Chemerin enhances the adhesion and migration of human endothelial progenitor cells and increases lipid accumulation in mice with atherosclerosis

Background The role of adipokines in the development of atherosclerosis (AS) has received increasing attention in recent years. This study aimed to explore the effects of chemerin on the functions of human endothelial progenitor cells (EPCs) and to investigate its role in lipid accumulation in ApoE-knockout (ApoE−/−) mice. Methods EPCs were cultured and treated with chemerin together with the specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 in a time- and dose-dependent manner. Changes in migration, adhesion, proliferation and the apoptosis rate of EPCs were detected. ApoE−/− mice with high-fat diet-induced AS were treated with chemerin with or without SB 203580. Weights were recorded, lipid indicators were detected, and tissues sections were stained. Results The data showed that chemerin enhanced the adhesion and migration abilities of EPCs, and reduced the apoptosis ratio and that this effect might be mediated through the p38 MAPK pathway. Additionally, chemerin increased the instability of plaques. Compared with the control group and the inhibitor group, ApoE−/− mice treated with chemerin protein had more serious arterial stenosis, higher lipid contents in plaques and decreased collagen. Lipid accumulation in the liver and kidney and inflammation in the hepatic portal area were enhanced by treatment with chemerin, and the size of adipocytes also increased after chemerin treatment. In conclusion, chemerin can enhance the adhesion and migration abilities of human EPCs and reduce the apoptosis ratio. In animals, chemerin can increase lipid accumulation in atherosclerotic plaques and exacerbate plaques instability. At the same time, chemerin can cause abnormal lipid accumulation in the livers and kidneys of model animals. After specifically blocking the p38 MAPK pathway, the effect of chemerin was reduced. Conclusions In conclusion, this study showed that chemerin enhances the adhesion and migration abilities of EPCs and increases the instability of plaques and abnormal lipid accumulation in ApoE−/− mice. Furthermore, these effects might be mediated through the p38 MAPK pathway.

Effect of p38 MAPK on oxidative phosphorylation in regeneration of muscle tissue

Цель исследования: определить влияние блокады р38 МАРК на систему окислительного фосфорилирования при репарации мышечной ткани. Материалы и методы: Контрольной группе животных (n = 21) наносили рану длиннейшей мышцы спины, в рану вносили лекарственную плёнку без активного вещества, основной группе (n = 21) пленку с блокатором р38 МАР-киназы SB 203580. Выведение из эксперимента осуществляли на 1, 3, 7, 14 и 30 сутки. При гистологическом исследовании подсчитывали количество миосателлитов и количество образующихся «мышечных почек» в поле зрения в зоне травмы. Иммунофлюоресцентным методом изучали активность экспрессии OxPhos Complex IV. Результаты. Установлено, что блокада р38 МАРК каскада при помощи локального введения активного вещества в составе лекарственной пленки позволила «затормозить» деление миосателлитов в ранние сроки репаративного процесса, обусловила позднее начало образования «мышечных» почек в зоне регенерации - на 7 сутки в сравнение с 3 сутками в контрольной группе, а также привела к резкому усилению окислительного фосфорилирования в зоне регенерации мышечной ткани. The aim of the study was to determine the effect of p38 MAPK inhibition on the oxidative phosphorylation system during repair of muscle tissue. Materials and methods. In the control animal group (n = 21), a wound was inflicted on the longest back muscle; then a medicinal film free of active substance was applied to the wound. In the main group (n = 21), the applied film contained a p38 MAP-kinase inhibitor, SB 203580. The animals were euthanized on days 1, 3, 7, 14, and 30. In the histological study, the number of myosatellite cells per field of view was counted in the trauma zone. The immunofluorescence method was used to study the intensity of OxPhos Complex IV expression. Results. Inhibition of the p38 MAPK cascade by local administration of the active substance formulated into the medicated film allowed «putting the brakes» on division of myosatellite cells during the early reparative process, on day 7 vs. day 3 in the control group, and also resulted in a sharp increase in oxidative phosphorylation in the regeneration zone of muscle tissue.

