Enhanced angiogenic function in response to fibroblasts from psoriatic arthritis synovium compared to rheumatoid arthritis.

INTRODUCTION: Angiogenesis is an early event in the pathogenesis of both psoriatic arthritis (PsA) and rheumatoid arthritis (RA); however, there are striking differences in blood vessel morphology and activation between the two arthropathies. The aim of this study was to assess if the PsA and RA joint microenvironments differentially regulate endothelial cell function. METHODS: PsA and RA primary synovial fibroblasts (SFC) were isolated from synovial biopsies, grown to confluence, and supernatants harvested and termed 'conditioned media' (CM). Human umbilical vein endothelial cells (HUVEC) were cultured with PsA SFC or RA SFC-CM (20%). HUVEC tube formation, migration, and PBMC adhesion were assessed by matrigel tube formation, wound repair, and PBMC adhesion assays. HUVEC cell surface expression of ICAM, VCAM, and E-Selectin was assessed by flow cytometry. Transcriptome analysis of genes promoting angiogenesis was performed by real-time PCR. Finally, a MSD multiplex angiogenic assay was performed on PsA SFC and RA SFC supernatants. RESULTS: Macroscopic synovitis and vascularity were similar in PsA and RA patients; however, significant differences in vascular morphological pattern were recorded with tortuous, elongated vessels observed in PsA compared to straight regular branching vessels observed in RA. Transcriptome analysis showed strong upregulation of the pro-angiogenic signature in HUVEC primed with PsA SFC-CM compared to RA SFC-CM and basal control. In parallel, paired PsA SFC-CM significantly induced HUVEC tube formation compared to that of RA SFC-CM. Furthermore, PsA SFC-CM induced HUVEC migration was paralleled by a significant induction in VEGFA, PFKFB3, ICAM-1, and MMP3 mRNA expression. A significant increase in PBMC adhesion and cell surface expression of VCAM-1, ICAM-1, and E-Selectin expression was also demonstrated in PsA SFC-CM-primed HUVEC compared to RA SFC-CM. Finally, VEGF, TSLP, Flt-1, and Tie-2 expression was elevated in PsA SFC-CM compared to RA SFC-CM, with no significant difference in other pro-angiogenic mediators including MIP-3, bFGF, PIGF, and MCP-1. CONCLUSION: PsA SFC and RA SFC secreted factors differentially regulate endothelial cell function, with soluble mediators in the PsA joint microenvironment inducing a more pro-angiogenic phenotype compared to the RA.

Altered metabolic pathways regulate synovial inflammation in rheumatoid arthritis.

Rheumatoid arthritis is characterized by synovial proliferation, neovascularization and leucocyte extravasation leading to joint destruction and functional disability. The blood vessels in the inflamed synovium are highly dysregulated, resulting in poor delivery of oxygen; this, along with the increased metabolic demand of infiltrating immune cells and inflamed resident cells, results in the lack of key nutrients at the site of inflammation. In these adverse conditions synovial cells must adapt to generate sufficient energy to support their proliferation and activation status, and thus switch their cell metabolism from a resting regulatory state to a highly metabolically active state. This alters redox-sensitive signalling pathways and also results in the accumulation of metabolic intermediates which, in turn, can act as signalling molecules that further exacerbate the inflammatory response. The RA synovium is a multi-cellular tissue, and while many cell types interact to promote the inflammatory response, their metabolic requirements differ. Thus, understanding the complex interplay between hypoxia-induced signalling pathways, metabolic pathways and the inflammatory response will provide better insight into the underlying mechanisms of disease pathogenesis.

Inverse agonist activity at the alpha(2A)-adrenergic receptor.

