A Structure-Function-Inhibition Analysis of the Pseudomonas aeruginosa Type III Secretion Needle Protein PscF.

The Pseudomonas aeruginosa type III secretion system (T3SS) needle comprised of multiple PscF subunits is essential for the translocation of effector toxins into human cells, facilitating the establishment and dissemination of infection. Mutations in the pscF gene provide resistance to the phenoxyacetamide (PhA) series of T3SS inhibitory chemical probes. To better understand PscF functions and interactions with PhA, alleles of pscF with 71 single mutations altering 49 of the 85 residues of the encoded protein were evaluated for their effects on T3SS phenotypes. Of these, 37% eliminated and 63% maintained secretion, with representatives of both evenly distributed across the entire protein. Mutations in 14 codons conferred a degree of PhA resistance without eliminating secretion, and all but one were in the alpha-helical C-terminal 25% of PscF. PhA-resistant mutants exhibited no cross-resistance to two T3SS inhibitors with different chemical scaffolds. Two mutations caused constitutive T3SS secretion. The pscF allele at its native locus, whether wild type (WT), constitutive, or PhA resistant, was dominant over other pscF alleles expressed from nonnative loci and promoters, but mixed phenotypes were observed in chromosomal ΔpscF strains with both WT and mutant alleles at nonnative loci. Some PhA-resistant mutants exhibited reduced translocation efficiency that was improved in a PhA dose-dependent manner, suggesting that PhA can bind to those resistant needles. In summary, these results are consistent with a direct interaction between PhA inhibitors and the T3SS needle, suggest a mechanism of blocking conformational changes, and demonstrate that PscF affects T3SS regulation, as well as carrying out secretion and translocation.IMPORTANCEP. aeruginosa effector toxin translocation into host innate immune cells is critical for the establishment and dissemination of P. aeruginosa infections. The medical need for new anti-P. aeruginosa agents is evident by the fact that P. aeruginosa ventilator-associated pneumonia is associated with a high mortality rate (40 to 69%) and recurs in >30% of patients, even with standard-of-care antibiotic therapy. The results described here confirm roles for the PscF needle in T3SS secretion and translocation and suggest that it affects regulation, possibly by interaction with T3SS regulatory proteins. The results also support a model of direct interaction of the needle with PhA and suggest that, with further development, members of the PhA series may prove useful as drugs for P. aeruginosa infection.

Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ.

Peroxisome proliferator-activated receptor δ (PPARδ) regulates many genes involved in lipid metabolism. Hepatic lysophosphatidylcholine acyltransferase 3 (LPCAT3) has critical functions in triglycerides transport and endoplasmic reticulum stress response due to its unique ability to catalyze the incorporation of polyunsaturated fatty acids into phospholipids. Previous studies identified liver X receptor as the transcription factor controlling LPCAT3 expression in mouse liver tissue. Here we show that the hepatic LPCAT3 gene is transcriptionally regulated by PPARδ. Adenovirus-mediated knockdown of PPARδ in cultured hepatic cells and liver tissue reduced LPCAT3 mRNA levels, and exogenous overexpression of PPARδ increased LPCAT3 mRNA expression. Activation of PPARδ in HepG2, Huh7, and Hepa 1-6 cells with its specific agonists increased LPCAT3 mRNA levels in all three hepatic cell lines. Through conducting sequence analysis, LPCAT3 promoter assays, and direct DNA binding assays, we have mapped the functional PPAR-responsive element to a proximal region from -135 to -123 of the LPCAT3 promoter that plays an essential role in mediating PPARδ-induced transactivation of the LPCAT3 gene. Finally, we have provided in vivo evidence showing that activation of PPARδ by agonist L165041 in mice increased hepatic LPCAT3 mRNA abundance and LPCAT enzymatic activity, which is associated with increased incorporations of arachidonate into liver phosphatidylcholine and phosphatidylethanolamine. Furthermore, transient liver-specific knockdown of LPCAT3 in mice affected PPARδ-mediated activation of several hepatic genes involving in FA metabolism. Altogether, our new findings identify LPCAT3 as a direct PPARδ target gene and suggest a novel function of PPARδ in regulation of phospholipid metabolism through LPCAT3.

Inhibition of thyroid hormone receptor locally in the retina is a therapeutic strategy for retinal degeneration.

