Angiogenesis: a new physiological role for N-arachidonoyl serine and GPR55?

N-arachidonoyl serine (ARA-S) is one of a number of acyl amino acids recently identified in mammalian tissues. It has been referred to as an endocannabinoid-like lipid largely based on its structural similarities with the endocannabinoid, N-arachidonoyl ethanolamide (anandamide). However, little is known about its potential physiological functions and receptor targets. In this issue of the British Journal of Pharmacology, Zhang and colleagues show that ARA-S is a potent inducer of endothelial cell proliferation and migration, and angiogenesis in vitro. Furthermore, this pro-angiogenic action is mediated, at least partly, by activation of the poorly characterized, G-protein-coupled GPR55 receptor. ARA-S, via GPR55, increases phosphorylation of extracellular signal-regulated kinases and Akt, and vascular endothelial growth factor signalling. These exciting findings highlight the endothelium as an endogenous target for ARA-S and GPR55.     

The orphan receptor GPR55 is a novel cannabinoid receptor

BACKGROUND: The endocannabinoid system functions through two well characterized receptor systems, the CB1 and CB2 receptors. Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological processes. EXPERIMENTAL APPROACH: Cells transfected with the human cDNA for GPR55 were tested for their ability to bind and to mediate GTPgammaS binding by cannabinoid ligands. Using an antibody and peptide blocking approach, the nature of the G-protein coupling was determined and further demonstrated by measuring activity of downstream signalling pathways. KEY RESULTS: We demonstrate that GPR55 binds to and is activated by the cannabinoid ligand CP55940. In addition endocannabinoids including anandamide and virodhamine activate GTPgammaS binding via GPR55 with nM potencies. Ligands such as cannabidiol and abnormal cannabidiol which exhibit no CB1 or CB2 activity and are believed to function at a novel cannabinoid receptor, also showed activity at GPR55. GPR55 couples to Galpha13 and can mediate activation of rhoA, cdc42 and rac1. CONCLUSIONS: These data suggest that GPR55 is a novel cannabinoid receptor, and its ligand profile with respect to CB1 and CB2 described here will permit delineation of its physiological function(s).     

Nitric oxide： a newly discovered function on wound healing

Wound healing impairment represents a particularly challenging clinical problem to which no efficacious treatment regimens currently exist. The factors ensuring appropriate intercellular communication during wound repair are not completely understood. Although protein-type mediators are well-established players in this process, emerging evidence from both animal and human studies indicates that nitric oxide (NO) plays a key role in wound repair. The beneficial effects of NO on wound repair may be attributed to its functional influences on angiogenesis, inflammation, cell proliferation, matrix deposition, and remodeling. Recent findings from in vitro and in vivo studies of NO on wound repair are summarized in this review. The unveiled novel mechanisms support the use of NO-containing agents and/or NO synthase gene therapy as new therapeutic regimens for impaired wound healing.     

Thioredoxin interacting protein is a novel mediator of retinal inflammation and neurotoxicity

BACKGROUND AND PURPOSE

Up-regulation of thioredoxin interacting protein (TXNIP), an endogenous inhibitor of thioredoxin (Trx), compromises cellular antioxidant and anti-apoptotic defences and stimulates pro-inflammatory cytokines expression, implying a role for TXNIP in apoptosis. Here we have examined the causal role of TXNIP expression in mediating retinal neurotoxicity and assessed the neuroprotective actions of verapamil, a calcium channel blocker and an inhibitor of TXNIP expression.

EXPERIMENTAL APPROACH

Retinal neurotoxicity was induced by intravitreal injection of NMDA in Sprague–Dawley rats, which received verapamil (10 mg·kg⁻¹, p.o.) or vehicle. Neurotoxicity was examined by terminal dUTP nick-end labelling assay and ganglion cell count. Expression of TXNIP, apoptosis signal-regulating kinase 1 (ASK-1), NF-κB, p38 MAPK, JNK, cleaved poly-ADP-ribose polymerase (PARP), caspase-3, nitrotyrosine and 4-hydroxy-nonenal were examined by Western and slot-blot analysis. Release of TNF-α and IL-1β was examined by elisa.

