1. There is considerable interest in elucidating potential endogenously derived agonists of the vanilloid receptor and the role of anandamide in this regard has received considerable attention. In the present study, we have used an electrophysiological technique to investigate the mechanism of activation of vanilloid receptors in an isolated vagal preparation. 2. Both capsaicin and anandamide depolarized de-sheathed whole vagal nerve preparations that was antagonized by the VR1 antagonist, capsazepine (P<0.05) whilst this response was unaltered by the cannabinoid (CB1) selective antagonist SR141716A or the CB2 selective antagonist, SR144528, thereby ruling out a role for cannabinoid receptors in this response. 3. The PKC activator, phorbol-12-myristate-13-acetate (PMA) augmented depolarization to both anandamide and capsaicin and this response was significantly inhibited with the PKC inhibitor, bisindolylmaleimide (BIM) (P<0.05). 4. The role of lipoxygenase products in the depolarization to anandamide was investigated in the presence of the lipoxygenase inhibitor, 5,8,11-Eicosatriynoic acid (ETI). Depolarization to anandamide and arachidonic acid was significantly inhibited in the presence of ET1 (P<0.05). However, in the absence of calcium depolarization to anandamide was not inhibited by ETI. 5. Using confocal microscopy we have demonstrated the presence of vanilloid receptors on both neuropeptide containing nerves and nerves that did not stain for sensory neuropeptides. 6. These results demonstrate that anandamide evokes depolarization of guinea-pig vagus nerve, following activation of vanilloid receptors, a component of which involves the generation of lipoxygenase products. Furthermore, these receptors are distributed in both neuropeptide and non-neuropeptide containing nerves.

Under hypoxic conditions eukaryotic cells and tissues undergo adaptive responses involving glycolysis, angiogenesis, vasoconstriction and inflammation. The underlying molecular mechanisms are not yet fully elucidated and are most likely cell and tissue specific. In the lung, alveolar epithelial cells and microvascular endothelial cells are highly sensitive to hypoxia and together orchestrate a rapid and sustained adaptive response. We examined the effect of different oxygen tensions on cell viability, glucose metabolism, key transcription factors and signaling molecules, in alveolar epithelial cells (A549) and microvascular endothelial cells (HMEC-1). Both cell types tolerated hypoxia without detectable cell injury. Hypoxia induced glycolysis in both epithelial and microvascular endothelial cells, although A549 cells exhibited a higher rate of glucose consumption. The transcription factor CREB (cAMP response element binding protein) was activated with decreasing oxygen tensions in both cell types. This effect was again more marked in A549 cells, demonstrating epithelial cells to be more oxygen sensitive. Activating Transcription Factor 3 (ATF-3) was heavily induced by hypoxia in A549 cells but not in HMEC-1 cells. Both cell types exhibited hypoxia induced secretion of VEGF and IL-6. Secretion of the vasoconstrictor endothelin-1 (ET1) was increased by hypoxia in HMEC-1 cells but decreased in A549 cells. These data reveal that both cell types exhibit an adaptive response to hypoxia but alveolar epithelial cells are generally more sensitive. ET-1 was oppositely regulated by decreased oxygen tensions in the investigated cell types. The present study further elucidates the adaptive molecular mechanisms in pulmonary hypoxia and demonstrates cell specific responses.

Bacteria were isolated from flowers and bark of apple and pear trees at three places in Australia. In Victoria, Tasmania and Queensland, strains with white colonies on nutrient agar were screened for dome-shaped colony morphology on agar with sucrose and were found to be closely related by several criteria. The isolates were not pathogenic on apples or pears. They were characterized by a polyphasic approach including microbiological and API assays as well as fatty acid methyl ester analysis, DNA-DNA hybridization and DNA sequencing. For molecular classification, the 16S rRNA cistron and the conserved genes gpd and recA of these bacteria were investigated. Together with other taxonomic criteria, the results of these studies indicate that the bacteria belong to a novel separate species, which we propose to name Erwinia tasmaniensis sp. nov., with the type strain Et1/99(T) (=DSM 17950(T)=NCPPB 4357(T)). From DNA-DNA hybridization kinetics, microbiological characteristics and nucleotide sequence analyses, this species is related to pathogenic Erwinia species, but also to the epiphytic species Erwinia billingiae.

