Emerging role of angiotensin type 2 receptor (AT2R)/Akt/NO pathway in vascular smooth muscle cell in the hyperthyroidism

Hyperthyroidism is characterized by increased vascular relaxation and decreased vascular contraction and is associated with augmented levels of triiodothyronine (T3) that contribute to the diminished systemic vascular resistance found in this condition. T3 leads to augmented NO production via PI3K/Akt signaling pathway, which in turn causes vascular smooth muscle cell (VSMC) relaxation; however, the underlying mechanisms involved remain largely unknown. Evidence from human and animal studies demonstrates that the renin-angiotensin system (RAS) plays a crucial role in vascular function and also mediates some of cardiovascular effects found during hyperthyroidism. Thus, in this study, we hypothesized that type 2 angiotensin II receptor (AT2R), a key component of RAS vasodilatory actions, mediates T3 induced-decreased vascular contraction. Marked induction of AT2R expression was observed in aortas from T3-induced hyperthyroid rats (Hyper). These vessels showed decreased protein levels of the contractile apparatus: α-actin, calponin and phosphorylated myosin light chain (p-MLC). Vascular reactivity studies showed that denuded aortic rings from Hyper rats exhibited decreased maximal contractile response to angiotensin II (AngII), which was attenuated in aortic rings pre-incubated with an AT2R blocker. Further study showed that cultured VSMC stimulated with T3 (0.1 µmol/L) for 24 hours had increased AT2R gene and protein expression. Augmented NO levels and decreased p-MLC levels were found in VSMC stimulated with T3, both of which were reversed by a PI3K/Akt inhibitor and AT2R blocker. These findings indicate for the first time that the AT2R/Akt/NO pathway contributes to decreased contractile responses in rat aorta, promoted by T3, and this mechanism is independent from the endothelium.

Nonpeptide somatostatin receptor agonists specifically target ocular neovascularization via the somatostatin type 2 receptor

PURPOSE: To define the molecular pharmacology underlying the antiangiogenic effects of nonpeptide imidazolidine-2,4-dione somatostatin receptor agonists (NISAs) and evaluate the efficacy of NISA in ocular versus systemic delivery routes in ocular disease models. METHODS: Functional inhibitory effects of the NISAs and the somatostatin peptide analogue octreotide were evaluated in vitro by chemotaxis, proliferation, and tube-formation assays. The oxygen-induced retinopathy (OIR) model and the laser model of choroidal neovascularization (CNV) were used to test the in vivo efficacy of NISAs. Transscleral permeability of a candidate NISA was also measured. RESULTS: NISAs inhibited growth factor-induced HREC proliferation, migration and tube formation with submicromolar potencies (IC(50), 0.1-1.0 microM) comparable to octreotide. In the OIR model, systemic administration of the NISAs RFE-007 and RFE-011 inhibited retinal neovascularization in a dose-dependent manner, comparable to octreotide. In the CNV model, intravitreal RFE-011 resulted in a 56% reduction (P < 0.01) in CNV lesion area, whereas systemic administration resulted in a 35% reduction (P < 0.05) in lesion area. RFE-011 demonstrated transscleral penetration. CONCLUSIONS: Micromolar concentrations of octreotide and NISAs are necessary for antiangiogenic effects, whereas nanomolar concentrations are effective for endocrine inhibition. This suggests that the antiangiogenic activity of NISAs and octreotide is mediated by an overall much less efficient downstream coupling mechanism than is growth hormone release. As a result, the intravitreal or transscleral route of administration should be seriously considered for future clinical studies of SSTR2 agonists used for treatment of ocular neovascularization to ensure efficacious concentrations in the target retinal and choroidal tissue.

