IL-33 mast cell axis is central in LL-37 induced bladder inflammation and pain in a murine interstitial cystitis model.

Interstitial cystitis (IC), also known as painful bladder syndrome (PBS), is a debilitating chronic condition that afflicts over 3 million women above the age of 18 in the U.S., and most patients fail to respond to current treatment options. Mast cells have previously been implicated as both a diagnostic and prognostic marker in IC/PBS. Patients with IC/PBS have been shown to have elevated levels of IL-33, a cytokine released in response to tissue insult, in their urine. We hypothesize that mast cell-mediated inflammation induced from IL-33 may play an important role in initiating pain and inflammation in IC/PBS. A human cathelicidin, LL-37, which is found at elevated levels in IC/PBS patients, was used to induce an IC/PBS-like state of inflammation and bladder pain in mast cell deficient C-kit (-/-) and wild type C57Bl/6 (WT) mice. Inflammation was quantified using myeloperoxidase (MPO) expression in bladder tissues measured via ELISA. Response rate to suprapubic stimulation from von Frey filaments was used to assess the relative pain and discomfort. Both types of mice increased IL-33 expression in response to LL-37 exposure. However, mast cell deficient mice demonstrated significantly lower levels of inflammation (p < 0.001) and reduced pain response (p < 0.001) compared to WT mice. These findings implicate an IL-33-mast cell dependent axis with a potential etiology of pain and inflammation in IC/PBS. Future therapeutics aimed at targeting the IL-33 - mast cell axis could potentially serve as useful targets for treating IC/PBS.

The Life of Pi Star: Exploring the Exciting and Forbidden Worlds of the Benzophenone Photophore.

The widespread applications of benzophenone (BP) photochemistry in biological chemistry, bioorganic chemistry, and material science have been prominent in both academic and industrial research. BP photophores have unique photochemical properties: upon n-π* excitation at 365 nm, a biradicaloid triplet state is formed reversibly, which can abstract a hydrogen atom from accessible C-H bonds; the radicals subsequently recombine, creating a stable covalent C-C bond. This light-directed covalent attachment process is exploited in many different ways: (i) binding/contact site mapping of ligand (or protein)-protein interactions; (ii) identification of molecular targets and interactome mapping; (iii) proteome profiling; (iv) bioconjugation and site-directed modification of biopolymers; (v) surface grafting and immobilization. BP photochemistry also has many practical advantages, including low reactivity toward water, stability in ambient light, and the convenient excitation at 365 nm. In addition, several BP-containing building blocks and reagents are commercially available. In this review, we explore the "forbidden" (transitions) and excitation-activated world of photoinduced covalent attachment of BP photophores by touring a colorful palette of recent examples. In this exploration, we will see the pros and cons of using BP photophores, and we hope that both novice and expert photolabelers will enjoy and be inspired by the breadth and depth of possibilities.

LPA-producing enzyme PA-PLA1α regulates hair follicle development by modulating EGFR signalling.

Recent genetic studies of human hair disorders have suggested a critical role of lysophosphatidic acid (LPA) signalling in hair follicle development, mediated by an LPA-producing enzyme, phosphatidic acid-selective phospholipase A(1)α (PA-PLA(1)α, also known as LIPH), and a recently identified LPA receptor, P2Y5 (also known as LPA(6)). However, the underlying molecular mechanism is unknown. Here, we show that epidermal growth factor receptor (EGFR) signalling underlies LPA-induced hair follicle development. PA-PLA(1)α-deficient mice generated in this study exhibited wavy hairs due to the aberrant formation of the inner root sheath (IRS) in hair follicles, which resembled mutant mice defective in tumour necrosis factor α converting enzyme (TACE), transforming growth factor α (TGFα) and EGFR. PA-PLA(1)α was co-localized with TACE, TGFα and tyrosine-phosphorylated EGFR in the IRS. In PA-PLA(1)α-deficient hair follicles, cleaved TGFα and tyrosine-phosphorylated EGFR, as well as LPA, were significantly reduced. LPA, P2Y5 agonists and recombinant PA-PLA(1)α enzyme induced P2Y5- and TACE-mediated ectodomain shedding of TGFα through G12/13 pathway and consequent EGFR transactivation in vitro. These data demonstrate that a PA-PLA(1)α-LPA-P2Y5 axis regulates differentiation and maturation of hair follicles via a TACE-TGFα-EGFR pathway, thus underscoring the physiological importance of LPA-induced EGFR transactivation.