OBJECTIVE

To assess the effect of two hypoglycemic drugs on ischemic preconditioning (IPC) patients with type 2 diabetes and coronary artery disease (CAD).

METHODS

We performed a prospective study of 96 consecutive patients allocated into two groups: 42 to group repaglinide (R) and 54 to group vildagliptin (V). All patients underwent two consecutive exercise tests (ET1 and ET2) in phase 1 without drugs. In phase 2, 1 day after ET1 and -2, 2 mg repaglinide three times daily or 50 mg vildagliptin twice daily was given orally to patients in the respective group for 6 days. On the seventh day, 60 min after 6 mg repaglinide or 100 mg vildagliptin, all patients underwent two consecutive exercise tests (ET3 and ET4).

RESULTS

In phase 1, IPC was demonstrated by improvement in the time to 1.0 mm ST-segment depression and rate pressure product (RPP). All patients developed ischemia in ET3; however, 83.3% of patients in group R experienced ischemia earlier in ET4, without significant improvement in RPP, indicating the cessation of IPC (P < 0.0001). In group V, only 28% of patients demonstrated IPC cessation, with 72% still having the protective effect (P < 0.0069).

CONCLUSIONS

Repaglinide eliminated myocardial IPC, probably by its effect on the KATP channel. Vildagliptin did not damage this protective mechanism in a relevant way in patients with type 2 diabetes and CAD, suggesting a good alternative treatment in this population.

Only few clinical factors predict the prognosis of patients with Ewing tumors. Unfavorable outcome is associated with primary metastatic disease, age>> 15 years, tumor volume above 200 ml, and the histological response to chemotherapy. The aim of this study was to elucidate the prevalence and clinical impact of microsatellite instability (MSI) together with the relation between MSI and mismatch repair protein expression in Ewing tumors. DNA from 61 primary Ewing tumors and 11 Ewing tumor cell lines was extracted and microsatellite analysis for the detection of instability or loss of heterozygosity was performed for the five markers of the Bethesda panel BAT25, BAT26, D5S346, D2S123, and D17S250, which represents the established marker panel for the analysis of hereditary non-polyposis colorectal carcinoma (HNPCC) patients. In addition, single nucleotide repeat regions of the two tumor genes BAX and transforming growth factor receptor II (TGFBR2) were also included. All of the 61 samples were suitable for LOH analysis and 55 for the determination of MSI-status. LOH of these microsatellite markers was detected in 9 of the 61 patients (14.8%). Over all, genetic instability, i.e. MSI and/or LOH, was detected in 17 tumors (27.9%). One out of the 11 tumor cell lines (STA ET1) was characterized by instability of all the five Bethesda markers, while from primary tumor samples, only one showed MSI in more than one microsatellite marker (D5S346 and D17S250, MSI-high). Eight of the fifty-five patients (14.5%) showed instability of one microsatellite locus (MSI-low). No instability was detected in BAT26, D2S123, BAX and TGFBR2. There was no significant correlation between MSI and loss of expression of mismatch repair proteins MLH1, MSH2, or MSH6. The impairment of the p53 signaling pathway (expression of TP53 and/or MDM2 by immunohistochemistry) was significantly associated with reduced overall survival (15 of 49 patients (30.6%), P = 0.0410, log-rank test). We conclude that MSI is not prevalent in Ewing tumor and that the nature of instability differs from the form observed in colorectal carcinoma, the model tumor of MSI. This is documented by the different pattern of MSI (no BAT26 instability) in Ewing tumors and the lack of a strict correlation between MSI-high and loss of expression of MSH2, MSH6 and MLH1.