Abstract 172: tPA Variant tPA-A296-299 Prevents Impairment of Cerebral Autoregulation After Stroke Through LRP Dependent Increase in cAMP and p38 MAPK

Introduction: The sole FDA approved treatment for acute stroke is tissue type plasminogen activator (tPA). However, its brief therapeutic window and post-treatment complications markedly constrain its use. The limited efficacy of tPA may be explained in part by our finding that it potentiates impairment of cerebral autoregulation after stroke in pigs. Upregulation of JNK MAPK after thrombotic stroke contributes to tPA mediated impairment of autoregulation whereas p38 is protective. Vasodilation is mediated by elevation of cAMP. However, cAMP falls after stroke due to over activation of NMDA receptors (NMDA-Rs) by toxic levels of glutamate, which is exacerbated by tPA. Hypothesis: Wild type (wt) tPA can bind to either the lipoprotein-related receptor (LRP), which mediates vasodilation, or NMDA-Rs. We propose that wt tPA given after stroke primarily binds to NMDA-Rs, reduces cAMP, which impairs ability of cerebral vessels to vasodilate. tPA-A296-299, a variant that is fibrinolytic but cannot bind to NMDA-Rs, preferentially binds LRP, which increases cAMP and p38 and limits impairment of autoregulation after stroke. Methods: Anesthetized pigs equipped with a closed cranial window were used. Stroke was induced by photothrombosis, CBF determined by radiolabeled microspheres, and CSF cAMP and p38 determined by ELISA. Results: CBF was unchanged during hypotension (mean arterial pressure decreased by 45%) in sham animals. CBF was reduced in brain tissue surrounding the infarct and was reduced further during hypotension, indicating impairment of autoregulation. Autoregulation was further impaired by wt tPA which was prevented by MK801 and tPA-A296-299. Protection by tPA-A296-299 was blocked by anti LRP Ab, the LRP antagonist RAP, the p38 inhibitor SB 203580, but not by IgG. Stroke reduced CSF cAMP, which was reduced further by wt tPA, but augmented by tPA-A296-299. CSF p38 was unchanged by wt tPA, robustly increased by tPA-A296-299, and decreased by co-administered anti LRP Ab and RAP but not by IgG. Conclusions: tPA-A296-299 prevents impairment of cerebral autoregulation after stroke through LRP dependent increase in cAMP and p38.

Role of p38 MAPK pathway in 17β-estradiol-mediated attenuation of hemorrhagic shock-induced hepatic injury

Although 17β-estradiol (E2) treatment following hemorrhagic shock or ischemic reperfusion prevents organs from dysfunction and injury, the precise mechanism remains unknown. We hypothesize that the E2-mediated attenuation of liver injury following hemorrhagic shock and fluid resuscitation occurs via the p38 mitogen-activated protein kinase (MAPK)-dependent heme oxygenase (HO)-1 pathway. After a 5-cm midline laparotomy, male rats underwent hemorrhagic shock (mean blood pressure ∼40 mmHg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, E2 (1 mg/kg) alone, or E2 plus p38 MAPK inhibitor SB-203580 (2 mg/kg), HO-1 inhibitor chromium mesoporphyrin-IX chloride (2.5 mg/kg) or estrogen receptor antagonist ICI 182,780 (3 mg/kg). At 2 h after hemorrhagic shock and fluid resuscitation, the liver injury markers were significantly increased compared with sham-operated control. Hemorrhagic shock resulted in a significant decrease in p38 MAPK phosphorylation compared with the shams. Administration of E2 following hemorrhagic shock normalized liver p38 MAPK phosphorylation, further increased HO-1 expression, and reduced cleaved caspase-3 levels. Coadministration of SB-203580 abolished the E2-mediated attenuation of the shock-induced liver injury markers. In addition, administration of chromium mesoporphyrin-IX chloride or ICI 182,780 abolished E2-mediated increases in liver HO-1 expression or p38 MAPK activation following hemorrhagic shock. Our results collectively suggest that the salutary effects of E2 on hepatic injury following hemorrhagic shock and resuscitation are in part mediated through an estrogen-receptor-related p38 MAPK-dependent HO-1 upregulation.