Vinyl sulfone analogs of lysophosphatidylcholine irreversibly inhibit autotaxin and prevent angiogenesis in melanoma.

Autotaxin (ATX) is an enzyme discovered in the conditioned medium of cultured melanoma cells and identified as a protein that strongly stimulates motility. This unique ectonucleotide pyrophosphatase and phosphodiesterase facilitates the removal of a choline headgroup from lysophosphatidylcholine (LPC) to yield lysophosphatidic acid (LPA), which is a potent lipid stimulator of tumorigenesis. Thus, ATX has received renewed attention because it has a prominent role in malignant progression with significant translational potential. Specifically, we sought to develop active site-targeted irreversible inhibitors as anti-cancer agents. Herein we describe the synthesis and biological activity of an LPC-mimetic electrophilic affinity label that targets the active site of ATX, which has a critical threonine residue that acts as a nucleophile in the lysophospholipase D reaction to liberate choline. We synthesized a set of quaternary ammonium derivative-containing vinyl sulfone analogs of LPC that function as irreversible inhibitors of ATX and inactivate the enzyme. The analogs were tested in cell viability assays using multiple cancer cell lines. The IC50 values ranged from 6.74 to 0.39 µM, consistent with a Ki of 3.50 µM for inhibition of ATX by the C16H33 vinyl sulfone analog CVS-16 (10b). A phenyl vinyl sulfone control compound, PVS-16, lacking the choline-like quaternary ammonium mimicking head group moiety, had little effect on cell viability and did not inhibit ATX. Most importantly, CVS-16 (10b) significantly inhibited melanoma progression in an in vivo tumor model by preventing angiogenesis. Taken together, this suggests that CVS-16 (10b) is a potent and irreversible ATX inhibitor with significant biological activity both in vitro and in vivo.

Autotaxin production of lysophosphatidic acid mediates allergic asthmatic inflammation.

RATIONALE: Bioactive lipid mediators, derived from membrane lipid precursors, are released into the airway and airspace where they bind high-affinity cognate receptors and may mediate asthma pathogenesis. Lysophosphatidic acid (LPA), a bioactive lipid mediator generated by the enzymatic activity of extracellular autotaxin (ATX), binds LPA receptors, resulting in an array of biological actions on cell proliferation, migration, survival, differentiation, and motility, and therefore could mediate asthma pathogenesis. OBJECTIVES: To define a role for the ATX-LPA pathway in human asthma pathogenesis and a murine model of allergic lung inflammation. METHODS: We investigated the profiles of LPA molecular species and the level of ATX exoenzyme in bronchoalveolar lavage fluids of human patients with asthma subjected to subsegmental bronchoprovocation with allergen. We interrogated the role of the ATX-LPA pathway in allergic lung inflammation using a murine allergic asthma model in ATX-LPA pathway-specific genetically modified mice. MEASUREMENTS AND MAIN RESULTS: Subsegmental bronchoprovocation with allergen in patients with mild asthma resulted in a remarkable increase in bronchoalveolar lavage fluid levels of LPA enriched in polyunsaturated 22:5 and 22:6 fatty acids in association with increased concentrations of ATX protein. Using a triple-allergen mouse asthma model, we showed that ATX-overexpressing transgenic mice had a more severe asthmatic phenotype, whereas blocking ATX activity and knockdown of the LPA2 receptor in mice produced a marked attenuation of Th2 cytokines and allergic lung inflammation. CONCLUSIONS: The ATX-LPA pathway plays a critical role in the pathogenesis of asthma. These preclinical data indicate that targeting the ATX-LPA pathway could be an effective antiasthma treatment strategy.

Exploiting differential surface display of chondroitin sulfate variants for directing neuronal outgrowth.