Thyroid hormone (TH) signaling regulates cell proliferation, differentiation, and metabolism. Recent studies have implicated TH signaling in cone photoreceptor viability. Using mouse models of retinal degeneration, we demonstrated that antithyroid drug treatment and targeting iodothyronine deiodinases (DIOs) to suppress cellular tri-iodothyronine (T3) production or increase T3 degradation preserves cones. In this work, we investigated the effectiveness of inhibition of the TH receptor (TR). Two genes, THRA and THRB, encode TRs; THRB2 has been associated with cone viability. Using TR antagonists and Thrb2 deletion, we examined the effects of TR inhibition. Systemic and ocular treatment with the TR antagonists NH-3 and 1-850 increased cone density by 30-40% in the Rpe65-/- mouse model of Leber congenital amaurosis and reduced the number of TUNEL+ cells. Cone survival was significantly improved in Rpe65-/- and Cpfl1 (a model of achromatopsia with Pde6c defect) mice with Thrb2 deletion. Ventral cone density in Cpfl1/Thrb2-/- and Rpe65-/- /Thrb2-/- mice was increased by 1- to 4-fold, compared with age-matched controls. Moreover, the expression levels of TR were significantly higher in the cone-degeneration retinas, suggesting locally elevated TR signaling. This work shows that the effects of antithyroid treatment or targeting DIOs were likely mediated by TRs and that suppressing TR protects cones. Our findings support the view that inhibition of TR locally in the retina is a therapeutic strategy for retinal degeneration management.-Ma, H., Yang, F., Butler, M. R., Belcher, J., Redmond, T. M., Placzek, A. T., Scanlan, T. S., Ding, X.-Q. Inhibition of thyroid hormone receptor locally in the retina is a therapeutic strategy for retinal degeneration.

Antihypertensive effects of peroxisome proliferator-activated receptor-ß/δ activation.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors, which is composed of three members encoded by distinct genes: PPARα, PPARß/δ, and PPARγ. The biological actions of PPARα and PPARγ and their potential as a cardiovascular therapeutic target have been extensively reviewed, whereas the biological actions of PPARß/δ and its effectiveness as a therapeutic target in the treatment of hypertension remain less investigated. Preclinical studies suggest that pharmacological PPARß/δ activation induces antihypertensive effects in direct [spontaneously hypertensive rat (SHR), ANG II, and DOCA-salt] and indirect (dyslipemic and gestational) models of hypertension, associated with end-organ damage protection. This review summarizes mechanistic insights into the antihypertensive effects of PPARß/δ activators, including molecular and functional mechanisms. Pharmacological PPARß/δ activation induces genomic actions including the increase of regulators of G protein-coupled signaling (RGS), acute nongenomic vasodilator effects, as well as the ability to improve the endothelial dysfunction, reduce vascular inflammation, vasoconstrictor responses, and sympathetic outflow from central nervous system. Evidence from clinical trials is also examined. These preclinical and clinical outcomes of PPARß/δ ligands may provide a basis for the development of therapies in combating hypertension.

PPARδ activation induces hepatic long-chain acyl-CoA synthetase 4 expression in vivo and in vitro.

The arachidonic acid preferred long-chain acyl-CoA synthetase 4 (ACSL4) is a key enzyme for fatty acid metabolism in various metabolic tissues. In this study, we utilized hamsters fed a normal chow diet, a high-fat diet or a high cholesterol and high fat diet (HCHFD) as animal models to explore novel transcriptional regulatory mechanisms for ACSL4 expression under hyperlipidemic conditions. Through cloning hamster ACSL4 homolog and tissue profiling ACSL4 mRNA and protein expressions we observed a selective upregulation of ACSL4 in testis and liver of HCHFD fed animals. Examination of transcriptional activators of the ACSL family revealed an increased hepatic expression of PPARδ but not PPARα in HCHFD fed hamsters. To explore a role of PPARδ in dietary cholesterol-mediated upregulation of ACSL4, we administered a PPARδ specific agonist L165041 to normolipidemic and dyslipidemic hamsters. We observed significant increases of hepatic ACSL4 mRNA and protein levels in all L165041-treated hamsters as compared to control animals. The induction of ACSL4 expression by L165041 in liver tissue in vivo was recapitulated in human primary hepatocytes and hepatocytes isolated from hamster and mouse. Moreover, employing the approach of adenovirus-mediated gene knockdown, we showed that depletion of PPARδ in hamster hepatocytes specifically reduced ACSL4 expression. Finally, utilizing HepG2 as a model system, we demonstrate that PPARδ activation leads to increased ACSL4 promoter activity, mRNA and protein expression, and consequently higher arachidonoyl-CoA synthetase activity. Taken together, we have discovered a novel PPARδ-mediated regulatory mechanism for ACSL4 expression in liver tissue and cultured hepatic cells.

