Foteini Kiagiadaki

Estrogen anti-inflammatory activity on human monocytes is mediated through cross-talk between estrogen receptor ERα36 and GPR30/GPER1

Estrogens are known modulators of monocyte/macrophage functions; however, the underlying mechanism has not been clearly defined. Recently, a number of estrogen receptor molecules and splice variants were identified that exert different and sometimes opposing actions. We assessed the expression of estrogen receptors and explored their role in mediating estrogenic anti‐inflammatory effects on human primary monocytes. We report that the only estrogen receptors expressed are estrogen receptor‐α 36‐kDa splice variant and G‐protein coupled receptor 30/G‐protein estrogen receptor 1, in a sex‐independent manner. 17‐β‐Estradiol inhibits the LPS‐induced IL‐6 inflammatory response, resulting in inhibition of NF‐κB transcriptional activity. This is achieved via a direct physical interaction of ligand‐activated estrogen receptor‐α 36‐kDa splice variant with the p65 component of NF‐κB in the nucleus. G‐protein coupled receptor 30/G‐protein estrogen receptor 1, which also physically interacts with estrogen receptor‐α 36‐kDa splice variant, acts a coregulator in this process, because its inhibition blocks the effect of estrogens on IL‐6 expression. However, its activation does not mimic the effect of estrogens, on neither IL‐6 nor NF‐κB activity. Finally, we show that the estrogen receptor profile observed in monocytes is not modified during their differentiation to macrophages or dendritic cells in vitro and is shared in vivo by macrophages present in atherosclerotic plaques. These results position estrogen receptor‐α 36‐kDa splice variant and G‐protein coupled receptor 30 as important players and potential therapeutic targets in monocyte/macrophage‐dependent inflammatory processes.

Somatostatin receptors (sst2) regulate cGMP production in rat retina

The present study investigated the effect of somatostatin in the regulation of cGMP levels in rat retina and the mechanisms involved in this process. Isolated rat retinas were treated alone or in the presence of somatostatin (0.01-10 microM), BIM23014 (sst2 agonist, 0.01-10 microM), L-796,778 (sst3 agonist, 10 microM), somatostatin (0.1 microM) in combination with CYN154806 (sst2 antagonist, 1 microM), N(G)-methyl-L-arginine acetate salt (NMMA, inhibitor of the nitric oxide synthase (NOS), 250 microM), orthovanadate (inhibitor of tyrosine phosphatase, SHP-1, 1 microM), and arginine alone (250 microM). cGMP levels were quantified by ELISA. Immunohistochemistry studies were performed for the detection of cGMP and nNOS, while Western blot analysis was employed for the detection of SHP-1. Somatostatin increased cGMP levels in a concentration-dependent manner. This increase was inhibited by CYN154806. BIM23014 increased cGMP levels only at the concentration of 10 microM, while L-796,778 had no effect. NMMA blocked completely the somatostatin stimulated increase of cGMP levels and nNOS was detected in rat retina. cGMP immunoreactivity was observed primarily in bipolar cells only of nitroprusside-treated retinas. SHP-1 inhibition by orthovanadate reduced the somatostatin effect in a statistically significant manner. These results suggest that a SRIF/SHP-1/NO/cGMP mechanism underlies the actions of somatostatin in the retina and in its influence of retinal circuitry.

Activation of somatostatin receptor (sst5) protects the rat retina from AMPA-induced neurotoxicity