Constitutive activation of G protein-coupled receptors (GPCRs) is now well recognized and many classical GPCR antagonists have been found to be inverse agonists. For the alpha(2A)-adrenergic receptor (AR) we determine the relative inverse efficacies of a series of antagonists and utilize the extended ternary complex model to estimate the fraction of constitutively active mutant (CAM) receptors in the active state. Stable Chinese hamster ovary cell lines expressing the porcine alpha(2A)-AR in its wild-type (WT) and constitutively activated (CAM-T373K) form were isolated. Activation of both G(i) and G(s) was enhanced for CAM receptors. cAMP production was suppressed in cells with the CAM alpha(2A)-AR and this suppression was reversed by alpha(2)-adrenergic antagonists with an order of inverse efficacy of rauwolscine > yohimbine > RX821002 > MK912, whereas phentolamine and idazoxan were essentially neutral antagonists. This striking difference in inverse efficacy between idazoxan and RX821002 may account for in vivo pharmacological differences between these two alpha(2)-adrenergic antagonists. Agonist binding affinity to the non-G protein-coupled CAM receptor was 3- to 9-fold higher than to WT, whereas binding of the most efficacious inverse agonists, yohimbine and rauwolscine, was 1.7- and 2.1-fold weaker. Analysis of this difference by the extended ternary complex model indicates that approximately 50% of the CAM alpha(2A)-AR is in the active (R*) state although there is no detectable constitutive activity of the WT receptor in the absence of agonist.

Agonist-directed trafficking of porcine alpha(2A)-adrenergic receptor signaling in Chinese hamster ovary cells: l-isoproterenol selectively activates G(s).

In this study, we investigated the hypothesis of agonist-directed trafficking of receptor signaling for the alpha(2A)-adrenergic receptor (alpha(2A)-AR). alpha(2A)-ARs couple to both G(s) and G(i) to stimulate or inhibit adenylyl cyclase activity. Chinese hamster ovary-K1 cell lines expressing the porcine alpha(2A)-AR at high (alpha(2A)-H) and low (alpha(2A)-L) levels were used to estimate the relative efficacies (R.e.s) of a series of agonists for the G(s) and G(i) pathways. G(s)-mediated responses were measured after pertussis toxin treatment to inactivate G(i) in alpha(2A)-H, whereas G(i) responses were measured in alpha(2A)-L, where G(s) responses were absent. The full agonist UK-14,304 showed a large receptor reserve for G(i) responses in alpha(2A)-H but little receptor reserve for G(s) responses in alpha(2A)-H or for G(i) responses in alpha(2A)-L. With the exception of l-isoproterenol (ISO), all agonists showed similar R.e.s at the alpha(2A)-AR for G(s) and G(i) responses, with rank orders of R.e.s as follows: l-epinephrine = l-norepinephrine = UK-14,304 > p-aminoclonidine > or = BHT-920 > or = BHT-933 > clonidine = p-iodoclonidine > or = xylazine > or = guanabenz. Interestingly, ISO had the highest efficacy at the alpha(2A)-AR for activating G(s) versus G(i) (9-fold higher); however, it had low potency for both. By several criteria, the ISO response was mediated by the alpha(2A)-AR, supporting the hypothesis of agonist-directed trafficking of receptor signaling or agonist-specific G protein selectivity. In contrast, the apparent G(i) pathway selectivity of oxymetazoline appears to be mediated by an endogenous serotonergic receptor. It is intriguing that a classic beta-AR agonist that activates G(s) through beta(2)-ARs also appears to produce a G(s)-selective conformation of the G(i)-coupled alpha(2A)-AR.

G(i) activator region of alpha(2A)-adrenergic receptors: distinct basic residues mediate G(i) versus G(s) activation.

The structural determinants of G protein coupling versus activation by G protein-coupled receptors are not well understood. We examine the role of two distinct basic regions in the carboxyl terminal portion of the third intracellular loop of the alpha(2A)-adrenergic receptor to dissect these aspects of function. Changing three arginines to alanines by mutagenesis and stable expression in Chinese hamster ovary-K1 cells impaired the alpha(2)-adrenergic receptor G(s)-mediated stimulation of cyclic AMP (cAMP) accumulation, whereas G(i)-mediated inhibition was normal. When two (B2) or three (B3) basic residues closer to transmembrane span 6 were mutated to alanine, normal ligand binding was observed, but G(i)-mediated inhibition of cAMP accumulation showed 20-fold and 50-fold decreases in agonist potency for the B2 and B3 mutants, respectively. Surprisingly, a normal G(s) response was seen for the B2 mutant, and the B3 mutant showed only a 6-fold decrease in agonist potency. Mutation of both the three alanines and B3 residues to alanines showed a 200-fold decrease in agonist potency for G(i)-mediated inhibition of cAMP accumulation, whereas the G(s) response was nearly completely eliminated. The three basic residues (which include the BB of the BBXXF motif) play a role as G(i) activators rather than in receptor-G protein coupling, because high-affinity agonist binding is intact. Thus, we have identified three basic residues required for activation of G(i) but not required for receptor-G protein coupling. Also, distinct basic residues are required for optimal G(i) and G(s) responses, defining a microspecificity determinant within the carboxyl terminal portion of the third intracellular loop of the alpha(2a) adrenergic receptor.