Alkylamides from Echinacea are a new class of cannabinomimetics. Cannabinoid type 2 receptor-dependent and -independent immunomodulatory effects

Alkylamides (alkamides) from Echinacea modulate tumor necrosis factor alpha mRNA expression in human monocytes/macrophages via the cannabinoid type 2 (CB2) receptor (Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. (2004) FEBS Lett. 577, 563-569). Here we show that the alkylamides dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and dodeca-2E,4E-dienoic acid isobutylamide (A2) bind to the CB2 receptor more strongly than the endogenous cannabinoids. The Ki values of A1 and A2 (CB2 approximately 60 nM; CB1 >1500 nM) were determined by displacement of the synthetic high affinity cannabinoid ligand [3H]CP-55,940. Molecular modeling suggests that alkylamides bind in the solvent-accessible cavity in CB2, directed by H-bonding and pi-pi interactions. In a screen with 49 other pharmacologically relevant receptors, it could be shown that A1 and A2 specifically bind to CB2 and CB1. A1 and A2 elevated total intracellular Ca2+ in CB2-positive but not in CB2-negative promyelocytic HL60 cells, an effect that was inhibited by the CB2 antagonist SR144528. At 50 nM, A1, A2, and the endogenous cannabinoid anandamide (CB2 Ki >200 nM) up-regulated constitutive interleukin (IL)-6 expression in human whole blood in a seemingly CB2-dependent manner. A1, A2, anandamide, the CB2 antagonist SR144528 (Ki <10 nM), and also the non-CB2-binding alkylamide undeca-2E-ene,8,10-diynoic acid isobutylamide all significantly inhibited lipopolysaccharide-induced tumor necrosis factor alpha, IL-1beta, and IL-12p70 expression (5-500 nM) in a CB2-independent manner. Alkylamides and anandamide also showed weak differential effects on anti-CD3-versus anti-CD28-stimulated cytokine expression in human whole blood. Overall, alkylamides, anandamide, and SR144528 potently inhibited lipopolysaccharide-induced inflammation in human whole blood and exerted modulatory effects on cytokine expression, but these effects are not exclusively related to CB2 binding.

T-cell reprogramming through targeted CD4-coreceptor and T-cell receptor expression on maturing thymocytes by latent Circoviridae family member porcine circovirus type 2 cell infections in the thymus

Although porcine circovirus type 2 (PCV2)-associated diseases have been evaluated for known immune evasion strategies, the pathogenicity of these viruses remained concealed for decades. Surprisingly, the same viruses that cause panzootics in livestock are widespread in young, unaffected animals. Recently, evidence has emerged that circovirus-like viruses are also linked to complex diseases in humans, including children. We detected PCV2 genome-carrying cells in fetal pig thymi. To elucidate virus pathogenicity, we developed a new pig infection model by in vivo transfection of recombinant PCV2 and the immunosuppressant cofactor cyclosporine A. Using flow cytometry, immunofluorescence and fluorescence in situ hybridization, we found evidence that PCV2 dictates positive and negative selection of maturing T cells in the thymus. We show for the first time that PCV2-infected cells reside at the corticomedullary junction of the thymus. In diseased animals, we found polyclonal deletion of single positive cells (SPs) that may result from a loss of major histocompatibility complex class-II expression at the corticomedullary junction. The percentage of PCV2 antigen-presenting cells correlated with the degree of viremia and, in turn, the severity of the defect in thymocyte maturation. Moreover, the reversed T-cell receptor/CD4-coreceptor expression dichotomy on thymocytes at the CD4(+)CD8(interm) and CD4SP cell stage is viremia-dependent, resulting in a specific hypo-responsiveness of T-helper cells. We compare our results with the only other better-studied member of Circoviridae, chicken anemia virus. Our data show that PCV2 infection leads to thymocyte selection dysregulation, adding a valuable dimension to our understanding of virus pathogenicity.