Chondroitin sulfate (CS) proteoglycans (CSPGs) are known to be primary inhibitors of neuronal regeneration at scar sites. However, a variety of CSPGs are also involved in neuronal growth and guidance during other physiological stages. Sulfation patterns of CS chains influence their interactions with various growth factors in the central nervous system (CNS), thus influencing neuronal growth, inhibition, and pathfinding. This report demonstrates the use of differentially sulfated CS chains for neuronal navigation. Surface-immobilized patterns of CS glycosaminoglycan chains were used to determine neuronal preference toward specific sulfations of five CS variants: CS-A, CS-B (dermatan sulfate), CS-C, CS-D, and CS-E. Neurons preferred CS-A, CS-B, and CS-E and avoided CS-C containing lanes. In addition, significant alignment of neurites was observed using underlying lanes containing CS-A, CS-B, and CS-E chains. To utilize differential preference of neurons toward the CS variants, a binary combinations of CS chains were created by backfilling a neuro-preferred CS variant between the microcontact printed lanes of CS-C stripes, which are avoided by neurons. The neuronal outgrowth results demonstrate for the first time that a combination of sulfation variants of CS chains without any protein component of CSPG is sufficient for directing neuronal outgrowth. Biomaterials with surface immobilized GAG chains could find numerous applications as bridging devices for tackling CNS injuries where directional growth of neurons is critical for recovery.

Physiological relevance of LL-37 induced bladder inflammation and mast cells.

PURPOSE: We established the physiological relevance of LL-37 induced bladder inflammation. We hypothesized that 1) human urinary LL-37 is increased in pediatric patients with spina bifida, 2) LL-37 induced inflammation occurs in our mouse model via urothelial binding and is dose dependent and 3) LL-37 induced inflammation involves mast cells. MATERIALS AND METHODS: To test our first hypothesis, we obtained urine samples from 56 pediatric patients with spina bifida and 22 normal patients. LL-37 was measured by enzyme-linked immunosorbent assay. Our second hypothesis was tested in C57Bl/6 mice challenged with 7 LL-37 concentrations intravesically for 1 hour. At 24 hours tissues were examined histologically and myeloperoxidase assay was done to quantitate inflammation. In separate experiments fluorescent LL-37 was instilled and tissues were obtained immediately (time = 0) and at 24 hours (time = 24). To test our final hypothesis, we performed immunohistochemistry for mast cell tryptase and evaluated 5 high power fields per bladder to determine the mean number of mast cells per mm(2). RESULTS: Urinary LL-37 was 89-fold higher in patients with spina bifida. Mouse LL-37 dose escalation experiments revealed increased inflammation at higher LL-37 concentrations. Fluorescent LL-37 demonstrated global urothelial binding at time = 0 but was not visible at time = 24. Immunohistochemistry for tryptase revealed mast cell infiltration in all tissue layers. At higher concentrations the LL-37 challenge led to significantly greater mast cell infiltration. CONCLUSIONS: Urinary LL-37 was significantly increased in pediatric patients with spina bifida. To our knowledge we report for the first time that LL-37 can elicit profound, dose dependent bladder inflammation involving the urothelium. Finally, inflammation propagation involves mast cells.

Phosphorothioate analogs of sn-2 radyl lysophosphatidic acid (LPA): metabolically stabilized LPA receptor agonists.

We describe an efficient synthesis of metabolically stabilized sn-2 radyl phosphorothioate analogs of lysophosphatidic acid (LPA), and the determination of the agonist activity of each analog for the six LPA receptors (LPA1-6) using a recently developed TGFα shedding assay. In general, the sn-2 radyl OMPT analogs showed similar agonist activities to the previous 1-oleoyl-2-O-methyl-glycerophosphothioate (sn-1 OMPT) analogs for LPA1-6 receptors. In most cases, the sn-2 radyl-OMPT analogs were more potent agonists than LPA itself. Most importantly, sn-2 alkyl OMPT analogs were very potent LPA5 and LPA6 agonists. The availability of sn-2 radyl OPMT analogs further refines the structure-activity relationships for ligand-receptor interactions for this class of GPCRs.

Measuring translational research impact requires reaching beyond current metrics.

Translational impact assessment is key to selecting those biomedical research discoveries most likely to be converted into viable new products to improve human health. However, metrics for translational success are variable, are not limited to commercial success, and may not be relevant to every case or institution. Societal impact is a top translational priority in a globalized society.

