vascularization assessed by real-time 3-dimensional Vevo ultrasound imaging and CD31 staining was evident in Trail_/_ mice after ischemia, and associated with reduced capillary formation and increased apoptosis. Notably, adenoviral TRAIL administration significantly improved limb perfusion, capillary density, and vascular smooth-muscle cell content in both Trail_/_ and wildtype mice. Fibroblast growth factor-2, a potent angiogenic factor, increased TRAIL expression in human microvascular endothelial cell-1, with fibroblast growth factor-2-mediated proliferation, migration, and tubule formation inhibited with TRAIL siRNA. Both fibroblast growth factor-2 and TRAIL significantly increased NADPH oxidase 4 (NOX4) expression. TRAIL-inducible angiogenic activity in vitro was inhibited with siRNAs targeting NOX4, and consistent with this, NOX4 mRNA was reduced in 3-day ischemic hindlimbs of Trail_/_ mice. Furthermore, TRAIL-induced proliferation, migration, and tubule formation was blocked by scavenging H2O2, or by inhibiting nitric oxide synthase activity. Importantly, TRAIL-inducible endothelial nitric oxide synthase phosphorylation at Ser-1177 and intracellular human microvascular endothelial cell-1 cell nitric oxide levels were NOX4 dependent. Conclusions — This is the first report demonstrating that TRAIL can promote angiogenesis following hindlimb ischemia in vivo. The angiogenic effect of TRAIL on human microvascular endothelial cell-1 cells is downstream of fibroblast growth factor-2, involving NOX4 and nitric oxide signaling. These data have significant therapeutic implications, such that TRAIL may improve the angiogenic response to ischemia and increase perfusion recovery in patients with cardiovascular disease and diabetes

Repetitive hypoglycemia reduces activation of glucose-responsive neurons in C1 and C3 medullary brain regions to subsequent hypoglycemia

The impaired ability of the autonomic nervous system to respond to hypoglycemia is termed "hypoglycemia-associated autonomic failure" (HAAF). This life-threatening phenomenon results from at least two recent episodes of hypoglycemia, but the pathology underpinning HAAF remains largely unknown. Although naloxone appears to improve hypoglycemia counterregulation under controlled conditions, hypoglycemia prevention remains the current mainstay therapy for HAAF. Epinephrine-synthesizing neurons in the rostroventrolateral (C1) and dorsomedial (C3) medulla project to the subset of sympathetic preganglionic neurons that regulate peripheral epinephrine release. Here we determined whether or not C1 and C3 neuronal activation is impaired in HAAF and whether or not 1 wk of hypoglycemia prevention or treatment with naloxone could restore C1 and C3 neuronal activation and improve HAAF. Twenty male Sprague-Dawley rats (250-300 g) were used. Plasma epinephrine levels were significantly increased after a single episode of hypoglycemia (n = 4; 5,438 ± 783 pg/ml vs. control 193 ± 27 pg/ml, P < 0.05). Repeated hypoglycemia significantly reduced the plasma epinephrine response to subsequent hypoglycemia (n = 4; 2,179 ± 220 pg/ml vs. 5,438 ± 783 pg/ml, P < 0.05). Activation of medullary C1 (n = 4; 50 ± 5% vs. control 3 ± 1%, P < 0.05) and C3 (n = 4; 45 ± 5% vs. control 4 ± 1%, P < 0.05) neurons was significantly increased after a single episode of hypoglycemia. Activation of C1 (n = 4; 12 ± 3%, P < 0.05) and C3 (n = 4; 19 ± 5%, P < 0.05) neurons was significantly reduced in the HAAF groups. Hypoglycemia prevention or treatment with naloxone did not restore the plasma epinephrine response or C1 and C3 neuronal activation. Thus repeated hypoglycemia reduced the activation of C1 and C3 neurons mediating adrenal medullary responses to subsequent bouts of hypoglycemia.Zohra M. Kakall, Mary M. Kavurma, E. Myfanwy Cohen, Peter R. Howe, Polina E. Nedoboy, and Paul M. Pilowsk