In situ activation of the type 2 ryanodine receptor in pancreatic beta cells requires cAMP-dependent phosphorylation

Molecular mechanisms that regulate in situ activation of ryanodine receptors (RY) in different cells are poorly understood. Here we demonstrate that caffeine (10 mM) released Ca2+ from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loaded beta cells from ob/ob mice. Surprisingly, when forskolin, an activator of adenylyl cyclase was present, caffeine induced larger Ca2+ spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release Ca2+ from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca2+ from ER, giving rise to a slow and small increase in [Ca2+]i in beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered Ca2+ release by 4-CEP, giving rise to Ca2+ spikes. In glucose-stimulated beta cells forskolin induced Ca2+ spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting βTC-3-cells by RNase protection assay, using type-specific RY probes, revealed low-level expression of mRNA for the type 2 isoform of the receptor (RY2). We conclude that in situ activation of RY2 in beta cells requires cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.

Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II

The classically recognized functions of the renin–angiotensin system are mediated by type 1 (AT1) angiotensin receptors. Whereas man possesses a single AT1 receptor, there are two AT1 receptor isoforms in rodents (AT1A and AT1B) that are products of separate genes (Agtr1a and Agtr1b). We have generated mice lacking AT1B (Agtr1b −/−) and both AT1A and AT1B receptors (Agtr1a −/−Agtr1b −/−). Agtr1b −/− mice are healthy, without an abnormal phenotype. In contrast, Agtr1a −/−Agtr1b −/− mice have diminished growth, vascular thickening within the kidney, and atrophy of the inner renal medulla. This phenotype is virtually identical to that seen in angiotensinogen-deficient (Agt−/−) and angiotensin-converting enzyme-deficient (Ace −/−) mice that are unable to synthesize angiotensin II. Agtr1a −/−Agtr1b −/− mice have no systemic pressor response to infusions of angiotensin II, but they respond normally to another vasoconstrictor, epinephrine. Blood pressure is reduced substantially in the Agtr1a −/− Agtr1b −/− mice and following administration of an angiotensin converting enzyme inhibitor, their blood pressure increases paradoxically. We suggest that this is a result of interruption of AT2-receptor signaling. In summary, our studies suggest that both AT1 receptors promote somatic growth and maintenance of normal kidney structure. The absence of either of the AT1 receptor isoforms alone can be compensated in varying degrees by the other isoform. These studies reaffirm and extend the importance of AT1 receptors to mediate physiological functions of the renin–angiotensin system.

Angiotensin II type 2 receptor mediates programmed cell death.

The function of the recently discovered angiotensin II type 2 (AT2) receptor remains elusive. This receptor is expressed abundantly in fetus, but scantily in adult tissues except brain, adrenal medulla, and atretic ovary. In this study, we demonstrated that this receptor mediates programmed cell death (apoptosis). We observed this effect in PC12W cells (rat pheochromocytoma cell line) and R3T3 cells (mouse fibroblast cell line), which express abundant AT2 receptor but not AT1 receptor. The cellular mechanism appears to involve the dephosphorylation of mitogen-activated protein kinase (MAP kinase). Vanadate, a protein-tyrosine-phosphatase inhibitor, attenuated the dephosphorylation of MAP kinases by the AT2 receptor and restored the apoptotic changes. Antisense oligonucleotide to MAP kinase phosphatase 1 inhibited the AT2 receptor-mediated MAP kinase dephosphorylation and blocked the AT2 receptor-mediated apoptosis. These results suggest that protein-tyrosine-phosphatase, including MAP kinase phosphatase 1 activated by the AT2 receptor, is involved in apoptosis. We hypothesize that this apoptotic function of the AT2 receptor may play an important role in developmental biology and pathophysiology.

The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer.

The type 1 angiotensin II (AT1) receptor is well characterized but the type 2 (AT2) receptor remains an enigma. We tested the hypothesis that the AT2 receptor can modulate the growth of vascular smooth muscle cells by transfecting an AT2 receptor expression vector into the balloon-injured rat carotid artery and observed that overexpression of the AT2 receptor attenuated neointimal formation. In cultured smooth muscle cells, AT2 receptor transfection reduced proliferation and inhibited mitogen-activated protein kinase activity. Furthermore, we demonstrated that the AT2 receptor mediated the developmentally regulated decrease in aortic DNA synthesis at the latter stages of gestation. These results suggest that the AT2 receptor exerts an antiproliferative effect, counteracting the growth action of AT1 receptor.