KEY RESULTS

NMDA injection enhanced TXNIP expression, decreased Trx activity, causing increased oxidative stress, glial activation and release of TNF-α and IL-1β. Enhanced TXNIP expression disrupted Trx/ASK-1 inhibitory complex leading to release of ASK-1 and activation of the pro-apoptotic p38 MAPK/JNK pathway, as indicated by cleaved PARP and caspase-3 expression. Treatment with verapamil blocked these effects.

CONCLUSION AND IMPLICATIONS

Elevated TXNIP expression contributed to retinal neurotoxicity by three different mechanisms, inducing release of inflammatory mediators such as TNF-α and IL-1β, altering antioxidant status and disrupting the Trx-ASK-1 inhibitory complex leading to activation of the p38 MAPK/JNK apoptotic pathway. Targeting TXNIP expression is a potential therapeutic target for retinal neurodegenerative disease.     

Beta-cell M3 muscarinic acetylcholine receptors as potential targets for novel antidiabetic drugs

A key feature of type 2 diabetes (T2D) is that beta-cells of the pancreatic islets fail to release sufficient amounts of insulin to overcome peripheral insulin resistance. Glucose-stimulated insulin secretion (GSIS) is regulated by the activity of numerous neurotransmitters, hormones and paracrine factors that act by stimulating specific G protein-coupled receptors expressed by pancreatic beta-cells. Studies with both mouse and human islets suggest that acetylcholine (ACh) acts on beta-cell M₃ muscarinic receptors (M3Rs) to promote GSIS. In mouse islets, beta-cell M3Rs are thought to be activated by ACh released from parasympathetic nerve endings. Interestingly, studies with human pancreatic islets suggest that ACh is synthesized, stored and released by alpha-cells, which, in human pancreatic islets, are intermingled with beta-cells. Independent of the source of pancreatic islet ACh, recent studies indicate that beta-cell M3Rs represent a potential target for drugs capable of promoting insulin release for therapeutic purposes. In this review, we will provide an overview about signaling pathways and molecules that regulate the activity of beta-cell M3Rs. We will also discuss a novel pharmacological strategy to stimulate the activity of these receptors to reduce the metabolic impairments associated with T2D.     

GM-CSF及其动员的骨髓源性细胞在创伤愈合中的作用研究

目的 研究粒细胞巨噬细胞集落刺激因子（granulocyte macrophage colony-stimulating factor,GM-CSF）及其动员的骨髓源性细胞促进创伤愈合的作用途径。方法 采用正常小鼠及化疗、放疗引起的骨髓抑制小鼠模型,背部形成创面,分别给予皮下注射高、低剂量（50,17 μg·kg^-1）GM-CSF,测量创伤愈合程度;采用MTT观察不同浓度下猪髋动脉内皮细胞（pig iliac endothelium cells,PIECs）增殖状况;采用Matrigel基质胶培养人脐静脉内皮细胞和小鼠动脉环,并给予100 ng·mL^-1 GM-CSF,观察其微管结构形成情况。结果 在小鼠创伤愈合模型中,高剂量GM-CSF（50 μg·kg^-1）组创面修复较低剂量更为明显;在小鼠骨髓抑制模型中,低剂量GM-CSF促进骨髓抑制小鼠的创伤愈合;GM-CSF在0.01~781.25 ng·mL^-1明显促进PIECs的增殖;GM-CSF 100 ng·mL^-1明显促进人脐静脉内皮细胞和动脉环周围微管形成。结论 GM-CSF通过动员骨髓源性细胞和作用于内皮细胞促进创伤愈合。     

Epigallocatechin‐3‐gallate augments therapeutic effects of mesenchymal stem cells in skin wound healing

In non‐healing wounds, mesenchymal stem cell (MSC)‐based therapies have the potential to activate a series of coordinated cellular processes, including angiogenesis, inflammation, cell migration, proliferation and epidermal terminal differentiation. As pro‐inflammatory reactions play indispensable roles in initiating wound repair, sustained and prolonged inflammation exhibit detrimental effects on skin wound closure. We investigated the feasibility of using an antioxidant agent epigallocatechin‐3‐gallate (EGCG), along with MSCs, to improve wound repair through their immunomodulatory actions. In a rat model of wound healing, a single dose of EGCG at 10 mg/kg increased the efficiency of MSC‐induced skin wound closure. Twenty days after the wound induction, MSC treatment significantly enhanced the epidermal thickness, which was further increased by EGCG administration. Consistently, the highest extent of growth factors upregulation for neovascularization induction was seen in the animals treated by both MSCs and EGCG, associated with a potent anti‐scarring effect throughout the healing process. Finally, expression levels of pro‐inflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β) and IL‐6, in the wound area were reduced by MSCs, and this reduction was further potentiated by EGCG co‐administration. EGCG, together with MSCs, can promote skin wound healing likely through their combinational effects in modulating chronic inflammation.     