TRAIL-deficiency accelerates vascular calcification in atherosclerosis via modulation of RANKL

The osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) cytokine system, not only controls bone homeostasis, but has been implicated in regulating vascular calcification. TNF-related apoptosis-inducing ligand (TRAIL) is a second ligand for OPG, and although its effect in vascular calcification in vitro is controversial, its role in vivo is not yet established. This study aimed to investigate the role of TRAIL in vascular calcification in vitro using vascular smooth muscle cells (VSMCs) isolated from TRAIL(-/-) and wild-type mice, as well as in vivo, in advanced atherosclerotic lesions of TRAIL(-/-)ApoE(-/-) mice. The involvement of OPG and RANKL in this process was also examined. TRAIL dose-dependently inhibited calcium-induced calcification of human VSMCs, while TRAIL(-/-) VSMCs demonstrated accelerated calcification induced by multiple concentrations of calcium compared to wild-type cells. Consistent with this, RANKL mRNA was significantly elevated with 24 h calcium treatment, while OPG and TRAIL expression in human VSMCs was inhibited. Brachiocephalic arteries from TRAIL(-/-)ApoE(-/-) and ApoE(-/-) mice fed a high fat diet for 12 w demonstrated increased chondrocyte-like cells in atherosclerotic plaque, as well as increased aortic collagen II mRNA expression in TRAIL(-/-)ApoE(-/-) mice, with significant increases in calcification observed at 20 w. TRAIL(-/-)ApoE(-/-) aortas also had significantly elevated RANKL, BMP-2, IL-1β, and PPAR-γ expression at 12 w. Our data provides the first evidence that TRAIL deficiency results in accelerated cartilaginous metaplasia and calcification in atherosclerosis, and that TRAIL plays an important role in the regulation of RANKL and inflammatory markers mediating bone turn over in the vasculature.Belinda A. Di Bartolo, Siân P. Cartland, Hanis H. Harith, Yuri V. Bobryshev, Michael Schoppet, Mary M. Kavurm

Sp1, Acetylated Histone-3 and p300 Regulate TRAIL Transcription: Mechanisms of PDGF-BB-mediated VSMC Proliferation and Migration

We recently reported that TNF-related apoptosis-inducing ligand (TRAIL) is important in atherogenesis, since it can induce vascular smooth muscle cell (VSMC) proliferation and arterial thickening following injury. Here we show the first demonstrate that TRAIL siRNA reduces platelet-derived growth factor-BB (PDGF-BB)-stimulated VSMC proliferation and migration. PDGF-BB-inducible VSMC proliferation was completely inhibited in VSMCs isolated from aortas of TRAIL(-/-) mice; whereas inducible migration was blocked compared to control VSMCs. TRAIL transcriptional control mediating this response is not established. TRAIL mRNA, protein and promoter activity was increased by PDGF-BB and subsequently inhibited by dominant-negative Sp1, suggesting that the transcription factor Sp1 plays a role. Sp1 bound multiple Sp1 sites on the TRAIL promoter, including two established (Sp1-1 and -2) and two novel Sp1-5/6 and -7 sites. PDGF-BB-inducible TRAIL promoter activity by Sp1 was mediated through these sites, since transverse mutations to each abolished inducible activity. PDGF-BB stimulation increased acetylation of histone-3 (ac-H3) and expression of the transcriptional co-activator p300, implicating chromatin remodelling. p300 overexpression increased TRAIL promoter activity, which was blocked by dominant-negative Sp1. Furthermore, PDGF-BB treatment increased the physical interaction of Sp1, p300 and ac-H3, while chromatin immunoprecipitation studies revealed Sp1, p300 and ac-H3 enrichment on the TRAIL promoter. Taken together, our studies demonstrate for the first time that PDGF-BB-induced TRAIL transcriptional activity requires the cooperation of Sp1, ac-H3 and p300, mediating increased expression of TRAIL which is important for VSMC proliferation and migration. Our findings have the promising potential for targeting TRAIL as a new therapeutic for vascular proliferative disorders