Peroxisome Proliferator-Activated Receptor-γ Coactivator 1-α (PPARGC1A) Genetic Associations with Type 2 Diabetes in Three Ethnicities

Genetic heterogeneity, lifestyle factors, gene-gene or gene-environment interactions are the determinants of T2D which puts Hispanics and populations with African ancestry at higher risk of developing T2D. In this dissertation, the genetic associations of PPARGC1A polymorphisms with T2D and its related phenotypes (metabolic markers) in Haitian Americans (cases=110, controls=116), African Americans (cases=120, controls=124) and Cuban Americans (cases=160, controls=181) of South Florida were explored. Five single nucleotide polymorphisms of gene PPARGC1A were evaluated in each ethnicity for their disease association. In Haitian Americans, rs7656250 (OR= 0.22, pp=0.03) had significant protective association with T2D but had risk association in African Americans for rs7656250 (OR=1.02, p=0.96) and rs4235308 (OR=2.53, p=0.03). We found that in Haitian American females, both rs7656250 (OR=0.23, pp=0.03) had protective association with T2D. In African American females, rs7656250 (OR=1.14, p=0.78) had risk association whereas in males, it had significant protective effect (OR=0.37, p=0.04). However, the risk association exhibited by rs4235308 was stronger in African American females (OR=2.69, p=0.03) than males (OR=1.16, p=0.72). In Cuban Americans, only rs7656250 showed significant risk association with T2D (OR=6.87, p=0.02) which was stronger in females alone (OR=7.67, p=0.01). We also observed significant differences among correlations of PPARGC1A SNPs and T2D phenotypes. Positive correlation was observed for log Hs-CRP with rs3774907 (pp=0.03) in Cuban Americans respectively. Correlation of log A1C with rs7656250 (p=0.02) was positive in Cuban Americans while it was negative for rs3774907 in Haitian Americans (ppPPARGC1A correlations with T2D and its phenotypes among the three ethnicities studied (ii) the associations of PPARGC1A SNPs showed significant effect modification by sex. The findings suggest that variations in effects of PPARGC1A gene polymorphisms among three ethnicities and between sexes may have biomedical implications for the development of T2D as well as the phenotypes related to T2D.

Is liraglutide or exenatide better in type 2 diabetes?

Background: Subjects with type 2 diabetes have high circulating levels of glucose. Glucagon-like peptide-1 (GLP-1) is an intestinal hormone that has a major role in glucose homeostasis. Exenatide and liraglutide are both agonists at the GLP-1 receptor, and are effective at reducing circulating glucose levels (measured as HbA1c levels), but they have not been compared. Objectives/methods: This evaluation is of a clinical trial comparing liraglutide once a day with exenatide twice a day in subjects with type 2 diabetes. Results: In the Liraglutide Effect and Action in Diabetes (LEAD)-6 trial, subcutaneous liraglutide 1.8 mg once a day was compared with exenatide 10 μg twice a day. The primary efficacy outcome was change in HbA1c levels, and this was significantly greater with liraglutide (1.12%) than with exenatide (0.79%). Liraglutide and exenatide had similar small abilities to reduce body weight, blood pressure and LDL-cholesterol. Conclusions: Liraglutide was more effective than exenatide for overall glycaemic control in subjects with type 2 diabetes. However, this is only true for the preparations and doses tested, that is liraglutide 1.8 mg once weekly and exenatide 10 μg b.i.d., and may not apply when the comparison is undertaken with the new longer-lasting preparation of exenatide once weekly.

A role for IGF-1R-targeted therapies in small-cell lung cancer?