Chronic Ethanol-Induced Impairment of Wnt/ß-Catenin Signaling is Attenuated by PPAR-δ Agonist.

BACKGROUND: The Wnt/ß-catenin pathway regulates liver growth, repair, and regeneration. Chronic ethanol (EtOH) exposure blunts normal liver regenerative responses, in part by inhibiting insulin/IGF signaling, and correspondingly, previous studies showed that EtOH-impaired liver regeneration could be restored by insulin sensitizer (proliferator-activated receptor [PPAR]-δ agonist) treatment. As Wnt/ß-catenin functions overlap and cross talk with insulin/IGF pathways, we investigated the effects of EtOH exposure and PPAR-δ agonist treatment on Wnt pathway gene expression in relation to liver regeneration. METHODS: Adult male Long Evans rats were fed with isocaloric liquid diets containing 0 or 37% EtOH for 8 weeks and also treated with vehicle or a PPAR-δ agonist during the last 3 weeks of the feeding regimen. The rats were then subjected to 70% partial hepatectomy (PH) and livers harvested at various post-PH time points were used to quantitate expression of 19 Wnt pathway genes using Quantigene 2.0 Multiplex Assay. RESULTS: EtOH broadly inhibited expression of Wnt/ß-catenin signaling-related genes, including down-regulation of Wnt1, Fzd3, Lef1, and Bcl9 throughout the post-PH time course (0 to 72 hours), and suppression of Wnt7a, Ccnd1, Fgf4, Wif1, Sfrp2, and Sfrp5 at 18- and 24-hour post-PH time points. PPAR-δ agonist treatments rescued the EtOH-induced suppression of Wnt1, Wnt7a, Fzd3, Lef1, Bcl9, Ccnd1, and Sfrp2 gene expression in liver, corresponding with the improvements in DNA synthesis and restoration of hepatic architecture. CONCLUSIONS: Chronic high-dose EtOH exposures inhibit Wnt signaling, which likely contributes to the impairments in liver regeneration. Therapeutic effects of PPAR-δ agonists extend beyond restoration of insulin/IGF signaling mechanisms and are mediated in part by enhancement of Wnt pathway signaling. Future studies will determine the degree to which targeted restoration of Wnt signaling is sufficient to improve liver regeneration and remodeling in the context of chronic EtOH exposure.

Finding new elicitors that induce resistance in rice to the white-backed planthopper Sogatella furcifera.

Herein we report a new way to identify chemical elicitors that induce resistance in rice to herbivores. Using this method, by quantifying the induction of chemicals for GUS activity in a specific screening system that we established previously, 5 candidate elicitors were selected from the 29 designed and synthesized phenoxyalkanoic acid derivatives. Bioassays confirmed that these candidate elicitors could induce plant defense and then repel feeding of white-backed planthopper Sogatella furcifera.

Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) protects against ceramide-induced cellular toxicity in rat brain astrocytes and neurons by activation of ceramide kinase.

Peroxisome proliferator-activated receptors (PPARs) are important members of the nuclear receptor superfamily. Ligands of these nuclear receptors (PPARα, ß/δ and γ) belong to a wide range of lipophilic substances. In spite of the proven neuroprotective efficacy of PPARß/δ in models of neurological diseases, the biology of PPARß/δ in the brain has been much less investigated than that of PPARα and PPARγ. In the present study, we test the hypothesis that neuroprotection induced by PPARß/δ could rely on the regulation of ceramide metabolism. We found that preincubation of neural cells with the PPARß/δ agonist L-165041 exerts significant protection against ceramide-induced cell death. Most importantly, L-165041 protects against ceramide-induced cell death not only before the insult, but also after the onset of the insult. To identify the mechanism of protection, we show that L-165041 upregulates ceramide kinase (CerK) expression levels in neural cells. Consistent with that, we detected that pharmacological inhibition of CerK reduces the protective effects of L-165041. To further decipher the mechanism of protection, gene knockdown in astrocytes was studied. Knockdown of PPARß/δ and CerK in astrocytes was used to verify that the protective effects of L-165041 are CerK- and PPARß/δ-dependent. We demonstrate that in CerK- or PPARß/δ-knockdown astrocytes, addition of L-165041 has no protective effect. Thus, we conclude that PPARß/δ protects neural cells against ceramide-induced cell death via induction and activation of CerK.