O55 is one of the most frequent enteropathogenic Escherichia coli (EPEC) O serogroups implicated in infantile diarrhea in developing countries. Multilocus enzyme electrophoresis analysis showed that this serogroup includes two major electrophoretic types (ET), designated ET1 and ET5. ET1 corresponds to typical EPEC, whilst ET5 comprises strains with different combinations of virulence genes, including those for localized adherence (LA) and diffuse adherence (DA). Here we report that ET5 DA strains possess a DA adhesin, designated EPEC Afa. An 11.6-kb chromosomal region including the DA adhesin operon from one O55:H(-) ET5 EPEC strain was sequenced and found to encode a protein with 98% identity to AfaE-1, an adhesin associated with uropathogenic E. coli. Although described as an afimbrial adhesin, we show that both AfaE-1 and EPEC Afa possess fine fibrillar structures. This is the first characterization and demonstration of an Afa adhesin associated with EPEC.

Protein kinase D (PKD) targets several proteins in the heart, including cardiac troponin I (cTnI) and class II histone deacetylases, and regulates cardiac contraction and hypertrophy. In adult rat ventricular myocytes (ARVM), PKD activation by endothelin-1 (ET1) occurs via protein kinase Cε and is attenuated by cAMP-dependent protein kinase (PKA). Intracellular compartmentalisation of cAMP, arising from localised activity of distinct cyclic nucleotide phosphodiesterase (PDE) isoforms, may result in spatially constrained regulation of the PKA activity that inhibits PKD activation. We have investigated the roles of the predominant cardiac PDE isoforms, PDE2, PDE3 and PDE4, in PKA-mediated inhibition of PKD activation. Pretreatment of ARVM with the non-selective PDE inhibitor isobutylmethylxanthine (IBMX) attenuated subsequent PKD activation by ET1. However, selective inhibition of PDE2 [by erythro-9-(2-hydroxy-3-nonyl) adenine, EHNA], PDE3 (by cilostamide) or PDE4 (by rolipram) individually had no effect on ET1-induced PKD activation. Selective inhibition of individual PDE isoforms also had no effect on the phosphorylation status of the established cardiac PKA substrates phospholamban (PLB; at Ser16) and cTnI (at Ser22/23), which increased markedly with IBMX. Combined administration of cilostamide and rolipram, like IBMX alone, attenuated ET1-induced PKD activation and increased PLB and cTnI phosphorylation, while combined administration of EHNA and cilostamide or EHNA and rolipram was ineffective. Thus, cAMP pools controlled by PDE3 and PDE4, but not PDE2, regulate the PKA activity that inhibits ET1-induced PKD activation. Furthermore, PDE3 and PDE4 play redundant roles in this process, such that inhibition of both isoforms is required to achieve PKA-mediated attenuation of PKD activation.

Recent genetic studies have uncovered a link between familial and idiopathic pulmonary arterial hypertension (PAH) and germline mutations in the bone morphogenetic protein type-II receptor (BMPRII). The pathology of PAH is characterized by remodeling of the pulmonary arteries due to pulmonary artery smooth muscle cell (PASMC) hyperproliferation. Although increased endothelial injury and impaired suppression of PASMC proliferation are both critical for the cellular pathogenesis of PAH, a detailed molecular mechanism underlying PAH has yet to be elucidated. In the present study, we investigated the roles of the BMP system and other vasoactive factors associated with PAH (including endothelin (ET), angiotensin II (Ang II) and aldosterone) in the mitotic actions of PASMCs isolated from idiopathic and secondary PAH lungs. ET1 and aldosterone stimulated PASMC proliferation of idiopathic PAH more effectively than secondary PAH, whereas Ang II and ET3 failed to activate mitosis in either of the PASMC cell type. The effects of ET1 and aldosterone were blocked by bosentan, an ET type-A/B receptor (ETA/BR) antagonist, and eplerenone, a selective mineralocorticoid receptor (MR) blocker, respectively. Among the BMP ligands examined, BMP-2 and BMP-7, but not BMP-4 or BMP-6, significantly increased cell mitosis in both PASMC cell types. Notably, ET1- and aldosterone-induced mitosis and mitogen-activated protein kinase phosphorylation were significantly increased in the presence of BMP-2 and BMP-7 in PASMCs isolated from idiopathic PAH, although additive effects were not observed in PASMCs isolated from secondary PAH. Inhibition of extracellular signal-regulated kinase 1 (ERK1)/ERK2 signaling suppressed basal-, ET1- and aldosterone-induced PASMC mitosis more potently than that of stress-activated protein kinase/c-Jun NH2-terminal kinase inhibition. Given the fact that BMP-2 and BMP-7 upregulated ETA/BR and MR expression and that BMP-2 decreased 11betaHSD2 (11beta-hydroxysteroid dehydrogenase type 2) levels in PASMCs isolated from idiopathic PAH, BMPR-Smad signaling may have a key role in amplifying the ETA/BR and/or MR-ERK signaling in PASMCs of the PAH lung. Collectively, the functional link between BMP and ET and/or the MR system may be involved in the progress of PASMC mitosis, ultimately leading to the development of clinical PAH.

