Vasculopathy in type 2 diabetes mellitus: role of specific angiogenic modulators

Type 2 diabetes mellitus (T2DM) is largely defined by hyperglycemia that promotes vascular complications. Abnormal angiogenesis has been claimed to have a role in this disease. This study aimed to investigate serum levels of both conventional and other markers of angiogenesis not well studied before in diabetes, and to correlate findings with age of the patients, glycemic control, presence of microvascular complications, and oxidative stress. Thirty-eight patients with T2DM and 13 age- and sex-matched healthy persons representing controls were recruited. Serum levels of basic fibroblast growth factor (b-FGF) was measured by immunosorbent assay kit; advanced glycosylation end products, platelet-derived endothelial cell growth factor (PD-ECGF), cathepsin-D (CD), gangliosides, hyaluronic acid (HA), nitric oxide (NO), lipid peroxides (LPER), superoxide dismutase, and total thiols by chemical methods; and copper (Cu) by atomic absorption flame photometry. Advanced glycosylation end products and angiogenic factors (b-FGF, PD-ECGF, CD, gangliosides, HA, and Cu) were significantly higher in patients than controls. Oxidative stress markers, NO, and LPER were significantly higher while total thiols were significantly lower in patients than controls. These changes were more pronounced with age, poor glycemic control, and presence of microvascular complications. Angiogenesis dysfunction coinciding with elevated levels of many angiogenic growth factors may point to their malfunctioning due to oxidative stress and/or protein glycation at the factor and the receptor levels. This necessitates further investigations.     

Unconventional ligands and modulators of nicotinic receptors

Evidence gathered from epidemiologic and behavioral studies have indicated that neuronal nicotinic receptors (nAChRs) are intimately involved in the pathogenesis of a number of neurologic disorders, including Alzheimer's disease, Parkinson's disease, and schizophrenia. In the mammalian brain, neuronal nAChRs, in addition to mediating fast synaptic transmission, modulate fast synaptic transmission mediated by the major excitatory and inhibitory neurotransmitters glutamate and GABA, respectively. Of major interest, however, is the fact that the activity of the different subtypes of neuronal nAChR is also subject to modulation by substances of endogenous origin such as choline, the tryptophan metabolite kynurenic acid, neurosteroids, and beta-amyloid peptides and by exogenous substances, including the so-called nicotinic allosteric potentiating ligands, of which galantamine is the prototype, and psychotomimetic drugs such as phencyclidine and ketamine. The present article reviews and discusses the effects of unconventional ligands on nAChR activity and briefly describes the potential benefits of using some of these compounds in the treatment of neuropathologic conditions in which nAChR function/expression is known to be altered.     

Differential expression of the angiogenic Tie receptor family in arthritic and normal synovial tissue

Angiopoietins (Ang) are vascular endothelial cell-specific growth factors that play important roles principally during the later stages of angiogenesis. We have compared the distribution of the receptor tyrosine kinase (Tie) and the Ang ligands in synovial tissues from normal subjects and those with rheumatoid arthritis (RA) and osteoarthritis (OA).     

Integrin receptors: the dynamic modulators of endometrial function

Cell-cell and cell-extracellular matrix (ECM) interactions play a critical role in various developmental processes, including differentiation, proliferation and migration of cells. ECM proteins can influence cellular function thus creating a complex feedback mechanism. The adhesion of cells to each other, their ECM proteins and endothelial surfaces is mediated by a variety of membrane proteins collectively known as adhesion molecules. Adhesion molecules have been further divided into five subfamilies, the integrins, the selectins, the cadherins, the mucins and the immunoglobulin superfamily. Members of the integrin family of cell surface adhesion receptors are important mediators of cell-ECM contact. Integrin receptors are alpha beta heterodimers with a transmembrane segment, a short cytoplasmic domain and a large extracellular domain. The role of integrins in reproduction has been established. Several reasons make these molecules very attractive due to their constant involvement from egg to birth. They participate in sperm-egg interaction, fertilization, implantation and placentation in many species including humans. Integrins provide signals to individual cells essential for growth and development of different tissues. In the present review, we describe (1) the regulatory pathways for controlling expression of integrins in the endometrium, (2) various biomarkers and their role in endometrial function, (3) reproductive disorders in women related to aberrant integrin expression in the endometrium and (4) the functional significance of integrins available from gene knockout studies.     