Pulmonary autotaxin expression contributes to the pathogenesis of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic form of diffuse lung disease occurring mainly in older adults. Increased lysophosphatidic acid (LPA) concentrations have been reported in the alveolar space of both idiopathic pulmonary fibrosis patients and a corresponding animal model, whereas the genetic deletion or pharmacological inhibition of LPA receptor 1 attenuated the development of the modeled disease, suggesting a direct involvement of LPA in disease pathogenesis. In this report, increased concentrations of autotaxin (ATX; ENPP2), the enzyme largely responsible for extracellular LPA production, were detected in both murine and human fibrotic lungs. The genetic deletion of ATX from bronchial epithelial cells or macrophages attenuated disease severity, establishing ATX as a novel player in IPF pathogenesis. Furthermore, the pharmacological inhibition of ATX attenuated the development of the modeled disease, suggesting that ATX is a possible therapeutic target in IPF.

Bis (monoacylglycero) phosphate interfacial properties and lipolysis by pancreatic lipase-related protein 2, an enzyme present in THP-1 human monocytes.

The interfacial physical properties of bis(monoacylglycero)phosphate (BMP) and its derivatives with three oleoyl chains (hemi-BDP) and four oleoyl chains (bis(diacylglycero)phosphate, BDP) were investigated using Langmuir monomolecular films. The mean molecular area of BMP at the collapse surface pressure (45mN m(-1)) was similar to those measured with other phospholipids bearing two acyl chains (66 and 59.6Å(2) molecule(-1) at pH 5.5 and 8.0, respectively). In Hemi-BDP and BDP, the mean molecular area increased by 26 and 35Å(2) molecule(-1) per additional acyl chain at pH 5.5 and 8.0, respectively. When BMP was added to a phospholipid mixture mimicking late endosome membrane composition at pH 8.0, the mean phospholipid molecular area increased by 7% regardless of the surface pressure. In contrast, the variation in molecular area was surface pressure-dependent at pH 5.5, a pH value close to that of intra-endosomal content. BMP and hemi-BDP, but not BDP, were hydrolyzed by pancreatic lipase-related protein 2 (PLRP2), which exhibits phospholipase A(1) activity. At pH 5.5, the maximum activities of PLRP2 on BMP were recorded at high surface pressures (25-35mN/m). At pH 8.0, the PLRP2 activity vs. surface pressure showed a bell-shaped curve with maximum activities at 15mN/m for both BMP and hemi-BDP. This is a new activity for this enzyme which could degrade cellular BMP since both human PLRP2 (HPLRP2) and BMP were localized in human monocytic THP-1 cells. This is the first report on the cellular localization of HPLRP2 in human monocytes.

Regulation of lung injury and repair by Toll-like receptors and hyaluronan.

Mechanisms that regulate inflammation and repair after acute lung injury are incompletely understood. The extracellular matrix glycosaminoglycan hyaluronan is produced after tissue injury and impaired clearance results in unremitting inflammation. Here we report that hyaluronan degradation products require MyD88 and both Toll-like receptor (TLR)4 and TLR2 in vitro and in vivo to initiate inflammatory responses in acute lung injury. Hyaluronan fragments isolated from serum of individuals with acute lung injury stimulated macrophage chemokine production in a TLR4- and TLR2-dependent manner. Myd88(-/-) and Tlr4(-/-)Tlr2(-/-) mice showed impaired transepithelial migration of inflammatory cells but decreased survival and enhanced epithelial cell apoptosis after lung injury. Lung epithelial cell-specific overexpression of high-molecular-mass hyaluronan was protective against acute lung injury. Furthermore, epithelial cell-surface hyaluronan was protective against apoptosis, in part, through TLR-dependent basal activation of NF-kappaB. Hyaluronan-TLR2 and hyaluronan-TLR4 interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity and promote recovery from acute lung injury.

A murine model of inflammatory bladder disease: cathelicidin peptide induced bladder inflammation and treatment with sulfated polysaccharides.