Bioactivity screening and mass spectrometric confirmation for the detection of PPARδ agonists that increase type 1 muscle fibres.

Sensitive and robust bioassays able to detect nuclear receptor activation are very useful for veterinary and doping control, pharmaceutical industry and environmental scientists. Here, we used bioassays based on human leukemic monocyte lymphoma U937 and human liver hepatocellular carcinoma HepG2 cell lines to detect the ligand-induced activation of the peroxisome proliferator-activated receptor delta (PPARδ). Exposure of U937 cells to the PPARδ agonist GW501516 resulted in a marked increase in mRNA expression of the PPARδ target gene Angptl4 which was quantified by qRT-PCR analysis. Exposure of HepG2 cells transiently transfected with a PPARδ expression plasmid and a PPAR-response element-driven luciferase reporter plasmid to PPARδ agonists GW501516, GW610742 and L-165041 resulted in clear dose-response curves. Although the qRT-PCR resulted in higher fold inductions, the luciferase assay with transfected HepG2 cells is cheaper and quicker and about ten times more sensitive to GW501516 compared to analysis of Angptl4 mRNA expression in U937 cells by qRT-PCR. The HepG2-based luciferase assay was therefore used to screen GW501516-spiked supplements and feed and water samples. After liquid extraction and clean-up by solid phase extraction using a weak anion exchange column, extracts were screened in the HepG2 bioassay followed by confirmation with a newly developed UPLC-MS/MS method, using two transitions for each compound, i.e., for GW501516, 454.07>188.15 (collision energy (CE) 46 V) and 454.07>257.08 (CE 30 V); for GW610742, 472.07>206.2 (CE 48 V) and 472.07>275.08 (CE 30 V); and for L-165041, 401.2>193.15 (CE 26 V) and 401.2>343.2 (CE 20 V).

The role of PPARß/δ in the regulation of glutamatergic signaling in the hamster suprachiasmatic nucleus.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily and function as transcription factors that regulate gene expression in numerous biological processes. Although the PPARß/δ subtype is highly expressed in the brain, its physiological roles in neuronal function remain to be elucidated. In this study, we examined the presence of PPARß/δ in the master circadian clock of the Syrian hamster and investigated its putative functional role in this structure. In mammals, the central circadian clock, located in the suprachiasmatic nucleus (SCN), is entrained by the light-dark (LD) cycle via photic6 signals conveyed by a direct pathway whose terminals release glutamate. Using immunocytochemical and qRT-PCR analysis, we demonstrated that the rhythmic expression of PPAR ß/δ within the SCN of hamsters raised under an LD cycle was detectable only at the transcriptional level when the hamsters were maintained under constant darkness (DD). The increase in the number of immunoreactive PPARß/δ cells observed under DD after light stimulation during the early subjective night (CT14), but not during the subjective day (CT06), demonstrated that the expression of PPARß/δ can be up-regulated according to the photosensitive phase of the circadian clock. All of the PPARß/δ-positive cells in the SCN also expressed the glutamate receptor NMDAR1. Moreover, we demonstrated that at the photosensitive point (CT14), the administration of L-16504, a specific agonist of PPARß/δ, amplified the phase delay of the locomotor response induced by a light pulse. Taken together, these data suggest that PPARß/δ activation modulates glutamate release that mediates entrainment of the circadian clock by light.

Synthesis and biological evaluation of 2-phenoxyacetamide analogues, a novel class of potent and selective monoamine oxidase inhibitors.

Monoamine oxidases (EC 1.4.3.4; MAOs), a family of FAD-containing enzymes, is an important target for antidepressant drugs. In this paper, a series of 2-phenoxyacetamide analogues were synthesized, and their inhibitory potency towards monoamine oxidases A (MAO-A) and B (MAO-B) were evaluated using enzyme and cancer cell lysate. 2-(4-Methoxyphenoxy)acetamide (compound 12) (SI=245) and (2-(4-((prop-2-ynylimino)methyl)phenoxy)acetamide (compound 21) (IC50MAO-A=0.018 µM, IC50MAO-B=0.07 µM) were successfully identified as the most specific MAO-A inhibitor, and the most potent MAO-A/-B inhibitor, respectively. The inhibitory activities of these two compounds in living cells were also further evaluated utilizing HepG2 and SHSY-5Y cell lysates.