P38 Is a Negative Regulator Of The Bortezomib-Induced Heat Shock Response In Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy with an estimated 22,350 new cases and 10,710 deaths in the United States in 2013. Novel treatments including autologous stem cell transplant, immunomodulatory drugs (IMiDs), and the proteasome inhibitor, bortezomib, have led to an increase in patient life span and long-term survival. Bortezomib is a highly selective and reversible 26S proteasome inhibitor. Proteasome inhibition can affect multiple signaling cascades and lead to a toxic buildup of misfolded proteins and eventually, cell death. As part of the response to this protein buildup following proteasome inhibition, myeloma cells activate the cytoprotective heat shock response. This includes upregulation of heat shock proteins (HSPs) such as HSP40, HSP70, and HSP90. Previous attempts at using HSP-specific inhibitors in combination with bortezomib have been disappointing. These results underscore the need to disrupt broad scale activation of the entire heat shock response. This can be achieved by inhibition of the master regulator, Heat Shock Factor 1 (HSF1). Here we show that in four human MM cell lines, MM1.s, KMS11, KMS18, and U266, HSF1 inhibition leads to downregulation of the bortezomib-induced heat shock response and ultimately, increased cell death. While HSF1 is activated by proteasome inhibition, the mechanism of activation has yet to be determined. HSF1 is regulated through a complex series of post-translational modifications. Here we show that bortezomib induces HSF1 phosphorylation in MM1.s and KMS18, and in freshly isolated patient samples. To determine which kinase pathways are responsible for HSF1 phosphorylation, we treated MM1.s and KMS18 with a non-lethal dose (10 μM) of PI3K, MEK, JNK, and p38 inhibitors in combination with bortezomib. Bortezomib-induced HSF1 phosphorylation was inhibited by the p38 inhibitor SB 203580, while inhibitors to PI3K, MEK, and JNK had no effect on bortezomib-induced HSF1 phosphorylation. To determine the consequence of p38 inhibition on HSF1 function, we performed RT-qPCR to probe for the expression of HSF1-dependent gene targets (HSPB1 [HSP27], HSP40B, HSPA1A [HSP70/72], HSPA1B [HSP70/72], HSP90AA1, HSP90A1B) following treatment with bortezomib with or without SB 203580. Surprisingly, gene expression for each of the targets increased when proteasome inhibition was combined with p38 inhibition compared to proteasome inhibition alone. The observed change ranged from 31% (HSPA1A) to 99% (HSP90AA1). Our results show a previously undescribed link between proteasome inhibition and HSF1 regulation; bortezomib-induced p38-dependent phosphorylation. This is consistent with studies in HeLa cells showing that the p38 effector MK2 negatively regulates HSF1 via phosphorylation of S121. Together these findings underscore the complexity of the cellular response to proteasome inhibition, and that understanding both the positive and negative regulatory events during HSF1 activation could lead to the development of novel partners for use with proteasome inhibitors. Disclosures: Lonial: Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy. Boise:Onyx Pharmaceuticals: Consultancy.

An Investigation Into the Molecular Pathways of Airway Smooth Muscle Relaxation

This paper investigates the effect of superimposed length oscillation (SILO) on tidal breathing on contracted airway smooth muscle (ASM) relaxation. The combined effect of SILO with each one of four inhibitors (Isoproterenol, Indomethacin, PD0980590 and SB 203580) is investigated to explore the molecular pathways involved in the tissue relaxation process.

ICAM-1-mediated leukocyte adhesion is critical for the activation of endothelial LSP1

Leukocyte-endothelial interaction triggers signaling events in endothelial cells prior to transendothelial migration of leukocytes. Leukocyte-specific protein 1 (LSP1), expressed in endothelial cells, plays a pivotal role in regulating subsequent recruitment steps following leukocyte adhesion. In neutrophils, LSP1 is activated by phosphorylation of its serine residues by molecules downstream of p38 MAPK and PKC. Whether leukocyte adhesion to endothelial cells is required for endothelial LSP1 activation remains elusive. In addition, discrepancies in the functions of endothelial and leukocyte LSP1 in leukocyte adhesion prevail. We demonstrate that adhesion of wild-type ( Lsp1+/+) neutrophils to LSP1-deficient ( Lsp1−/−) endothelial cells was significantly reduced compared with adhesion to Lsp1+/+endothelial cells. Immunoblotting revealed increased phosphorylated endothelial LSP1 in the presence of adherent Lsp1−/−neutrophils [stimulated by macrophage inflammatory protein-2 (CXCL2), TNF-α, or thapsigargin], but not cytokine or chemokine alone. Pharmacological inhibition of p38 MAPK by SB-203580 (10 μM) significantly blunted the phosphorylation of endothelial LSP1. Functionally blocking endothelial ICAM-1 or neutrophil β2-integrins diminished neutrophil adhesion and phosphorylation of endothelial LSP1. The engagement of endothelial ICAM-1 cross-linking, which mimics leukocyte adhesion, resulted in phosphorylation of endothelial LSP1. In neutrophil-depleted Lsp1+/+mice, administration of ICAM-1 cross-linking antibody resulted in increased phosphorylation of LSP1 and p38 MAPK in TNF-α-stimulated cremaster muscle. In conclusion, endothelial LSP1 participates in leukocyte adhesion in vitro, and leukocyte adhesion through ICAM-1 fosters the activation of endothelial LSP1, an effect at least partially mediated by the activation of p38 MAPK. Endothelial LSP1, in contrast to neutrophil LSP1, is not phosphorylated by cytokine or chemokine stimulation alone.