PURPOSE: Studies show that LL-37 is a naturally occurring urinary defensin peptide that is up-regulated during urinary tract infections. Although normal urinary LL-37 levels are antimicrobial, we propose that increased LL-37 may trigger bladder inflammation. We further suggest that anti-inflammatory sulfated polysaccharides known as semi-synthetic glycosaminoglycan ether compounds can treat/prevent LL-37 mediated bladder inflammation. MATERIALS AND METHODS: C57BL/6 mice were catheterized/instilled with LL-37 (320 µM, 150 µl) for 45 minutes. Animals were sacrificed at 12 and 24 hours, and tissues were examined using hematoxylin and eosin. Separate experiments were performed for myeloperoxidase to quantify inflammation. GM-1111 semi-synthetic glycosaminoglycan ether treatments involved instillation of 10 mg/ml for 45 minutes directly before or after LL-37. Tissues were harvested at 24 hours. To compare semi-synthetic glycosaminoglycan ether efficacy, experiments were performed using 10 mg/ml heparin. Finally, tissue localization of semi-synthetic glycosaminoglycan ether was examined using a fluorescent GM-1111-Alexa Fluor® 633 conjugate. RESULTS: Profound bladder inflammation developed after LL-37. Greater tissue inflammation occurred after 24 hours compared to that at 12 hours. Myeloperoxidase assays revealed a 21 and 61-fold increase at 12 and 24 hours, respectively. Semi-synthetic glycosaminoglycan ether treatment after LL-37 showed mild attenuation of inflammation with myeloperoxidase 2.5-fold below that of untreated bladders. Semi-synthetic glycosaminoglycan ether treatment before LL-37 demonstrated almost complete attenuation of inflammation. Myeloperoxidase results mirrored those in controls. In heparin treated bladders minimal attenuation of inflammation occurred. Finally, instillation of GM-1111-Alexa Fluor 633 revealed urothelial coating, significant tissue penetration and binding to endovasculature. CONCLUSIONS: We developed what is to our knowledge a new model of inflammatory bladder disease by challenge with the naturally occurring urinary peptide LL-37. We also noted that a new class of anti-inflammatory sulfated polysaccharides prevents and mitigates bladder inflammation.

Characterization of sphingosine-1-phosphate lyase activity by electrospray ionization-liquid chromatography/tandem mass spectrometry quantitation of (2E)-hexadecenal.

Sphingosine-1-phosphate (S1P) is a sphingolipid signaling molecule crucial for cell survival and proliferation. S1P-mediated signaling is largely controlled through its biosynthesis and degradation, and S1P lyase (S1PL) is the only known enzyme that irreversibly degrades sphingoid base-1-phosphates to phosphoethanolamine and the corresponding fatty aldehydes. S1PL-mediated degradation of S1P results in the formation of (2E)-hexadecenal, whereas hexadecanal is the product of dihydrosphingosine-1-phosphate (DHS1P) degradation. Fatty aldehydes can undergo biotransformation to fatty acids and/or alcohols, making them elusive and rendering the task of fatty aldehyde quantitation challenging. We have developed a simple, highly sensitive, and high-throughput protocol for (2E)-hexadecenal quantitation as a semicarbazone derivative by liquid chromatography-electrospray ionization-tandem mass spectrometry. The approach was applied to determining S1PL activity in vitro with the ability to use as low as 0.25µg of microsomal protein per assay. The method is also applicable to the use of total tissue homogenate as the source of S1PL. A correction for (2E)-hexadecenal disappearance due to its biotransformation during enzymatic reaction is required, especially at higher protein concentrations. The method was applied to confirm FTY720 as the inhibitor of S1PL with an IC50 value of 52.4µM.

A PtdInsP(3)- and Rho GTPase-mediated positive feedback loop regulates neutrophil polarity.