Cotransfection of second and third intracellular loop fragments inhibit angiotensin AT1a receptor activation of phospholipase C in HEK-293 cells.

Peptides from the intracellular regions of G protein-coupled receptors are useful probes of receptor-G protein coupling mechanisms. As a first step toward the genetic delivery of such "G protein inhibitors," we describe inhibition of angiotensin II (AII) receptor responses by expressed fragments of the second and third intracellular loops of the AT1a receptor (AT1a/i2 and AT1a/i3). Transient transfection of human embryonic kidney 293 cells with DNA encoding the rat AT1a receptor resulted in AII-dependent increases of inositol phosphates (maximum 4.5-fold). Cotransfection of AT1a/i2 and AT1a/i3 fragments raised the EC50 for AII stimulation of phospholipase C activity 5-fold (from 0.18 nM to 0.99 nM, n = 12, P < .001) and 3-fold (from 0.38 nM to 1.2 nM, n = 8, P < .002), respectively. The combined effect of AT1a/i2 and AT1a/3 was additive, and transfection of an alpha-1b adrenergic receptor third intracellular loop (alpha1b/i3) fragments also increased the EC50 for AII. Neither AT1a/i1 nor C-terminus (AT1a/Ct) constructs had significant effects on angiotensin responses. These data confirm a role for the second and third intracellular loops in angiotensin receptor responses and show the potential of this approach to blocking multiple phospholipase C-linked receptors.

Modulation of K+ and Ca2+ currents in cultured neurons by an angiotensin II type 1a receptor peptide.

Angiotensin II (ANG II) inhibits delayed rectifier K+ current (IK) and stimulates total Ca2+ current (ICa) in neurons cocultured from newborn rat hypothalamus and brain stem, effects mediated via ANG II type 1 (AT1) receptors. Here, we identify potential G protein activator regions of the AT1 receptor responsible for initiating the intracellular changes that lead to alterations in these currents. Intracellular application into cultured neurons of a peptide corresponding to the third cytoplasmic loop of the AT1 receptor (AT1a/i3) mimicked the actions of ANG II on IK and ICa, whereas application of a peptide corresponding to the second cytoplasmic loop (AT1a/i2) did not alter these currents. This modulation of IK and ICa by AT1a/i3 involves intracellular messengers (G alpha q, protein kinase C, and intracellular Ca2+) that are identical to those involved in the modulation of IK and ICa following ANG II activation of AT1 receptors. These data provide functional evidence for a role of the third cytoplasmic loop of the AT1 receptor in G protein coupling and subsequent modulation of ion channel effectors.

Structural requirements for G(o) activation by receptor-derived peptides: activation and modulation domains of the alpha 2-adrenergic receptor i3c region.