Biased signalling is structurally encoded as an autoproteolysis event in adhesion G protein-coupled receptor Latrophilin-3/ADGRL3

Adhesion G protein-coupled receptors (aGPCRs) possess a unique topology, including the presence of a GPCR proteolysis site (GPS), which, upon autoproteolysis, generates two functionally distinct fragments that remain non-covalently associated at the plasma membrane. A proposed activation mechanism for aGPCRs involves the exposure of a tethered agonist, which depends on cleavage at the GPS. However, this hypothesis has been challenged by the observation that non-cleavable aGPCRs exhibit constitutive activity, thus making the function of GPS cleavage widely enigmatic. In this study, we sought to elucidate the function of GPS-mediated cleavage through the study of G protein coupling with Latrophilin-3/ADGRL3, a prototypical aGPCR involved in synapse formation and function. Using BRET-based G protein biosensors, we reveal that an autoproteolysis-deficient mutant of ADGRL3 retains constitutive activity. Surprisingly, we uncover that cleavage deficiency leads to a signalling bias directed at potentiating the activity of select G proteins such as Gi2 and G12/13. These data unveil the underpinnings of biased signalling for aGPCRs defined by GPS autoproteolysis.     

Inhibition of Tubulogenesis and of Carcinogen-mediated Signaling in Brain Endothelial Cells Highlight the Antiangiogenic Properties of a Mumbaistatin Analog

A better understanding of the metabolic adaptations of the vascular endothelial cells (EC) that mediate tumor vascularization would help the development of new drugs and therapies. Novel roles in cell survival and metabolic adaptation to hypoxia have been ascribed to the microsomal glucose-6-phosphate translocase (G6PT). While antitumorigenic properties of G6PT inhibitors such as chlorogenic acid (CHL) have been documented, those of the G6PT inhibitor and semi-synthetic analog AD4-015 of the polyketide mumbaistatin are not understood. In the present study, we evaluated the in vitro antiangiogenic impact of AD4-015 on human brain microvascular endothelial cells (HBMEC), which play an essential role as structural and functional components in tumor angiogenesis. We found that in vitro HBMEC migration and tubulogenesis were reduced by AD4-015 but not by CHL. The mumbaistatin analog significantly inhibited the phorbol 12-myristate 13-acetate (PMA)-induced matrix-metalloproteinase (MMP)-9 secretion and gene expression as assessed by zymography and RT-PCR. PMA-mediated cell signaling leading to cyclooxygenase (COX)-2 expression and IκB downregulation was also inhibited, further confirming AD4-015 as a cell signaling inhibitor in tumor promoting conditions. G6PT functions may therefore account for the metabolic flexibility that enables EC-mediated neovascularization. This process could be specifically targeted within the vasculature of developing brain tumors by G6PT inhibitors.     

The therapeutic potential of novel antiangiogenic therapies

Promising new antiangiogenic strategies are emerging for the treatment of cancer. Numerous candidate drugs that target vascular endothelial growth factor, vascular endothelial growth factor receptors, integrins, matrix metalloproteinases and other blood vessel targets are being developed and tested in clinical trials. This review highlights the numerous drugs in clinical trials and expands on potential new approaches to inhibiting angiogenesis. These approaches include gene therapy, vaccine strategies and antiangiogenic radioligands. New insight has been gained from completed Phase III trials with antiangiogenic drugs and some of the major obstacles include design of trials, dosing, toxicities and resistance. This review will discuss these barriers and methods by which they can be overcome.     

Sphingosine kinase 1 is critically involved in nitric oxide-mediated human endothelial cell migration and tube formation

Background and purpose:

Sphingosine kinases (SKs) convert sphingosine to sphingosine 1-phosphate (S1P), which is a bioactive lipid that regulates a variety of cellular processes including proliferation, differentiation and migration.