When presented with a gradient of chemoattractant, many eukaryotic cells respond with polarized accumulation of the phospholipid PtdIns(3,4,5)P(3). This lipid asymmetry is one of the earliest readouts of polarity in neutrophils, Dictyostelium discoideum and fibroblasts. However, the mechanisms that regulate PtdInsP(3) polarization are not well understood. Using a cationic lipid shuttling system, we have delivered exogenous PtdInsP(3) to neutrophils. Exogenous PtdInsP(3) elicits accumulation of endogenous PtdInsP(3) in a positive feedback loop that requires endogenous phosphatidylinositol-3-OH kinases (PI(3)Ks) and Rho family GTPases. This feedback loop is important for establishing PtdInsP(3) polarity in response to both chemoattractant and to exogenous PtdInsP(3); it may function through a self-organizing pattern formation system. Emergent properties of positive and negative regulatory links between PtdInsP(3) and Rho family GTPases may constitute a broadly conserved module for the establishment of cell polarity during eukaryotic chemotaxis.

Aromatic phosphonates inhibit the lysophospholipase D activity of autotaxin.

Autotaxin (ATX) is an attractive target for the anticancer therapeutics that inhibits angiogenesis, invasion and migration. ATX is an extracellular lysophospholipase D that hydrolyzes lysophosphatidylcholine to form the bioactive lipid lysophosphatidic acid. The aromatic phosphonate S32826 was the first described nanomolar inhibitor of ATX. However, the tridecylamide substituent on aromatic ring contributed to its poor solubility and bioavailability, severely limiting its utility in vivo. cLogP calculations revealed that the lipophilicity of S32826 could be lowered by shortening its hydrophobic chain and by introducing substituents alpha to the phosphonate. Herein, we describe the synthesis of a small set of α-substituted phosphonate analogs of S32826, and we show that shortening the chain and adding α-halo or α-hydroxy substituents increased solubility; however, ATX inhibition was reduced by most substitutions. An optimal compound was identified for examination of biological effects of ATX inhibition in vivo.

Low anticoagulant heparin targets multiple sites of inflammation, suppresses heparin-induced thrombocytopenia, and inhibits interaction of RAGE with its ligands.

While heparin has been used almost exclusively as a blood anticoagulant, important literature demonstrates that it also has broad anti-inflammatory activity. Herein, using low anti-coagulant 2-O,3-O-desulfated heparin (ODSH), we demonstrate that most of the anti-inflammatory pharmacology of heparin is unrelated to anticoagulant activity. ODSH has low affinity for anti-thrombin III, low anti-Xa, and anti-IIa anticoagulant activities and does not activate Hageman factor (factor XII). Unlike heparin, ODSH does not interact with heparin-platelet factor-4 antibodies present in patients with heparin-induced thrombocytopenia and even suppresses platelet activation in the presence of activating concentrations of heparin. Like heparin, ODSH inhibits complement activation, binding to the leukocyte adhesion molecule P-selectin, and the leukocyte cationic granular proteins azurocidin, human leukocyte elastase, and cathepsin G. In addition, ODSH and heparin disrupt Mac-1 (CD11b/CD18)-mediated leukocyte adhesion to the receptor for advanced glycation end products (RAGE) and inhibit ligation of RAGE by its many proinflammatory ligands, including the advanced glycation end-product carboxymethyl lysine-bovine serum albumin, the nuclear protein high mobility group box protein-1 (HMGB-1), and S100 calgranulins. In mice, ODSH is more effective than heparin in reducing selectin-mediated lung metastasis from melanoma and inhibits RAGE-mediated airway inflammation from intratracheal HMGB-1. In humans, 50% inhibitory concentrations of ODSH for these anti-inflammatory activities can be achieved in the blood without anticoagulation. These results demonstrate that the anticoagulant activity of heparin is distinct from its anti-inflammatory actions and indicate that 2-O and 3-O sulfate groups can be removed to reduce anticoagulant activity of heparin without impairing its anti-inflammatory pharmacology.

Targeting melanoma growth and viability reveals dualistic functionality of the phosphonothionate analogue of carba cyclic phosphatidic acid.