PPAR beta/delta regulates the immune response mechanisms in the porcine endometrium during LPS-induced inflammation - An in vitro study.

Inflammation in the reproductive tract has become a serious threat to animal fertility. Recently, the role of peroxisome proliferator-activated receptor gamma (PPARγ) in the context of reproduction and the inflammatory response has been highlighted, but the role of PPARß/δ has not been fully elucidated. The aim of the present study was to investigate the in vitro effect of PPARß/δ ligands (agonist: L-165,041 and antagonist: GSK 3787) on the transcriptome profile of porcine endometrium during LPS-induced inflammation in the mid-luteal and follicular phases of the oestrous cycle (days 10-12 and 18-20, respectively) using the RNA-Seq method. During the mid-luteal phase of the oestrous cycle, the current study identified 145 and 143 differentially expressed genes (DEGs) after treatment with an agonist or antagonist, respectively. During the follicular phase of the oestrous cycle, 55 and 207 DEGs were detected after treatment with an agonist or antagonist, respectively. The detected DEGs are engaged in the regulation of various processes, such as the complement and coagulation cascade, NF-κB signalling pathway, or the pathway of 15-eicosatetraenoic acid derivatives synthesis. The results of the current study indicate that PPARß/δ ligands are involved in the control of the endometrial inflammatory response.

Peroxisome proliferator-activated receptor ß/δ induces myogenesis by modulating myostatin activity.

Classically, peroxisome proliferator-activated receptor ß/δ (PPARß/δ) function was thought to be restricted to enhancing adipocyte differentiation and development of adipose-like cells from other lineages. However, recent studies have revealed a critical role for PPARß/δ during skeletal muscle growth and regeneration. Although PPARß/δ has been implicated in regulating myogenesis, little is presently known about the role and, for that matter, the mechanism(s) of action of PPARß/δ in regulating postnatal myogenesis. Here we report for the first time, using a PPARß/δ-specific ligand (L165041) and the PPARß/δ-null mouse model, that PPARß/δ enhances postnatal myogenesis through increasing both myoblast proliferation and differentiation. In addition, we have identified Gasp-1 (growth and differentiation factor-associated serum protein-1) as a novel downstream target of PPARß/δ in skeletal muscle. In agreement, reduced Gasp-1 expression was detected in PPARß/δ-null mice muscle tissue. We further report that a functional PPAR-responsive element within the 1.5-kb proximal Gasp-1 promoter region is critical for PPARß/δ regulation of Gasp-1. Gasp-1 has been reported to bind to and inhibit the activity of myostatin; consistent with this, we found that enhanced secretion of Gasp-1, increased Gasp-1 myostatin interaction and significantly reduced myostatin activity upon L165041-mediated activation of PPARß/δ. Moreover, we analyzed the ability of hGASP-1 to regulate myogenesis independently of PPARß/δ activation. The results revealed that hGASP-1 protein treatment enhances myoblast proliferation and differentiation, whereas silencing of hGASP-1 results in defective myogenesis. Taken together these data revealed that PPARß/δ is a positive regulator of skeletal muscle myogenesis, which functions through negatively modulating myostatin activity via a mechanism involving Gasp-1.

[Design, synthesis and PPAR agonist activities of novel L-tyrosine derivatives containing phenoxyacetyl moiety].

The design, synthesis and bioevaluation of a series of novel L-tyrosine derivatives as peroxisome proliferator-activated receptor (PPAR) agonists are reported. Four intermediates and twenty L-tyrosine derivatives containing phenoxyacetyl moiety TM1 were synthesized starting from L-tyrosine via four step reactions including the esterification of carboxyl group, phenoxyacetylation of a-amino group, bromoalkylation of phenolic hydroxyl group and then nucleophilic substitution reaction with various heterocyclic amines in 21%-75% overall yield. Subsequently TM1 were hydrolyzed to give sixteen corresponding target compounds TM2 in 77%-99% yield. The chemical structures of the thirty-nine new compounds were identified using 1H NMR, 13C NMR techniques and thirty-five were confirmed by HR-MS techniques. Screening results in vitro showed that the PPAR relative activation activities of the target molecules are weak overall, while compound TM2i reaches 50.01%, which hints that the molecular structures of these obtained compounds need to be modified further.