The Angiopoietin ligands and Tie receptors: potential diagnostic biomarkers of vascular disease

Abstract

The Angiopoietin-1 (Angpt1)/Tie2 signaling pathway is important in regulating vascular function. Angpt1-induced Tie2 activation promotes vascular endothelial cell survival and reduces vascular leakage. Angiopoietin-2 (Angpt2), a weak agonist/antagonist of Tie2, opposes and regulates Angpt1 action. The Tie family of receptor tyrosine kinases, Tie2 and Tie1, exist as either homo-or heterodimers. The molecular complex between the receptors is also crucial in controlling Angpt1 signaling; hence, the molecular balance between Angpt1:Angpt2 and Tie2:Tie1 is important in determining endothelial integrity and vascular stability. This review presents evidence of the change observed in the Angiopoietin/Tie molecules in various pathophysiological conditions and discusses the potential clinical applications of these molecules in vascular complications.     

Decidual stromal cell response to paracrine signals from the trophoblast: amplification of immune and angiogenic modulators

During the invasive phase of implantation, trophoblasts and maternal decidual stromal cells secrete products that regulate trophoblast differentiation and migration into the maternal endometrium. Paracrine interactions between the extravillous trophoblast and the maternal decidua are important for successful embryonic implantation, including establishing the placental vasculature, anchoring the placenta to the uterine wall, and promoting the immunoacceptance of the fetal allograph. To our knowledge, global crosstalk between the trophoblast and the decidua has not been elucidated to date, and the present study used a functional genomics approach to investigate these paracrine interactions. Human endometrial stromal cells were decidualized with progesterone and further treated with conditioned media from human trophoblasts (TCM) or, as a control, with control conditioned media (CCM) from nondecidualized stromal cells for 0, 3, and 12 h. Total RNA was isolated and processed for analysis on whole-genome, high-density oligonucleotide arrays containing 54,600 genes. We found that 1374 genes were significantly upregulated and that 3443 genes were significantly downregulated after 12 h of coincubation of stromal cells with TCM, compared to CCM. Among the most upregulated genes were the chemokines CXCL1 (GRO1) and IL8,CXCR4, and other genes involved in the immune response (CCL8 [SCYA8], pentraxin 3 (PTX3), IL6, and interferon-regulated and -related genes) as well as TNFAIP6 (tumor necrosis factor alpha-induced protein 6) and metalloproteinases (MMP1, MMP10, and MMP14). Among the downregulated genes were growth factors, e.g., IGF1, FGF1, TGFB1, and angiopoietin-1, and genes involved in Wnt signaling (WNT4 and FZD). Real-time RT-PCR and ELISAs, as well as immunohistochemical analysis of human placental bed specimens, confirmed these data for representative genes of both up- and downregulated groups. The data demonstrate a significant induction of proinflammatory cytokines and chemokines, as well as angiogenic/static factors in decidualized endometrial stromal cells in response to trophoblast-secreted products. The data suggest that the trophoblast acts to alter the local immune environment of the decidua to facilitate the process of implantation and ensure an enriched cytokine/chemokine environment while limiting the mitotic activity of the stromal cells during the invasive phase of implantation.     

Toll-like receptor, RIG-I-like receptors and the NLRP3 inflammasome: key modulators of innate immune responses to double-stranded RNA viruses

Double-stranded RNA (dsRNA), the genetic material for many RNA viruses, induces robust host immune responses via pattern recognition receptors, which include Toll-like receptor 3 (TLR3), retinoic acid-inducible gene-I-like receptors (RLRs) and the multi-protein NLRP3 inflammasome complex. The engagement of dsRNA receptors or inflammasome activation by viral dsRNA initiates complex intracellular signaling cascades that play essential roles in inflammation and innate immune responses, as well as the resultant development of adaptive immunity. This review focuses on signaling pathways mediated by TLR3, RLRs and the NLRP3 inflammasome, as well as the potential use of agonists and antagonists that target these pathways to treat disease.     

Syntheses and optimization of new GS39783 analogues as positive allosteric modulators of GABA B receptors

The optimization of GS39783 into potent, selective, and safe positive allosteric modulators of GABA(B) receptors is presented.     

Fibroblast nemosis induces angiogenic responses of endothelial cells

Increasing evidence points to a central link between inflammation and activation of the stroma, especially of fibroblasts therein. However, the mechanisms leading to such activation mostly remain undescribed. We have previously characterized a novel type of fibroblast activation (nemosis) where clustered fibroblasts upregulated the production of cyclooxygenase-2, secretion of prostaglandins, proteinases, chemotactic cytokines, and hepatocyte growth factor (HGF), and displayed activated nuclear factor-κB. Now we show that nemosis drives angiogenic responses of endothelial cells. In addition to HGF, nemotic fibroblasts secreted vascular endothelial growth factor (VEGF), and conditioned medium from spheroids promoted sprouting and networking of human umbilical venous endothelial cells (HUVEC). The response was partly inhibited by function-blocking antibodies against HGF and VEGF. Conditioned nemotic fibroblast medium promoted closure of HUVEC and human dermal microvascular endothelial cell monolayer wounds, by increasing the motility of the endothelial cells. Wound closure in HUVEC cells was partly inhibited by the antibodies against HGF. The stromal microenvironment regulates wound healing responses and often promotes tumorigenesis. Nemosis offers clues to the activation process of stromal fibroblasts and provides a model to study the part they play in angiogenesis-related conditions, as well as possibilities for therapeutical approaches desiring angiogenesis in tissue.     