Experimental approach:

We used the human endothelial cell line EA.hy926 to investigate the effect of nitric oxide (NO) donors on SK-1 expression, and on cell migration and tube formation.

Key results:

We showed that exposure of EA.hy926 cells to Deta-NO (125–1000 µM) resulted in a time- and concentration-dependent up-regulation of SK-1 mRNA and protein expression, and activity with a first significant effect at 250 µM of Deta-NO. The increased SK-1 mRNA expression resulted from an enhanced SK-1 promoter activity. A similar effect was also seen with various other NO donors. In mechanistic terms, the NO-triggered effect occurred independently of cGMP, but involved the classical mitogen-activated protein kinase cascade because the MEK inhibitor U0126 abolished the NO-induced SK-1 expression. The effect of NO was also markedly reduced by the thiol-reducing agent N-acetylcysteine, suggesting a redox-dependent mechanism. Functionally, Deta-NO triggered an increase in the migration of endothelial cells in an adapted Boyden chamber assay, and also increased endothelial tube formation in a Matrigel assay. These responses were both abolished in cells depleted of SK-1.

Conclusions and implications:

These data show that NO donors up-regulate specifically SK-1 expression and activity in human endothelial cells, and SK-1 in turn critically contributes to the migratory capability and tube formation of endothelial cells. Thus, SK-1 may be considered an attractive novel target to interfere with pathological processes involving angiogenesis.     

Fasiglifam as a new potential treatment option for patients with type 2 diabetes

Introduction: Fasiglifam, a novel G protein-coupled receptor 40 (GPR40) agonist, has demonstrated glucose-lowering effects in type 2 diabetes mellitus (T2DM) through stimulation of glucose-dependent insulin secretion.

Areas covered: This review is based on a PubMed search for all articles on fasiglifam and TAK-875. The pharmacology of fasiglifam is reviewed, focusing on studies in human volunteers and patients with T2DM. All published clinical trials with fasiglifam in T2DM are summarized, including two 12-week dose-ranging studies (one from Japan and the other from Central and North America), both of which employed glimepiride as an active comparator.

Expert opinion: Fasiglifam, a novel glucose-dependent insulin secretagogue, is the first GPR40 agonist to enter Phase III clinical evaluation. It has been shown to produce statistically significant and clinically relevant improvements in glycemic control in patients with early-stage T2DM. Furthermore, its tolerability and safety profile was comparable to placebo and no dose-related adverse effects were observed. Importantly, fasiglifam was comparable to placebo with regards to the incidence of hypoglycemia and it produced significantly fewer episodes compared with glimepiride. Fasiglifam is an interesting and novel oral anti-diabetic agent which may offer new avenues for treating T2DM, but clearly more thorough clinical evaluation is still needed.     

Methyl tert butyl ether is anti‐angiogenic in both in vitro and in vivo mammalian model systems

Methyl‐tertiary butyl ether (MTBE), a well known gasoline oxygenate, and US Food and Drug Administration approved gallstone treatment, has been previously shown to specifically target teleost embryonic angiogenesis. The studies reported here were to determine whether similar vascular disrupting effects occur in higher vertebrate models. Rat brain endothelial cells were isolated and allowed to form microcapillary‐like tubes on Matrigel. MTBE (0.34–34.0 mm ) exposure resulted in a dose‐dependent reduction of tube formation, with the LOAEL at 0.34 mm , while MTBE's primary metabolite, tertiary butyl alcohol had no effect on tube formation. HUVECs, a primary cell line representing macrovascular cells, were able to form tubes on Matrigel in the presence of MTBE (1.25–80 mm ), but the tubes were narrower than those formed in the absence of MTBE. In a mouse Matrigel plug implantation assay, 34.0 mm MTBE completely inhibited vessel invasion into plugs containing endothelial cell growth supplement (ECGS) compared with control plugs with ECGS alone. When timed‐pregnant Fisher 344 rats were gavaged with MTBE (500–1500 mg kg^?1) from day 6 of organogenesis through 10 days post‐parturition, no organ toxicity or histological changes in pup vasculature were observed. Results of the in vitro cell culture studies show that MTBE is anti‐angiogenic at mm concentrations and has potential use as an anti‐angiogenic treatment for solid tumors with minimal toxicity. Copyright © 2012 John Wiley & Sons, Ltd.     