Angiotensin II increases the permeability and PV-1 expression of endothelial cells

Angiotensin II (ANG II), the major effector molecule of the renin-angiotensin system (RAS), is a powerful vasoactive mediator associated with hypertension and renal failure. In this study the permeability changes and its morphological attributes in endothelial cells of human umbilical vein (HUVECs) were studied considering the potential regulatory role of ANG II. The effects of ANG II were compared with those of vascular endothelial growth factor (VEGF). Permeability was determined by 40 kDa FITC-Dextran and electrical impedance measurements. Plasmalemmal vesicle-1 (PV-1) mRNA levels were measured by PCR. Endothelial cell surface was studied by atomic force microscopy (AFM), and caveolae were visualized by transmission electron microscopy (TEM) in HUVEC monolayers. ANG II (10−7 M), similarly to VEGF (100 ng/ml), increased the endothelial permeability parallel with an increase in the number of cell surface openings and caveolae. AT1 and VEGF-R2 receptor blockers (candesartan and ZM-323881, respectively) blunted these effects. ANG II and VEGF increased the expression of PV-1, which could be blocked by candesartan or ZM-323881 pretreatments and by the p38 mitogem-activated protein (MAP) kinase inhibitor SB-203580. Additionally, SB-203580 blocked the increase in endothelial permeability and the number of surface openings and caveolae. In conclusion, we have demonstrated that ANG II plays a role in regulation of permeability and formation of cell surface openings through AT1 receptor and PV-1 protein synthesis in a p38 MAP kinase-dependent manner in endothelial cells. The surface openings that increase in parallel with permeability may represent transcellular channels, caveolae, or both. These morphological and permeability changes may be involved in (patho-) physiological effects of ANG II.

Ginsenoside Rb1 Prevents MPP+-Induced Apoptosis in PC12 Cells by Stimulating Estrogen Receptors with Consequent Activation of ERK1/2, Akt and Inhibition of SAPK/JNK, p38 MAPK

Ginsenoside Rb1 shows neuroprotective effects in various neurons, including dopaminergic cells. However, the precise mechanisms of action are uncertain. In this paper, we examine whether Rb1 has a neuroprotective effect on MPP+-induced apoptosis and attempt to clarify the signaling pathway in PC12 cells. Apoptosis of PC12 cells was determined by DNA fragmentation assay, the activation of caspase-3, or by the inactivation of Bcl-xL. Rb1 inhibited MPP+-induced caspase-3 activation and DNA fragmentation and activated Bcl-xL in MPP+-treated PC12 cells. These antiapoptotic effect was abrogated in PC12 cells transfected with estrogen receptor siRNA. Levels of DNA fragmentation were increased by wortmannin or PD 98059, while they were decreased by SB 203580 or SP 600125 in MPP+-treated PC12 cells. Rb1 increased phosphorylation levels of ERK1/2 or Akt in MPP+-treated PC12 cells, while it reduced phosphorylated p38 or SAPK/JNK. The increased phosphorylation of ERK/1/2 or Akt by Rb1 was abrogated by estrogen receptor siRNA. Rb1-induced inhibition of SAPK/JNK or p38 phosphorylation was also abolished by estrogen receptor siRNA. These results suggest that ginsenoside Rb1 protects PC12 cells from caspase-3-dependent apoptosis through stimulation of estrogen receptor with consequent activation of ERK1/2 and Akt and inhibition of SAPK/JNK and p38.

Original article. The role of p38MAPK in asymmetric dimethylarginineinduced cytoskeleton and cellular permeability changes in cultured endothelial cells

Background: Asymmetric dimethylarginine (ADMA) induces endothelial cell barrier dysfunction via cytoskeleton activation and contraction. It is supposed that activated p38 mitogen-activated protein kinase (MAPK) would trigger the formation of stress fibers and increase cellular permeability. Objective: Explore p38 MAPK as a potentially important enzyme in ADMA-mediated endothelial cell contractile response and permeability change. Methods: Human umbilical endothelial cells (HUVECs) were cultured, where ADMA and/or SB203580 (the specific inhibitor of p38MAPK) were used to stimulate HUVECs. Immunofluorescent staining was carried out to examine the expression and distribution of F-actin, flow cytometry was used to quantify F-actin, and Transwell was applied to test cellular permeability with FITC-labelled human serum albumin (HSA). Scanning electronic microscopy (SEM) was utilized to observe the changes of intercellar contact. Results: ADMA induced significant p38MAPK activation in a dose-dependent manner, which correlated with increased stress fibers. SB-203580 attenuated the formation of actin stress fiber and the increase of cellular permeability induced ADMA in the HUVECs (p<0.01, LSCM; p<0.01, cytometry; p<0.05, Transwell). Widened intercellular space induced by ADMA was detected and could be inhibited by SB-203580 (SEM). SB-203580 alone had no effect on cytoskeleton and cellular permeability. Conclusion: p38MAPK activation participated in cytoskeleton and cellular permeability changes induced by ADMA in HUVECs.