Synthetic peptides are important tools for understanding the sites and mechanisms of receptor/G protein interactions. We examined the structural determinants of receptor-fragment peptides for G protein binding and activation. A dimer of peptides from the carboxyl-terminal (i3c) and amino-terminal (i3n) regions of the alpha 2A-adrenergic receptor is most potent in stimulating guanine-nucleotide exchange of any peptides studied. Stimulation of GTPase by i3n is partially blocked by pertussis toxin treatment, whereas stimulation by i3c is not, which is consistent with action of i3c at the amino terminus of Gi. Both peptides inhibit adenylyl cyclase in Chinese hamster ovary cell membranes, but only the i3c effect is consistent with a pure Gi stimulation. We also examined the mechanism and defined a minimal structural subset of i3c required for G protein activation. Residues 361-365 from the receptor were essential for GTPase stimulation, whereas determinants in the region 368-373 modulated that activity. A specific role for arginines is defined beyond just their positive charge. Complex effects of modifications of Thr373 suggest a regulatory or conformational role of that residue in the previously defined constitutive activation of the alpha 2-adrenergic receptor [J. Biol. Chem. 268:16483-16487 (1993)]. Thus, our data plus recent mutagenesis results support a role for hydrophobicity in the i3n region and a positively charged/arginine-rich region approximately 15-20 residues from the sixth transmembrane span in G protein activation. In contrast, the immediate perimembrane region of i3c seems to have largely conformational effects in producing constitutive activation of the receptors.

Multisite interactions of receptors and G proteins: enhanced potency of dimeric receptor peptides in modifying G protein function.

Synthetic peptides that activate or inhibit G proteins reveal structural determinants of receptor-G protein interactions and show promise as potential therapeutic agents. A cysteine-containing peptide from the carboxyl-terminal part of the third cytoplasmic loop of the alpha 2-adrenergic receptor (peptide Q) uncouples alpha 2-adrenergic receptors from Gi. Peptide Q readily forms disulfide-linked dimers (Qdimer), as detected by high performance liquid chromatography and mass spectrometry. Qdimer is > 100-fold more potent than monomeric Q peptide in inhibiting p-[125I] iodoclonidine binding to the human alpha 2a-adrenergic receptor in platelet membranes and transfected Chinese hamster ovary cells. In addition, Qdimer is 10-20 times more potent than monomeric Q peptide in inhibiting alpha 2 agonist-stimulated GTPase in cell membranes and in directly stimulating G(o)/Gi GTPase in lipid vesicles. The effect of Qdimer is reversible and not mimicked by cystine. Formylation of both tryptophans greatly reduces the potency of the dimer but a single formyl group is well tolerated, indicating an asymmetric interaction of the dimer with Gi in membranes. A mixed dimer of peptides from the amino- and carboxyl-terminal ends of the third cytoplasmic loop of the alpha 2-adrenergic receptor is most potent in all measures of G protein interactions, suggesting that the dimer of Q peptides mimics multiple intracellular portions of the alpha 2-adrenergic receptor with the G protein. These data confirm the importance of multiple receptor regions in G protein activation and suggest a strategy for examining the role of physically separated regions in protein-protein interactions.

Focus group interview with parents of children with medically complex needs: an intimate look at their perceptions and feelings.

The purpose of this paper was to identify the needs of parents of children with medically complex needs from their own perception. In order to provide in-depth information, the focus group interview technique was used. Several strong recurrent themes were identified. The most persistent need was for a general organization or framework with which the care providers could operate. Along these same lines, the fragmentation of training, needs and services was consistently stated. A general lack of information in terms of home care and how to plan for the future was identified. Support groups were universally lauded for the invaluable services provided to the care parents.

p-[125I]iodoclonidine is a partial agonist at the alpha 2-adrenergic receptor.