雌激素调节肿瘤血管生成研究进展

妇科肿瘤已成为全球妇女的头号恶性肿瘤疾病。尽管医学界迄今为止尚未完全清楚其明确的发病机制,但有一点可以肯定:妇科肿瘤的发生与雌激素有关。近年来关于妇科肿瘤与雌激素分泌表达异常之间的分子调控机制已成为国内外研究的重点。临床及实验研究均认为雌激素可通过雌激素受体调节机体的多种信号途径,引起内皮细胞增殖、凋亡、运动、芽生、粘附等特性改变,导致肿瘤血管生成的异常,从而最终影响到肿瘤发生、发展、转移过程。现对近年来国内外有关雌激素调节肿瘤血管生成信号转导通路的研究进行概述,并提出其在中药抗肿瘤研究中的可能影响,期望对肿瘤临床及实验研究者有所借鉴。     

Peptide G protein agonists from a phage display library

G proteins may serve as targets for pharmacological activation of signaling pathways bypassing the regulatory events that may counteract the effect of the external stimulus on the level of receptors. We sought to identify peptides as lead structures interacting with G proteins utilizing a commercially available phage-display library. The heptapeptide library was screened for binding to human G alpha(i1) which was modified with a hexahistidine tag and immobilized on a Ni(2+)-coated surface. After several rounds of phage selection a number of phage clones with consensus sequences were found. Peptides with such sequences were chemically synthesized and their effect on [35S]GTP gamma S binding to G alpha(i1) and other G protein alpha subunits was determined. Through this process two peptide 'families' with the capability to activate G proteins were identified. The peptides showed no structural similarity to known peptide or nonpeptide G protein activators with a cationic amphiphilic structure like mastoparan or alkylamines. The functional relevance of the peptide-G protein interaction was shown by an increased sensitivity for guanine nucleotides of high-affinity agonist binding to A(1) adenosine receptors. The peptide G protein activators may, therefore, serve as novel tools for further investigation of receptor-independent activation of G proteins.     

VEGF/VEGFR signalling as a target for inhibiting angiogenesis

VEGFs and a respective family of tyrosine kinases receptors (VEGFRs) are key proteins modulating angiogenesis, the formation of new vasculature from an existing vascular network. There has been considerable evidence in vivo, including clinical observations, that abnormal angiogenesis is implicated in a number of disease conditions, which include rheumatoid arthritis, inflammation, cancer, psoriasis, degenerative eye conditions and others. Antiangiogenic therapies based on inhibition of VEGF/VEGFR signalling were reported to be powerful clinical strategies in oncology and ophthalmology. Current efforts have yielded promising clinical data for several antiangiogenic therapeutics. In this review, the authors elucidate key aspects of VEGFR signalling, as well as clinically relevant strategies for the inhibition of VEGF-induced angiogenesis, with an emphasis on small-molecule VEGFR inhibitors.     

Monocytes: a novel drug delivery system targeting atherosclerosis

Abstract

Scavenging of reactive oxygen species (ROS) by antioxidants holds great promise to alleviate the symptoms of cardiovascular diseases and atherosclerosis. In atherosclerosis, damaged endothelial cells (EC) generate more ROS and inflammatory mediators, which recruit more monocytes to the EC. Antioxidants are good therapeutic drug candidates; however, antioxidant enzymes such as catalase are easily degraded by proteases in vivo and chemical mimetics of superoxide dismutase such as tempol and tempo require a target-specific delivery system since hydrophobic tempol or tempo can diffuse into any type of cells non-specifically. Here, we report a novel monocyte-based drug delivery system encapsulating either catalase or tempol/tempo. Monocyte as a novel drug delivery vehicle offers advantages over other delivery systems due to its target specificity to damaged EC. The delivery system can also be easily fabricated in biological conditions and keeps antioxidants active. Membrane impermeable catalase with protease inhibitors was formulated in monocytes via a hypotonic/resealing method and membrane permeable tempol/tempo were encapsulated in monocytes via passive diffusion with 40–60% encapsulation efficiency. Antioxidant-loaded monocytes targeted EC and the antioxidants scavenged more than 90% intracellular ROS generated by cytokines or exogenous ROS. We anticipate numerous applications of the monocyte-based drug delivery system, given its target specificity to activated EC.