The Apoptotic Effect of the Hexane Extract of Rheum undulatum L. in Oral Cancer Cells through the Down-regulation of Specificity Protein 1 and Survivin

The hexane extract of Rheum undulatum L. (HERL) has been shown to have anti-cancer activity in several cancers in vivo and in vitro. However, the anti-cancer activity of HERL and its molecular mechanism in human oral cancer cells has not been explored. Thus, the aim of this study was to elucidate the growth-inhibitory and apoptosis-inducing effects of HERL in HN22 and SCC15 oral cancer cell lines. This study shows that HERL inhibits oral cancer growth, decreases cell viability, and causes apoptotic cell death in HN22 and SCC15 cells, as characterized by morphological changes, nuclear condensation and fragmentation, the cleavage of PARP and the accumulation of cells in the sub-G1 phase. The treatment of oral cancer cells with HERL also resulted in decreased expression of specificity protein (Sp1) and its downstream protein, survivin. Therefore, our results suggest that the regulation of Sp1 and survivin plays a critical role in HERL-induced apoptosis in human oral cancer cells

HDL improves cholesterol and glucose homeostasis and reduces Aatherosclerosis in diabetes-associated atherosclerosis

Background and Aims. Apolipoprotein A-I (ApoA-I), the main component of high-density lipoprotein (HDL), not only promotes reverse cholesterol transport (RCT) in atherosclerosis but also increases insulin secretion in pancreatic β-cells, suggesting that interventions which raise HDL levels may be beneficial in diabetes-associated cardiovascular disease (CVD). Previously, we showed that TNF-related apoptosis-inducing ligand (TRAIL) deletion in Apolipoprotein Eknockout (Apoe-/-) mice results in diabetes-accelerated atherosclerosis in response to a “Western” diet. Here, we sought to identify whether reconstituted HDL (rHDL) could improve features of diabetes-associated CVD in Trail-/-Apoe-/- mice. Methods and Results. Trail-/-Apoe-/- and Apoe-/- mice on a “Western” diet for 12 weeks received 3 weekly infusions of either PBS (vehicle) or rHDL (containing ApoA-I (20 mg/kg) and 1-palmitoyl-2-linoleoyl phosphatidylcholine). Administration of rHDL reduced total plasma cholesterol, triglyceride, and glucose levels in Trail-/-Apoe-/- but not in Apoe-/- mice, with no change in weight gain observed. rHDL treatment also improved glucose clearance in response to insulin and glucose tolerance tests. Immunohistological analysis of pancreata revealed increased insulin expression/production and a reduction in macrophage infiltration in mice with TRAIL deletion. Furthermore, atherosclerotic plaque size in Trail-/-Apoe-/- mice was significantly reduced associating with increased expression of the M2 macrophage marker CD206, suggesting HDL's involvement in the polarization of macrophages. rHDL also increased vascular mRNA expression of RCT transporters, ABCA1 and ABCG1, in Trail-/-Apoe-/- but not in Apoe-/- mice. Conclusions. rHDL improves features of diabetes-associated atherosclerosis in mice. These findings support the therapeutic potential of rHDL in the treatment of atherosclerosis and associated diabetic complications. More studies are warranted to understand rHDL’s mechanism of action.Belinda A. Di Bartolo, Siân P. Cartland, Scott Genner, Pradeep Manuneedhi Cholan, Melissa Vellozzi, Kerry-Anne Rye and Mary M. Kavurm

Repetitive hypoglycemia reduces activation of glucose-responsive neurons in C1 and C3 medullary brain regions to subsequent hypoglycemia