Background: Small-cell lung cancer (SCLC) is an aggressive disease with a poor prognosis. The insulin-like growth factor-1 receptor (IGF-1R) is an autocrine growth factor and an attractive therapeutic target in many solid tumors, but particularly in lung cancer. Patients and Methods: This study examined tumor samples from 23 patients diagnosed with SCLC, 11 resected specimens and 12 nodal biopsies obtained by mediastinoscopy, for expression of IGF-1R using the monoclonal rabbit anti-IGF-1R (clone G11, Ventana Medical Systems, Tucson, AZ) and standard immunohistochemistry (IHC). Results: All 23 tumor samples expressed IGF-1R with a range of stain intensity from weak (1+) to strong (3+). Ten tumors had a score of 3+, 7 tumors 2+, and 6 tumors 1+. Patient survival data were available for all 23 patients. Two patients died < 30 days post biopsy, therefore, the intensity of anti-IGF-1R immunostaining for 21 patients was correlated to survival. Patients with 3+ immunostaining had a poorer prognosis (P = .003). The overall survival of patients who underwent surgical resection was significantly better (median survival not reached) than patients who were not resected (median survival, 7.4 months) (P = .006). Conclusion: IGF-1R targeted therapies may have a role in the treatment of SCLC in combination with chemotherapy or as maintenance therapy. Further studies on the clinical benefit of targeting IGF-1R in SCLC are needed.

Evidence for an association with type 2 diabetes mellitus at the PPARG locus in a South Indian population

Peroxisome proliferator activated receptor-gamma 2 (PPARG2) is a nuclear hormone receptor of ligand-dependent ranscription factor involved in adipogenesis and a molecular target of the insulin sensitizers thiazolidinediones. We addressed the question of whether the 3 variants (-1279G/A, Pro12Ala, and His478His) in the PPARG2 gene are associated with type 2 diabetes mellitus and its related traits in a South Indian population. The study subjects (1000 type 2 diabetes mellitus and 1000 normal glucose-tolerant subjects) were chosen randomly from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in southern India. The variants were screened by single-stranded conformational variant, direct sequencing, and restriction fragment length polymorphism. Linkage disequilibrium was estimated from the estimates of haplotypic frequencies. The -1279G/A, Pro12Ala, and His478His variants of the PPARG2 gene were not associated with type 2 diabetes mellitus. However, the 2-loci analyses showed that, in the presence of Pro/Pro genotype of the Pro12Ala variant, the -1279G/A promoter variant showed increased susceptibility to type 2 diabetes mellitus (odds ratio, 2.092; 95% confidence interval, 1.22-3.59; P = .008), whereas in the presence of 12Ala allele, the -1279G/A showed a protective effect against type 2 diabetes mellitus (odds ratio, 0.270; 95% confidence interval, 0.15-0.49; P < .0001). The 3-loci haplotype analysis showed that the A-Ala-T (-1279G/A-Pro12Ala-His478His) haplotype was associated with a reduced risk of type 2 diabetes mellitus (P < .0001). Although our data indicate that the PPARG2 gene variants, independently, have no association with type 2 diabetes mellitus, the 2-loci genotype analysis involving -1279G/A and Pro12Ala variants and the 3-loci haplotype analysis have shown a significant association with type 2 diabetes mellitus in this South Indian population. (C) 2010 Elsevier Inc. All rights reserved.

Molecular identification and expression analysis of tumor necrosis factor receptor-associated factor 2 in grass carp Ctenopharyngodon idella