In a recent study, we employed an in vivo model of retinal excitotoxicity to investigate the neuroprotective effect of somatostatinergic agents. Intravitreal administration of somatostatin and sst(2) selective agonists protected the retina from (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA) induced excitotoxicity. The sst(1) and sst(4) selective ligands had no effect (Kiagiadaki and Thermos, 2008). The presence of sst(5) receptors in rat retina was only recently reported (Ke and Zhong, 2007). Synthetic agonists that activate sst(2) receptors also bind with high affinity to the sst(5) subtype. In the present study the putative neuroprotective effects of sst(5) receptor activation were investigated. Adult female and male Sprague-Dawley (250-350g) rats were employed. Groups of animals received intravitreally PBS (50mM) or AMPA (42 nmol/eye) alone or in combination with L-817,818 (sst(5), 10(-5), 10(-4)M). To exclude neuroprotective effects via the activation of sst(2) receptors, L-817,818 (10(-4)M) was coinjected with the sst(2) antagonist CYN-154806 (10(-4)M). Immunohistochemistry (IHC) studies using the anti-retinal marker choline acetyltransferase (ChAT) and TUNEL staining were employed to examine retinal cell loss and protection. IHC and Western blot analysis were also employed to assess whether the sst(5) receptors are viable in the AMPA treated tissue as compared to control retina. sst(5) receptors were not affected by AMPA. L-817,818 protected the retina from the AMPA insult in the dose of 10(-4)M, while CYN-154806 (10(-4)M) had no effect on the sst(5) neuroprotection. TUNEL staining confirmed the AMPA-induced retinal toxicity and the L-817,818 neuroprotection. These results demonstrate for the first time that sst(5) receptors are functional in the retina, and that sst(5) analogs administered intravitreally protect the retina from excitotoxicity. Further studies are essential to ascertain the therapeutic relevance of these results.

Effect of intravitreal administration of somatostatin and sst2 analogs on AMPA-induced neurotoxicity in rat retina.

PURPOSE The aim of the present study was to use an in vivo model of retinal excitotoxicity to investigate the neuroprotective effect of somatostatin (SRIF)-ergic agents. METHODS Adult Sprague-Dawley rats (weight range, 250-300 g) intravitreally received (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA; 21, 42, 84 nmol/eye) or PBS (50 mM). Time-dependent responses were examined in animals that received AMPA (42 nmol/eye). Animals received AMPA (42 nmol) alone or in combination with SRIF (10(-5), 10(-4) M) or the sst-selective ligands lanreotide (sst(2),10(-5),10(-4) M), L-779976 (sst(2,)10(-6),10(-5), 10(-4) M), L-797591 (sst(1),10(-4) M), and L-803087 (sst(4),10(-4) M). Immunohistochemistry and TUNEL studies were used to examine retinal cell loss and protection. Immunochemistry, Western blot analysis, and radioimmunoassay assessed the viability of sst(2A) receptors and SRIF levels, respectively, in control and AMPA-treated tissue. RESULTS AMPA (42 nmol) treatment resulted in total and major loss of ChAT and bNOS immunoreactivity, respectively, 24 hours after its administration. This loss was sustained up to 30 days for ChAT- and 8 days for bNOS-expressing amacrine cells. SRIF and the sst(2) receptors were not affected by AMPA. SRIF and the sst(2) analogs protected the retina from the AMPA insult in a dose-dependent manner, whereas activation of the sst(1) and sst(4) subtypes had no effect. TUNEL staining confirmed AMPA-induced retinal ischemia and L-779976 neuroprotection. CONCLUSIONS These results demonstrate for the first time that SRIF and the sst(2) analogs, administered intravitreally, protect the retina from excitotoxicity. Further studies are essential to ascertain the therapeutic relevance of these results.

The Role of Nitric Oxide and cGMP in Somatostatin’s Protection against Retinal Ischemia

PURPOSE To investigate whether nitric oxide (NO) and/or cGMP protects the retina from chemical ischemia and underlie somatostatins neuroprotective effects. METHODS Eyecups of female Sprague-Dawley rats were incubated with PBS or the chemical ischemia mixture [iodoacetic acid (5 mM)/sodium cyanate (25 mM)] in the absence or presence of (1) arginine (0.05-2.0 mM), the substrate of nitric oxide synthase (NOS); (2) the NO donors sodium nitroprusside (SNP; 0.25-4.0 mM), 3-morpholinosydnonimine (SIN-1; 0.1, 0.3, 1.0 mM), SIN-1 (0.1 mM)/L-cysteine (5 mM, peroxynitrite scavenger), and NONOate (1, 5, 10 microM, slow NO releaser); (3) 8-Br-cGMP (0.1, 0.5, 1.0 mM); (4) BIM23014 (sst(2) receptor agonist; 1 microM), alone or in the presence of (5) the NOS inhibitor N(gamma)-monomethyl-L-arginine (NMMA; 0.5 mM); or (6) the guanylyl cyclase inhibitors 1H-[1,2,4]oxadiazolol [4,3-a]quinoxalin-1-one (ODQ;100 microM) and NS2028 (50 microM) for 60 minutes, at 5%CO(2)/air in 37 degrees C. The effect of SIN-1 (0.1, 0.3, 1.0, or 3.0 mM) on the retina was also examined. Subsequently, the eyecups were fixed and sectioned for choline acetyltransferase (ChAT) immunoreactivity and TUNEL staining. RESULTS Arginine and SNP had no effect on the chemical ischemia-induced toxicity. SIN-1, NONOate, and 8-Br-cGMP produced a concentration-dependent protective effect, as shown by ChAT immunoreactivity. TUNEL staining also confirmed the neuroprotective effect of these agents. L-cysteine partially reduced the SIN-1-induced protective effect. SIN-1 alone was toxic only at the highest concentration used (3 mM). NMMA, ODQ, and NS2028 reversed the protective effect of BIM23014. CONCLUSIONS The results suggest that a NO/peroxynitrite/cGMP mechanism may be important in the protection of the retina from ischemic insult. Furthermore, the NO/sGC/cGMP pathway is involved in the neuroprotective effects of sst(2) ligands against retinal ischemia.