Endothelin-B receptor (ET(B))-deficient rats have low-renin, salt-sensitive hypertension. We hypothesized this was caused by an absence of renal ET(B) signaling and performed a series of experiments to examine the effect of dietary sodium (Na) on endothelin-1 (ET1) expression and renal function in wild-type (WT) and ET(B)-deficient rats. We found that ET(B) deficiency, but not dietary Na, increases circulating and tissue (kidney and aorta) ET1 levels. Quantitative reverse-transcription polymerase chain reaction reveals that aortic and renal ET1 and endothelin-A receptor (ET(A)) mRNA, however, are similarly increased by dietary Na in ET(B)-WT and ET(B)-deficient rats. We then determined the effect of chronic ET(A) blockade on blood pressure (direct conscious measurements), urinary protein excretion, and creatinine clearance (Crcl). On a Na-deficient diet, ET(B)-deficient rats have mild proteinuria and impaired Crcl. On a high-Na diet, severe hypertension and renal dysfunction develop in ET(B)-deficient rats. Chronic ET(A) blockade prevents hypertension and renal injury. To determine the role of the renal versus the extrarenal endothelin system, we performed renal cross-transplantation. We found that ET(B) deficiency in the body is associated with renal injury and an impaired ability to excrete an Na load. We also found that ET(B) deficiency in the body affects blood pressure response to dietary Na. Expression of ET1 and ET(A) are regulated by dietary Na. ET(B) receptors outside of the kidney, likely by functioning as a clearance receptor for ET1, limit salt-sensitivity in rats.

The peptide endothelin-1 (ET1), which was originally identified as a vasoconstrictor, has emerged as a critical regulator of a number of painful conditions, including inflammatory pain and tumor-associated pain. There is considerable pharmacological evidence supporting a role for endothelin A receptors (ET(A)) in mediating ET1-induced pro-algesic functions. ET(A) receptors are expressed in small-diameter nociceptive neurons, but also found in a variety of other cell types in peripheral tissues, including immune cells, keratinocytes, endothelial cells, which have the potential to modulate nociception. To elucidate the functional contribution of ET(A) receptors expressed in sensory neurons towards the functions of the ET1 axis in pathological pain states, we undertook a conditional gene deletion approach to selectively deplete expression of ET(A) in sensory nerves, preserving expression in non-neural peripheral tissues; the expression of ET(B) remained unchanged. Behavioural and pharmacological experiments showed that only late nociceptive hypersensitivity caused by ET1 is abrogated upon a loss of ET(A) receptors on nociceptors and further suggest that ET1-induced early nociceptive hypersensitivity involves activation of ET(A) as well as ET(B) receptors in non-neural peripheral cells. Furthermore, in the context of alleviation of cancer pain and chronic inflammatory pain by ET(A) receptor antagonists, we observed in corresponding mouse models that the contribution of ET(A) receptors expressed in nociceptors is most significant. These results help understand the role of ET(A) receptors in complex biological processes and peripheral cell-cell interactions involved in inflammatory and tumor-associated pain.