The impaired ability of the autonomic nervous system to respond to hypoglycemia is termed 'hypoglycemia-associated autonomic failure' (HAAF). This life-threatening phenomenon results from at least two recent episodes of hypoglycemia, but the pathology underpinning HAAF remains largely unknown. Although naloxone appears to improve hypoglycemia counterregulation under controlled conditions, hypoglycemia prevention remains the current mainstay therapy for HAAF. Epinephrine-synthesizing neurons in the rostroventrolateral (C1) and dorsomedial (C3) medulla project to the subset of sympathetic preganglionic neurons that regulate peripheral epinephrine release. Here we determined whether or not C1 and C3 neuronal activation is impaired in HAAF and whether or not 1 wk of hypoglycemia prevention or treatment with naloxone could restore C1 and C3 neuronal activation and improve HAAF. Twenty male Sprague-Dawley rats (250–300 g) were used. Plasma epinephrine levels were significantly increased after a single episode of hypoglycemia (n = 4; 5,438 ± 783 pg/ml vs. control 193 ± 27 pg/ml, P < 0.05). Repeated hypoglycemia significantly reduced the plasma epinephrine response to subsequent hypoglycemia (n = 4; 2,179 ± 220 pg/ml vs. 5,438 ± 783 pg/ml, P < 0.05). Activation of medullary C1 (n = 4; 50 ± 5% vs. control 3 ± 1%, P < 0.05) and C3 (n = 4; 45 ± 5% vs. control 4 ± 1%, P < 0.05) neurons was significantly increased after a single episode of hypoglycemia. Activation of C1 (n = 4; 12 ± 3%, P < 0.05) and C3 (n = 4; 19 ± 5%, P < 0.05) neurons was significantly reduced in the HAAF groups. Hypoglycemia prevention or treatment with naloxone did not restore the plasma epinephrine response or C1 and C3 neuronal activation. Thus repeated hypoglycemia reduced the activation of C1 and C3 neurons mediating adrenal medullary responses to subsequent bouts of hypoglycemia

Non-alcoholic fatty liver disease, vascular inflammation and insulin resistance are exacerbated by TRAIL deletion in mice

Non-alcoholic fatty liver disease (NAFLD) incorporates steatosis, non-alcoholic steato-hepatitis (NASH) and liver cirrhosis, associating with diabetes and cardiovascular disease (CVD). TNF-related apoptosis-inducing ligand (TRAIL) is protective of CVD. We aimed to determine whether TRAIL protects against insulin resistance, NAFLD and vascular injury. Twelve-week high fat diet (HFD)-fed Trail -/- mice had increased plasma cholesterol, insulin and glucose compared to wildtype. Insulin tolerance was impaired with TRAIL-deletion, with reduced p-Akt, GLUT4 expression and glucose uptake in skeletal muscle. Hepatic triglyceride content, inflammation and fibrosis were increased with TRAIL-deletion, with elevated expression of genes regulating lipogenesis and gluconeogenesis. Moreover, Trail -/- mice exhibited reduced aortic vasorelaxation, impaired insulin signaling, and >20-fold increased mRNA expression for IL-1β, IL-6, and TNF-α. In vitro, palmitate treatment of hepatocytes increased lipid accumulation, inflammation and fibrosis, with TRAIL mRNA significantly reduced. TRAIL administration inhibited palmitate-induced hepatocyte lipid uptake. Finally, patients with NASH had significantly reduced plasma TRAIL compared to control, simple steatosis or obese individuals. These findings suggest that TRAIL protects against insulin resistance, NAFLD and vascular inflammation. Increasing TRAIL levels may be an attractive therapeutic strategy, to reduce features of diabetes, as well as liver and vascular injury, so commonly observed in individuals with NAFLD.Siân P. Cartland, Hanis H. Harith, Scott W. Genner, Lei Dang, Victoria C. Cogger, Melissa Vellozzi, Belinda A. Di Bartolo, Shane R. Thomas, Leon A. Adams, Mary M. Kavurm