Hippocampal sst1 receptors are autoreceptors and do not affect seizures in rats

Somatostatin-14 (SRIF-14) exerts anticonvulsive effects in several rat seizure models, generally attributed to sst2 receptor activation. Whereas sst1 immunoreactivity has been localized to both polymorphic interneurons and principal cells in the rat hippocampus, its potential role as an inhibitory autoreceptor or as a receptor involved in mediating anticonvulsive actions remains unknown. We showed that intrahippocampal administration of the sst1 antagonist SRA880 (1 μM) induced a robust increase in hippocampal SST-14 levels without affecting gamma-aminobutyric acid levels in conscious rats, indicating that the sst1 receptor acts as an inhibitory autoreceptor. SRA880 did not affect seizure severity and did not reverse the anticonvulsive action of SRIF-14 (1 μM) against pilocarpine-induced seizures, suggesting that hippocampal sst1 receptors are not involved in the anticonvulsive effects of SRIF-14.

Octreotide modulates the effects on fibrosis of TNF-α, TGF-β and PDGF in activated rat hepatic stellate cells ☆

BACKGROUND AND AIMS Somatostatin and its analogs may influence hepatic fibrosis interfering through several mechanisms. The aim of this study was to investigate the effect of octreotide on cytokine activated hepatic stellate cells (HSC). METHODS Primary HSCs were isolated from rats and were cultured on plastic for activation. Expression of somatostatin receptors (SSTR) was investigated in cultured HSCs by immunofluorescence and western blot. The effect of octreotide on cellular proliferation was studied with the MTT assay and western blot for α1-procollagen (α1-PROC) production in TNFα, TGF-β1 or PDGF treated HSCs. Phosphotyrosine (PTP) and phosphoserine-phosphothreonine (STP) phosphatases inhibition was performed with sodium orthovanadate and okadaic acid respectively. RESULTS Activated HSC express SSTR subtypes 1, 2A, 2B, 3 and 4 and their expression is enhanced by further HSC activation. Octreotide did not have an effect on HSC proliferation but inhibited plastic induced α1-PROC production. Interestingly, it enhanced PDGF-induced HSC proliferation but inhibited PDGF and TGFβ1 dependent expression of α1-PROC, while an opposite effect was observed in TNFα-induced cell proliferation and collagen production. PTP inhibition reversed the inhibitory effect of octreotide on α1-PROC, but potentiated its effect on PDGF and TGFβ1 dependent α1-PROC production. Finally, STP inhibition profoundly inhibited α1-PROC expression in all cases suggesting that both STP and PTP phosphatases are important regulators of pro-fibrotic mechanisms. CONCLUSIONS The net effect of octreotide on HSCs and therefore liver fibrosis is subject to the cytokine microenvironment of these cells. This effect is modulated by PTPs and STPs inhibition. Especially in the case of STPs their profibrotic effects could be an interesting new therapeutic target in liver fibrosis.