We examined the genetic structure and symbiotic characteristics of Bradyrhizobium isolates recovered from four legume species (Lupinus albus [white lupine], Lupinus angustifolius [blue lupine], Ornithopus compressus [yellow serradella], and Macroptilium atropurpureum [sirato]) grown in an Oregon soil. We established that multilocus enzyme electrophoresis (MLEE) can provide insights into the genetic relatedness among Bradyrhizobium strains by showing a positive correlation (r =>>/=0.90) between the relatedness of Bradyrhizobium japonicum strains determined by MLEE at 13 enzyme loci and that determined by other workers using either DNA-DNA hybridization or DNA sequence divergence estimates. MLEE identified 17 electrophoretic types (ETs) among 95 Bradyrhizobium isolates recovered from the four hosts. Although the overall genetic diversity among the ETs (H = 0.69) is one of the largest measured to date in a local population of any soilborne bacterial species, there was no evidence of multilocus structure (linkage disequilibrium) within the population. The majority of the isolates (73%) were represented by two closely related ETs (2 and 3) which dominated the root nodules of white lupine, serradella, and siratro. In contrast, ET1 dominated nodules of blue lupine. Although representative isolates from all of the 17 ETs nodulated siratro, white lupine, blue lupine, and big trefoil (Lotus pedunculatus), they were either completely ineffective or poorly effective at fixing nitrogen on these hosts. Despite the widespread use of serradella as a surrogate host for lupine-nodulating bradyrhizobia, 7 of the 17 ETs did not nodulate this host, and the remaining 10 ETs were ineffective at fixing nitrogen.

Gap junctions contribute to important functions of communicating glial cells in brain physiology and pathology. Endothelins (ETs), a vasoactive family of peptides present in the brain, have been described as potent inhibitors of astrocyte gap junctional communication. Through dye-coupling studies we demonstrate here that this inhibition occurs rapidly and then successively reverses and returns to control levels after 90 min of continuous ET1 or ET3 exposure. In addition, long-term exposure of cells to ET3, which acts mainly on ETB receptors, also desensitized the acute action of ET1, which was previously shown to act through either ETA or ETB receptor sites, or both. The gap junction blocker carbenoxolone did not show any time-dependent desensitization and was fully effective also in cultures treated with ETs for prolonged times. The ETs inhibitory effects were partially prevented when blocking pertussis toxin-sensitive G-proteins, chelating intracellular Ca2+, or omitting extracellular Ca2+. We further show that ETs modulate gap junction-mediated transfer of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-Y1)amino]-2-deoxyglucose (2-NBDG), a fluorescent glucose molecule, indicating a role of astrocyte gap junction coupling in metabolic trafficking and suggesting the importance of these peptides in the control of intercellular diffusion of energetic compounds. These findings might have particular relevance in early tissue reactions after various cerebral injuries, which commonly involve increased cerebral ET levels.

BACKGROUND

Human mesenchymal stem cells (hMSCs) reside in a perivascular niche of the body, suggesting that they interact closely with vascular endothelial cells (ECs) through cell-cell interaction or paracrine signaling to maintain cell functions. Endothelin-1 (ET1) is a paracrine factor mainly secreted by ECs. We thus hypothesize that ECs can regulate cellular activities of hMSCs and direct their stem cell fate.

METHODS

We investigated whether co-cultured human aortic endothelial cells (HAECs) were able to regulate expression of potency- and lineage-related markers in bone marrow-derived hMSCs. We further explored the regulatory effects of ET1 on cell proliferation, expression of surface antigens and pluripotency-related markers, and multilineage differentiation in hMSCs. Activation of the AKT signaling pathway in hMSCs was also analyzed to identify its mechanistic role in the ET1-induced regulation.