The binding properties of p-[125I]iodoclonidine [( 125I]PIC) to human platelet membranes and the functional characteristics of PIC are reported. [125I]PIC bound rapidly and reversibly to platelet membranes, with a first-order association rate constant (kon) at room temperature of 8.0 +/- 2.7 x 10(6) M-1 sec-1 and a dissociation rate constant (koff) of 2.0 +/- 0.8 x 10(-3) sec-1. Scatchard plots of specific [125I]PIC binding (0.1-5 nM) were linear, with a Kd of 1.2 +/- 0.1 nM. [125I]PIC bound to the same number of high affinity sites as the alpha 2-adrenergic receptor (alpha 2-AR) full agonist [3H] bromoxidine (UK14,304), which represented approximately 40% of the sites bound by the antagonist [3H]yohimbine. Guanosine 5'-(beta, gamma-imido)triphosphate greatly reduced the amount of [125I]PIC bound (greater than 80%), without changing the Kd of the residual binding. In competition experiments, the alpha 2-AR-selective ligands yohimbine, bromoxidine, oxymetazoline, clonidine, p-aminoclonidine, (-)-epinephrine, and idazoxan all had Ki values in the low nanomolar range, whereas prazosin, propranolol, and serotonin yielded Ki values in the micromolar range. Epinephrine competition for [125I]PIC binding was stereoselective. Competition for [3H]bromoxidine binding by PIC gave a Ki of 1.0 nM (nH = 1.0), whereas competition for [3H]yohimbine could be resolved into high and low affinity components, with Ki values of 3.7 and 84 nM, respectively. PIC had minimal agonist activity in inhibiting adenylate cyclase in platelet membranes, but it potentiated platelet aggregation induced by ADP with an EC50 of 1.5 microM. PIC also inhibited epinephrine-induced aggregation, with an IC50 of 5.1 microM. Thus, PIC behaves as a partial agonist in a human platelet aggregation assay. [125I]PIC binds to the alpha 2B-AR in NG-10815 cell membranes with a Kd of 0.5 +/- 0.1 nM. [125I]PIC should prove useful in binding assays involving tissues with a low receptor density or in small tissue samples and in studies of cloned and expressed alpha 2-AR.

Immunity to leprosy. IV. Murine T-cell proliferative responses to mycobacteria.

The nature of antigens shared between Mycobacterium leprae and other species of mycobacteria has been examined using a murine T-cell proliferation assay. Mice were immunized with different mycobacteria, and lymph node cultures were prepared one week later and challenged with M. leprae antigen. The 13 species of mycobacteria tested as antigens in this assay revealed that several species shared antigens in common with M. leprae as recognized by T-cell responses. C57BL/10J mice and congenic strains exhibit differences in T-cell responsiveness to M. leprae. B10.M and B10.Q mice are high responders and C57BL/10J are low responders, while F1 (C57BL/10J X B10.M) and (C57BL/10J X B10.Q) hybrid progeny are also low responders. These genetic differences were not observed when six other mycobacterial species were used as T-cell antigens. An unexpected finding was that the genetic pattern of T-cell responsiveness to M. marinum was identical to that observed for M. leprae using these strains of mice. Helper T cells may recognize antigenic determinants shared by M. leprae and M. marinum. These antigens may initiate the induction of T-cell responses to these two species of mycobacteria.

Mammalian host- and fluid-mediated mutagenicity assays of captan and streptozotocin in Salmonella typhimurium.

The mutagenicity of captan and of streptozotocin was tested in vivo by reversion of hisG46 base-pair substitution histidine auxotrophs of Salmonella typhimurium in the peritoneal cavity or in blood, plasma or urine of rats or mice. Genetic response was determined by the frequency of revertants (quantitative test) or by the number of revertants per plate (semiquantitative test). In quantitative HMA captan gave negative results following 3 hourly 500 mg/kg s.c. doses or 1000 mg/kg oral dose in mice with the hisG46 mutant or 2000 mg/kg oral dose in rats with the hisG46, uvrB (TA1950) mutant. The positive control SZN induced many reversions at 0.5 mg/kg i.p. or 10 or 100 mg/kg oral doses. In semiquantitative in vivo blood or urine assays captan gave negative results after a 250 mg/kg oral dose with hisG46. SZN in the same experiment gave positive results in both semiquantitative and quantitative in vivo blood assays following 1000 mg/kg i.p. or 2000 mg/kg oral doses in the rat with TA1950. Rat blood mixed with captan for 45 min before adding TA1950 cells inactivated 1000 mug captan/ml but not 5000 mg/ml in the semiquantitative test. Corresponding figures in the quantitative test were 500 mu/ml and 1000 mug/ml. Rat plasma inactivated the mutagenicity of about 10 times less captan than rat blood. Human blood inactivated about as much captan as rat blood. The mutagenicity of captan was inactivated more efficiently than of SZN by blood. The results of the experiments suggested that captan's mutagenicity is probably inactivated by glutathione of the erythrocytes. Rat S-9 liver microsomal fraction also strongly decreased captan's mutagenicity in a semiquantitative test with the R factor, uvrB, hisG46 (TA100) mutant.