Endogenous and Synthetic Cannabinoids as Therapeutics in Retinal Disease

The functional significance of cannabinoids in ocular physiology and disease has been reported some decades ago. In the early 1970s, subjects who smoked Cannabis sativa developed lower intraocular pressure (IOP). This led to the isolation of phytocannabinoids from this plant and the study of their therapeutic effects in glaucoma. The main treatment of this disease to date involves the administration of drugs mediating either the decrease of aqueous humour synthesis or the increase of its outflow and thus reduces IOP. However, the reduction of IOP is not sufficient to prevent visual field loss. Retinal diseases, such as glaucoma and diabetic retinopathy, have been defined as neurodegenerative diseases and characterized by ischemia-induced excitotoxicity and loss of retinal neurons. Therefore, new therapeutic strategies must be applied in order to target retinal cell death, reduction of visual acuity, and blindness. The aim of the present review is to address the neuroprotective and therapeutic potential of cannabinoids in retinal disease.

Trans-Modulation of the Somatostatin Type 2A Receptor Trafficking by Insulin-Regulated Aminopeptidase Decreases Limbic Seizures

Within the hippocampus, the major somatostatin (SRIF) receptor subtype, the sst2A receptor, is localized at postsynaptic sites of the principal neurons where it modulates neuronal activity. Following agonist exposure, this receptor rapidly internalizes and recycles slowly through the trans-Golgi network. In epilepsy, a high and chronic release of somatostatin occurs, which provokes, in both rat and human tissue, a decrease in the density of this inhibitory receptor at the cell surface. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. In addition, IRAP ligands display anticonvulsive properties. We therefore sought to assess by in vitro and in vivo experiments in hippocampal rat tissue whether IRAP ligands could regulate the trafficking of the sst2A receptor and, consequently, modulate limbic seizures. Using pharmacological and cell biological approaches, we demonstrate that IRAP ligands accelerate the recycling of the sst2A receptor that has internalized in neurons in vitro or in vivo. Most importantly, because IRAP ligands increase the density of this inhibitory receptor at the plasma membrane, they also potentiate the neuropeptide SRIF inhibitory effects on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures and possibly for other neurological conditions in which downregulation of G-protein-coupled receptors occurs. SIGNIFICANCE STATEMENT The somatostatin type 2A receptor (sst2A) is localized on principal hippocampal neurons and displays anticonvulsant properties. Following agonist exposure, however, this receptor rapidly internalizes and recycles slowly. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. We therefore assessed by in vitro and in vivo experiments whether IRAP could regulate the trafficking of this receptor. We demonstrate that IRAP ligands accelerate sst2A recycling in hippocampal neurons. Because IRAP ligands increase the density of sst2A receptors at the plasma membrane, they also potentiate the effects of this inhibitory receptor on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures.

Activin-A causes Hepatic stellate cell activation via the induction of TNFα and TGFβ in Kupffer cells

BACKGROUND & AIMS TGFβ superfamily member Activin-A is a multifunctional hormone/cytokine expressed in multiple tissues and cells, where it regulates cellular differentiation, proliferation, inflammation and tissue architecture. High activin-A levels have been reported in alcoholic cirrhosis and non-alcoholic steatohepatitis (NASH). Our aim was to identify the cell types involved in the fibrotic processes induced by activin-A in liver and verify the liver diseases that this molecule can be found increased. METHODS We studied the effect of activin-A on mouse primary Kupffer cells (KCs) and Hepatic Stellate cells (HSCs) and the levels of activin-A and its inhibitor follistatin in the serum of patients from a large panel of liver diseases. RESULTS Activin-A is expressed by mouse hepatocytes, HSCs and Liver Sinusoid Endothelial cells but not KCs. Each cell type expresses different activin receptor combinations. HSCs are unresponsive to activin-A due to downregulation/desensitization of type-II activin receptors, while KCs respond by increasing the expression/production of TNFα και TGFβ1. In the presence of KCs or conditioned medium from activin-A treated KCs, HSCs switch to a profibrogenic phenotype, including increased collagen and αSMA expression and migratory capacity. Incubation of activin-A treated KC conditioned medium with antibodies against TNFα and TGFβ1 partially blocks its capacity to activate HSCs. Only patients with alcoholic liver diseases and NASH cirrhosis have significantly higher activin-A levels and activin-A/follistatin ratio. CONCLUSIONS Activin-A may induce fibrosis in NASH and alcoholic cirrhosis via activation of KCs to express pro-inflammatory molecules that promote HSC-dependent fibrogenesis and could be a target for future anti-fibrotic therapies.