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a crucial component of almost the entire tumor necrosis factor receptor superfamily signaling pathway. In the present study, a TRAF2 gene has been cloned from grass carp (Ctenopharyngodon idella) by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The full-length cDNA is 3162 bp, including a 60 bp 5' untranslated region (UTR), a 1611 bp open reading frame, and a 1491 bp 3' UTR. The polyadenylation signal (AATAAA) and the mRNA instability motifs (ATTTTA, ATTTA) were followed by a poly(A) tail in the 3' UTR. No signal peptide or transmembrane region has been found in the putative amino acids of grass carp TRAF2 (gcTRAF2). Phylogenetic tree analysis clearly showed that gcTRAF2 is nearest to the TRAF2 gene of goldfish. The identity of gcTRAF2 with its homologs in other vertebrates ranges from 56% to 97%. It is characterized by one RING-type signature at the N-terminus, one zinc finger in the middle part, and one conserved TRAF domain consisting of a C-proximal (TRAF-C) subdomain and a N-proximal (TRAF-N) subdomain. The identity of TRAF-C among all TRAF2 homologs in vertebrates varies from 78% to 97%, whereas the identity of TRAF-N ranges from 56% to 100%. The recombinant gcTRAF2 has been expressed in Escherichia coli using pET-32a expression vector. The rabbit anti-gcTRAF2 polyclonal antibody was obtained. The expression of gcTRAF2 in different organs was examined by real-time quantitative polymerase chain reaction and Western blot analysis. It was widely distributed in heart, head kidney, thymus, brain, gill, liver, spleen, and trunk kidney. This is the first report of a TRAF2 homolog molecule in fish.

A genome-wide association study identifies susceptibility variants for type 2 diabetes in Han Chinese.

To investigate the underlying mechanisms of T2D pathogenesis, we looked for diabetes susceptibility genes that increase the risk of type 2 diabetes (T2D) in a Han Chinese population. A two-stage genome-wide association (GWA) study was conducted, in which 995 patients and 894 controls were genotyped using the Illumina HumanHap550-Duo BeadChip for the first genome scan stage. This was further replicated in 1,803 patients and 1,473 controls in stage 2. We found two loci not previously associated with diabetes susceptibility in and around the genes protein tyrosine phosphatase receptor type D (PTPRD) (P = 8.54x10(-10); odds ratio [OR] = 1.57; 95% confidence interval [CI] = 1.36-1.82), and serine racemase (SRR) (P = 3.06x10(-9); OR = 1.28; 95% CI = 1.18-1.39). We also confirmed that variants in KCNQ1 were associated with T2D risk, with the strongest signal at rs2237895 (P = 9.65x10(-10); OR = 1.29, 95% CI = 1.19-1.40). By identifying two novel genetic susceptibility loci in a Han Chinese population and confirming the involvement of KCNQ1, which was previously reported to be associated with T2D in Japanese and European descent populations, our results may lead to a better understanding of differences in the molecular pathogenesis of T2D among various populations.

A novel, long-acting agonist of glucose-dependent insulinotropic polypeptide suitable for once-daily administration in type 2 diabetes

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone with a potentially therapeutic role in type 2 diabetes. Rapid degradation by dipeptidylpeptidase IV has prompted the development of enzyme-resistant N-terminally modified analogs, but renal clearance still limits in vivo bioactivity. In this study, we report long-term antidiabetic effects of a novel, N-terminally protected, fatty acid-derivatized analog of GIP, N-AcGIP(LysPAL(37)), in obese diabetic (ob/ob) mice. Once-daily injections of N-AcGIP(LysPAL(37)) over a 14-day period significantly decreased plasma glucose, glycated hemoglobin, and improved glucose tolerance compared with ob/ob mice treated with saline or native GIP. Plasma insulin and pancreatic insulin content were significantly increased by N-AcGIP(LysPAL(37)). This was accompanied by a significant enhancement in the insulin response to glucose together with a notable improvement of insulin sensitivity. No evidence was found for GIP receptor desensitization and the metabolic effects of NAcGIP(LysPAL(37)) were independent of any change in feeding or body weight. Similar daily injections of native GIP did not affect any of the parameters measured. These data demonstrate the ability of once-daily injections of N-terminally modified, fatty acid-derivatized analogs of GIP, such as N-AcGIP(LysPAL(37)), to improve diabetes control and to offer a new class of agents for the treatment of type 2 diabetes.