BACKGROUND: Although the incidence of melanoma in the U.S. is rising faster than any other cancer, the FDA-approved chemotherapies lack efficacy for advanced disease, which results in poor overall survival. Lysophosphatidic acid (LPA), autotaxin (ATX), the enzyme that produces LPA, and the LPA receptors represent an emerging group of therapeutic targets in cancer, although it is not known which of these is most effective. RESULTS: Herein we demonstrate that thio-ccPA 18:1, a stabilized phosphonothionate analogue of carba cyclic phosphatidic acid, ATX inhibitor and LPA1/3 receptor antagonist, induced a marked reduction in the viability of B16F10 metastatic melanoma cells compared with PBS-treated control by 80-100%. Exogenous LPA 18:1 or D-sn-1-O-oleoyl-2-O-methylglyceryl-3-phosphothioate did not reverse the effect of thio-ccPA 18:1. The reduction in viability mediated by thio-ccPA 18:1 was also observed in A375 and MeWo melanoma cell lines, suggesting that the effects are generalizable. Interestingly, siRNA to LPA3 (siLPA3) but not other LPA receptors recapitulated the effects of thio-ccPA 18:1 on viability, suggesting that inhibition of the LPA3 receptor is an important dualistic function of the compound. In addition, siLPA3 reduced proliferation, plasma membrane integrity and altered morphology of A375 cells. Another experimental compound designed to antagonize the LPA1/3 receptors significantly reduced viability in MeWo cells, which predominantly express the LPA3 receptor. CONCLUSIONS: Thus the ability of thio-ccPA 18:1 to inhibit the LPA3 receptor and ATX are key to its molecular mechanism, particularly in melanoma cells that predominantly express the LPA3 receptor. These observations necessitate further exploration and exploitation of these targets in melanoma.

Lysophosphatidic acid induces neointima formation through PPARgamma activation.

Neointimal lesions are characterized by accumulation of cells within the arterial wall and are a prelude to atherosclerotic disease. Here we report that a brief exposure to either alkyl ether analogs of the growth factor-like phospholipid lysophosphatidic acid (LPA), products generated during the oxidative modification of low density lipoprotein, or to unsaturated acyl forms of LPA induce progressive formation of neointima in vivo in a rat carotid artery model. This effect is completely inhibited by the peroxisome proliferator-activated receptor (PPAR)gamma antagonist GW9662 and mimicked by PPARgamma agonists Rosiglitazone and 1-O-hexadecyl-2-azeleoyl-phosphatidylcholine. In contrast, stearoyl-oxovaleryl phosphatidylcholine, a PPARalpha agonist and polypeptide epidermal growth factor, platelet-derived growth factor, and vascular endothelial growth factor failed to elicit neointima. The structure-activity relationship for neointima induction by LPA analogs in vivo is identical to that of PPARgamma activation in vitro and disparate from that of LPA G protein-coupled receptor activation. Neointima-inducing LPA analogs up-regulated the CD36 scavenger receptor in vitro and in vivo and elicited dedifferentiation of cultured vascular smooth muscle cells that was prevented by GW9662. These results suggest that selected LPA analogs are important novel endogenous PPARgamma ligands capable of mediating vascular remodeling and that activation of the nuclear transcription factor PPARgamma is both necessary and sufficient for neointima formation by components of oxidized low density lipoprotein.

5-Stabilized phosphatidylinositol 3,4,5-trisphosphate analogues bind Grp1 PH, inhibit phosphoinositide phosphatases, and block neutrophil migration.

Metabolically stabilized analogues of PtdIns(3,4,5)P3 have shown long-lived agonist activity for cellular events and selective inhibition of lipid phosphatase activity. We describe an efficient asymmetric synthesis of two 5-phosphatase-resistant analogues of PtdIns(3,4,5)P3, the 5-methylene phosphonate (MP) and 5-phosphorothioate (PT). Furthermore, we illustrate the biochemical and biological activities of five stabilized PtdIns(3,4,5)P3 analogues in four contexts. First, the relative binding affinities of the 3-MP, 3-PT, 5-MP, 5-PT, and 3,4,5-PT3 analogues to the Grp1 PH domain are shown, as determined by NMR spectroscopy. Second, the enzymology of the five analogues is explored, showing the relative efficiency of inhibition of SHIP1, SHIP2, and phosphatase and tensin homologue deleted on chromosome 10 (PTEN), as well as the greatly reduced ability of these phosphatases to process these analogues as substrates as compared to PtdIns(3,4,5)P3. Third, exogenously delivered analogues severely impair complement factor C5a-mediated polarization and migration of murine neutrophils. Finally, the new analogues show long-lived agonist activity in mimicking insulin action in sodium transport in A6 cells.