Netrin-4 regulates angiogenic responses and tumor cell growth

Netrin-4 is a 628 amino acid basement membrane component that promotes neurite elongation at low concentrations but inhibits neurite extension at high concentrations. There is a growing body of literature suggesting that several molecules, including netrins, are regulators of both neuronal and vascular growth. It is believed that molecules that guide neural growth and development are also involved in regulating morphogenesis of the vascular tree. Further, netrins have recently been implicated in controlling epithelial cell branching morphogenesis in the breast, lung and pancreas.Characterization of purified netrin-4 in in vitro angiogenesis assays demonstrated that netrin-4 markedly inhibits HMVEC migration and tube formation. Moreover, netrin-4 inhibits proliferation of a variety of human tumor cells in vitro. Netrin-4 has only modest effects on proliferation of endothelial and other non-transformed cells. Netrin-4 treatment results in phosphorylation changes of proteins that are known to control cell growth. Specifically, Phospho-Akt-1, Phospho-Jnk-2, and Phospho-c-Jun are reduced in tumor cells that have been treated with netrin-4. Together, these data suggest a potential role for netrin-4 in regulating tumor growth.     

Discovery of dual-action membrane-anchored modulators of incretin receptors

Background

The glucose-dependent insulinotropic polypeptide (GIP) and the glucagon-like peptide-1 (GLP-1) receptors are considered complementary therapeutic targets for type 2 diabetes. Using recombinant membrane-tethered ligand (MTL) technology, the present study focused on defining optimized modulators of these receptors, as well as exploring how local anchoring influences soluble peptide function.

Methodology/Principal Findings

Serial substitution of residue 7 in membrane-tethered GIP (tGIP) led to a wide range of activities at the GIP receptor, with [G⁷]tGIP showing enhanced efficacy compared to the wild type construct. In contrast, introduction of G⁷ into the related ligands, tGLP-1 and tethered exendin-4 (tEXE4), did not affect signaling at the cognate GLP-1 receptor. Both soluble and tethered GIP and GLP-1 were selective activators of their respective receptors. Although soluble EXE4 is highly selective for the GLP-1 receptor, unexpectedly, tethered EXE4 was found to be a potent activator of both the GLP-1 and GIP receptors. Diverging from the pharmacological properties of soluble and tethered GIP, the newly identified GIP-R agonists, (i.e. [G⁷]tGIP and tEXE4) failed to trigger cognate receptor endocytosis. In an attempt to recapitulate the dual agonism observed with tEXE4, we conjugated soluble EXE4 to a lipid moiety. Not only did this soluble peptide activate both the GLP-1 and GIP receptors but, when added to receptor expressing cells, the activity persists despite serial washes.

Conclusions

These findings suggest that conversion of a recombinant MTL to a soluble membrane anchored equivalent offers a means to prolong ligand function, as well as to design agonists that can simultaneously act on more than one therapeutic target.     

Class II cytokine receptors and their ligands: key antiviral and inflammatory modulators

Class II cytokine receptors were originally defined on the basis of sequence homologies in the extracellular domains of receptors for interferons (IFNs) and interleukin-10 (IL-10), and the ligands, known as class II cytokines, also have a common structure. More recently, a series of new receptors and cytokines that belong to this family have been discovered. The therapeutic potential of the 'old' members of this family, IFNs and IL-1, is recognized in the clinic, and the existence of structurally related molecules is raising expectations for additional clinical applications. In this review, I discuss both structural and biological data that are emerging about this family of receptors and ligands, to highlight the potential applications of modulating the activity of these cytokines.     

Allosteric modulators: the new generation of receptor antagonist

Allosteric antagonists modulate the affinity and/or efficacy of agonists for receptors. Although the manner in which this modulation can occur can mimic that of simple competitive antagonists, allosteric antagonists possess unique properties that can present seemingly capricious profiles of antagonism. These unique properties also offer potentially useful patterns for therapeutic utility. This review summarizes methods to detect allosteric antagonism and some special properties of these receptor modulators.     