[Impact of infusion therapy on the indices of endogenous intoxication in severe combined trauma of the abdominal organs].

The peculiarities of course of endogenous intoxication (EI) in severe combined abdominal trauma were studied up in experiment. There was established, that in the laboratory animals, to whom the blood loss compensation was done, using colloid-hyperosmolar solution, the El severity was trustworthy lesser than in theothergroups.

Combined toxicity of Cd and 2,4-dichlorophenoxyacetic acid on the earthworm Eisenia andrei under biochar amendment.

Due to anthropogenic activities, various pollutants can be found in agricultural soil, such as cadmium (Cd) and 2,4-dichlorophenoxyacetic acid (2,4-D). They are highly toxic and can have a negative impact on soil fertility. For remediation strategies, biochar has acquired considerable attention due to its benefits for agriculture. However, we should recognize the ecological risk posed by biochar use. In addition, little is known about its non-desirable effects on soil organisms such as earthworms, especially in the case of soil remediation. In this study, earthworms (Eisenia andrei) were exposed to soil contaminated with Cd (0.7 mg/kg), (2,4-D) (7 mg/kg), and a mixture of the two in the presence and absence of biochar (2%). A 7- and 14-day incubation experiment was carried out for this purpose. Cd and 2,4-D uptakes in earthworms' tissues, oxidative stress, cytotoxic response, DNA damage, histopathological changes, and gene expression level were assessed. Results suggested that biochar increased the bioavailability of Cd and 2,4-D and the frequency of micronuclei (MNi) and decreased the lysosomal membrane stability (LMS) in earthworms. Also, histopathological examination detected numerous alterations in animals exposed to the contaminants without any amelioration when biochar was added. The biochemical response of earthworms in terms of oxidative stress demonstrates that in the presence of biochar, animals tend to alleviate the toxicity of Cd and 2,4-D. This was also supported by transcriptomic analyses where expression gene levels related to oxidative stress were upregulated in earthworms exposed to Cd and 2,4-D + biochar. The present investigation brought new insights concerning the use of biochar in agriculture.

The PPARδ ligand L-165041 inhibits VEGF-induced angiogenesis, but the antiangiogenic effect is not related to PPARδ.

Peroxisome proliferator-activated receptor (PPAR)δ is known to be expressed ubiquitously and involved in lipid and glucose metabolism. Recent studies have demonstrated that PPARδ is expressed in endothelial cells (ECs) and plays a potential role in endothelial survival and proliferation. Although PPARα and PPARγ are well recognized to play anti-inflammatory, antiproliferative, and antiangiogenic roles in ECs, the general effect of PPARδ on angiogenesis in ECs remains unclear. Thus, we investigated the effect of the PPARδ ligand L-165041 on vascular EC proliferation and angiogenesis in vitro as well as in vivo. Our data show that L-165041 inhibited VEGF-induced cell proliferation and migration in human umbilical vein ECs (HUVECs). L-165041 also inhibited angiogenesis in the Matrigel plug assay and aortic ring assay. Flow cytometric analysis indicated that L-165041 reduced the number of ECs in the S phase and the expression levels of cell cycle regulatory proteins such as cyclin A, cyclin E, CDK2, and CDK4; phosphorylation of the retinoblastoma protein was suppressed by pretreatment with L-165041. We confirmed whether these antiangiogenic effects of L-165041 were PPARδ-dependent using GW501516 and PPARδ siRNA. GW501516 treatment did not inhibit VEGF-induced angiogenesis, and transfection of PPARδ siRNA did not reverse this antiangiogenic effect of L-165041, suggesting that the antiangiogenic effect of L-165041 on ECs is PPARδ-independent. Together, these data indicate that the PPARδ ligand L-165041 inhibits VEGF-stimulated angiogenesis by suppressing the cell cycle progression independently of PPARδ. This study highlights the therapeutic potential of L-165041 in the treatment of many disorders related to pathological angiogenesis.

Ligand activation of peroxisome proliferator-activated receptor delta suppresses cathepsin B expression in human endothelial cells in a posttranslational manner.