Tie receptors and their angiopoietin ligands are context-dependent regulators of vascular remodeling

Angiopoietins are ligands of the Tie2 receptor that control angiogenic remodeling in a context-dependent manner. Tie signaling is involved in multiple steps of the angiogenic remodeling process during development, including destabilization of existing vessels, endothelial cell migration, tube formation and the subsequent stabilization of newly formed tubes by mesenchymal cells. Beyond this critical role in blood vessel development, recent studies suggest a wider role for Tie2 and angiopoietins in lymphangiogenesis and the development of the hematopoietic system, as well as a possible role in the regulation of certain non-endothelial cells. The outcome of Tie signaling depends on which vascular bed is involved, and crosstalk between different VEGFs has an important modulating effect on the properties of the angiopoietins. Signaling through the Tie1 receptor is not well understood, but Tie1 may have both angiopoietin-dependent and ligand-independent functions. Changes in the expression of Tie receptors and angiopoietins occur in many pathological conditions, and mutations in the Tie2 gene are found in familial cases of vascular disease.     

Akt is a major angiogenic mediator downstream of the Ang1/Tie2 signaling pathway

Tie2 and VEGF receptors (VEGFRs) are tyrosine kinases that play essential roles in angiogenesis. Activation of both receptors leads to the activation of Akt, an important mediator of cell survival and cell motility. In this study, we compared the role of Akt in Tie2-mediated versus VEGF-mediated endothelial cell (EC) survival and EC sprouting. Our data show that Akt is required and sufficient to mediate Ang1-induced EC survival in response to growth factor depletion. Blocking Akt function abolishes angiopoietin 1 (Ang1), a ligand for Tie2, mediated EC survival, and activating Akt rescues a Tie2 blockade-induced EC apoptosis. In contrast, activating Akt rescues EC apoptosis induced by a VEGF blockade, but interestingly, blocking Akt function has no effects on VEGF-induced EC survival, demonstrating that Akt is sufficient but not required for VEGF-mediated EC survival. In addition, we show that both Ang1 and VEGF induce EC sprouting in a three-dimensional collagen gel, which depends on the activation of Akt. Blocking Akt action inhibited EC sprouting induced by Ang1 or VEGF. Therefore, the data show that Akt is the primary mediator of Ang1-induced EC survival while multiple pathways are involved downstream of VEGF responsible for EC survival. However, Akt is required and sufficient to mediate the EC sprouting induced by both Ang1 and VEGF.     

Development and SAR of functionally selective allosteric modulators of GABAA receptors

Positive modulators at the benzodiazepine site of α2- and α3-containing GABA(A) receptors are believed to be anxiolytic. Through oocyte voltage clamp studies, we have discovered two series of compounds that are positive modulators at α2-/α3-containing GABA(A) receptors and that show no functional activity at α1-containing GABA(A) receptors. We report studies to improve this functional selectivity and ultimately deliver clinical candidates. The functional SAR of cinnolines and quinolines that are positive allosteric modulators of the α2- and α3-containing GABA(A) receptors, while simultaneously neutral antagonists at α1-containing GABA(A) receptors, is described. Such functionally selective modulators of GABA(A) receptors are expected to be useful in the treatment of anxiety and other psychiatric illnesses.     

The thyroid hormone receptors as modulators of skin proliferation and inflammation

We have analyzed the role of the thyroid hormone receptors (TRs) in epidermal homeostasis. Reduced keratinocyte proliferation is found in interfollicular epidermis of mice lacking the thyroid hormone binding isoforms TRα1 and TRβ (KO mice). Similar results were obtained in hypothyroid animals, showing the important role of the liganded TRs in epidermal proliferation. In addition, KO and hypothyroid animals display decreased hyperplasia in response to 12-O-tetradecanolyphorbol-13-acetate. Both receptor isoforms play overlapping functional roles in the skin because mice lacking individually TRα1 or TRβ also present a proliferative defect but not as marked as that found in double KO mice. Defective proliferation in KO mice is associated with reduction of cyclin D1 expression and up-regulation of the cyclin-dependent kinase inhibitors p19 and p27. Paradoxically, ERK and AKT activity and expression of downstream targets, such as AP-1 components, are increased in KO mice. Increased p65/NF-κB and STAT3 phosphorylation and, as a consequence, augmented expression of chemokines and proinflammatory cytokines is also found in these animals. These results show that thyroid hormones and their receptors are important mediators of skin proliferation and demonstrate that TRs act as endogenous inhibitors of skin inflammation, most likely due to interference with AP-1, NF-κB, and STAT3 activation.     

Cell adhesion receptors, tyrosine kinases and actin modulators: a complex three-way circuitry

The interaction of cells with surrounding matrix and neighbouring cells governs many aspects of cell behaviour. Aside from transmitting signals from the external environment, adhesion receptors also receive signals from the cell interior. Here we review the interrelationship between adhesion receptors, tyrosine kinases (both growth factor receptor and non-receptor) and modulators of the actin cytoskeletal network. Deregulation of many aspects of these signalling pathways in cancer highlights the need for a better understanding of the complexities involved.