Peroxisome proliferator-activated receptor (PPAR) delta agonists are known to have distinct anti-inflammatory and antitumor effects; though, the knowledge regarding their mode of action has thus far been limited. Different cathepsins have been shown to be upregulated in a broad range of pathological events, such as rheumatoid arthritis, psoriasis, atherosclerosis and diverse tumor entities, for example melanoma. Recent work demonstrated that cathepsin B in particular is an important pro-angiogenic protease in various pathological conditions. We therefore analysed whether cathepsins are a valid target for PPARδ agonists. This study reveals an inhibitory effect of two commonly used PPARδ agonists, GW501516 and L-165,041, on the protein expression and enzyme activity of cathepsin B in human endothelial cells. In contrast, no inhibitory effects were observed on cathepsin L and cathepsin D protein expression after treatment with PPARδ agonists. Furthermore, the results substantiate that PPARδ activators mediate their inhibitory action in a PPARδ-dependent manner and that the underlying regulatory mechanism is not based on a transcriptional but rather on a posttranslational mode of action, via the reduction in the cathepsin B protein half-life. Mechanisms conveying the suppressive effect by 5'-alternative splicing, a 3'-UTR-dependent way or by miRNA could be excluded. The data of this study explore cathepsin B as a new valid target for PPARδ agonists in endothelial cells. The results bolster other studies demonstrating PPARδ agonists as anti-inflammatory and anticarcinogenic agents and thus might have the potential to help to develop new pharmaceutical drugs.

Predicting in vivo cardiovascular properties of ß-blockers from cellular assays: a quantitative comparison of cellular and cardiovascular pharmacological responses.

ß-Adrenoceptor antagonists differ in their degree of partial agonism. In vitro assays have provided information on ligand affinity, selectivity, and intrinsic efficacy. However, the extent to which these properties are manifest in vivo is less clear. Conscious freely moving rats, instrumented for measurement of heart rate (ß1; HR) and hindquarters vascular conductance (ß2; HVC) were used to measure receptor selectivity and ligand efficacy in vivo. CGP 20712A caused a dose-dependent decrease in basal HR (P<0.05, ANOVA) at 5 doses between 6.7 and 670 µg/kg (i.v.) and shifted the dose-response curve for isoprenaline to higher agonist concentrations without altering HVC responses. In contrast, at doses of 67 µg/kg (i.v.) and above, ICI 118551 substantially reduced the HVC response to isoprenaline without affecting HR responses. ZD 7114, xamoterol, and bucindolol significantly increased basal HR (ΔHR: +122 ± 12, + 129 ± 11, and + 59 ± 11 beats/min, respectively; n=6), whereas other ß-blockers caused significant reductions (all at 2 mg/kg i.v.). The agonist effects of xamoterol and ZD 7114 were equivalent to that of the highest dose of isoprenaline. Bucindolol, however, significantly antagonized the response to the highest doses isoprenaline. An excellent correlation was obtained between in vivo and in vitro measures of ß1-adrenoceptor efficacy (R(2)=0.93; P<0.0001).

2,4-Dichlorophenoxyacetic acid (2,4-D) affects DNA integrity and retina structure in zebrafish larvae.

Monitoring the potential risk of herbicides in non-target organisms is a crucial issue for environmental safety. 2,4-D is an herbicide of high environmental relevance that has been shown to exert toxic effects to soil and aquatic biota. In the present study, we investigated the possible genotoxic and retinal development effects of 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide in early life stages zebrafish (Danio rerio). Genotoxicity was evaluated by measuring DNA damage using the comet assay and also by the mRNA expression of genes implicated in apoptosis and/or DNA repair. Retinal development toxicity was evaluated with histological approach. The results obtained revealed that 2,4-D alters DNA integrity of zebrafish larvae. Moreover, transcriptomic data showed a significant induction of p-53 and casp-3 genes and a significant decrease of lig-4 in larvae exposed to the highest tested concentration of 2,4-D (0.8 mg/L). This suggested that p-53 gene regulates the process of DNA repair and apoptosis with increased levels of 2,4-D. The histopathological analysis revealed that early exposure to 2,4-D damaged the structure of larvae retina. Overall, this study is the first to report the DNA damage, casp-3, lig-4 and p-53 regulation, as well as the ocular developmental toxicity in zebrafish larvae at environmentally relevant concentrations of 2,4-D herbicide.