RESULTS

Co-cultured HAECs enhanced expression of mesenchymal lineage-related markers in hMSCs. Treatment of ET receptor antagonist downregulated the increased expression of CBFA1 in hMSCs cultured with HAEC-conditioned medium. hMSCs treated with ET1 showed cell proliferation and expression of surface antigens, CD73, CD90, and CD105, comparable with those without ET1 treatment. ET1-treated hMSCs also expressed upregulated mRNA transcript levels of OCT3/4, NANOG, CBFA1 and SOX9. When induced for lineage-specific differentiation, hMSCs pre-treated with ET1 showed enhanced osteogenesis and chondrogenesis. However, adipogenic differentiation of hMSCs was not affected by ET1 pretreatment. We further showed that the ET1-induced regulation was mediated by activation of AKT signaling.

CONCLUSIONS

Our results demonstrate that ET1 secreted by HAECs can direct bone marrow-derived hMSCs for osteo- and chondro-lineage differentiation through activation of the AKT signaling pathway, suggesting that ET1 plays a crucial role in regulation of hMSC activity. Our findings may help understand how hMSCs interact with ECs in a perivascular niche.

The Caenorhabditis elegans ortholog of the Fanconi anemia pathway component J (FANCJ) is DOG-1, which is essential for genome stability. Previous studies have shown that disruption of the dog-1 gene generates small deletions of poly-C/poly-G tracts detectable by PCR and results in a mutator phenotype. In this paper, we describe the isolation and characterization of lethal mutations resulting from the loss of dog-1 function. The mutant strains were analyzed using a combination of techniques including genetic mapping, SNP mapping, and oaCGH (oligo array Comparative Genome Hybridization). Using the eT1 balancer system to recover lethal mutants, we isolated, in addition to small deletions, large chromosomal rearrangements, including duplications, translocations and deficiencies. The forward mutation frequency was 10-fold higher than the spontaneous frequency for eT1, and equivalent to that observed for low doses of standard mutagens. From a screen for suppressors of mdf-1/MAD1 lethality, we previously had isolated such-4(h2168), shown here to be a large tandem duplication. Thus, the range of mutational events caused by lack of DOG-1/FANCJ is much broader than previously described.

The nematode Caenorhabditis elegans was exposed to natural space radiation using the ESA Biorack facility aboard Spacelab on International Microgravity Laboratory 1, STS-42. For the major experimental objective dormant animals were suspended in buffer or on agar or immobilized next to CR-39 plastic nuclear track detectors to correlate fluence of HZE particles with genetic events. This configuration was used to isolate mutations in a set of 350 essential genes as well as in the unc-22 structural gene. From flight samples 13 mutants in the unc-22 gene were isolated along with 53 lethal mutations from autosomal regions balanced by a translocation eT1(III;V). Preliminary analysis suggests that mutants from worms correlated with specific cosmic ray tracks may have a higher proportion of rearrangements than those isolated from tube cultures on a randomly sampled basis. Right sample mutation rate was approximately 8-fold higher than ground controls which exhibited laboratory spontaneous frequencies.

BACKGROUND

Up to 80% of patients dying from prostate carcinoma have developed bone metastases that are incurable. Castration is commonly used to treat prostate cancer. Although the disease initially responds to androgen blockade strategies, it often becomes castration-resistant (CRPC for Castration Resistant Prostate Cancer). Most of the murine models of mixed lesions derived from prostate cancer cells are androgen sensitive. Thus, we established a new model of CRPC (androgen receptor (AR) negative) that causes mixed lesions in bone.

METHODS

PC3 and its derived new cell clone PC3c cells were directly injected into the tibiae of SCID male mice. Tumor growth was analyzed by radiography and histology. Direct effects of conditioned medium of both cell lines were tested on osteoclasts, osteoblasts and osteocytes.

RESULTS

We found that PC3c cells induced mixed lesions 10 weeks after intratibial injection. In vitro, PC3c conditioned medium was able to stimulate tartrate resistant acid phosphatase (TRAP)-positive osteoclasts. Osteoprotegerin (OPG) and endothelin-1 (ET1) were highly expressed by PC3c while dikkopf-1 (DKK1) expression was decreased. Finally, PC3c highly expressed bone associated markers osteopontin (OPN), Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP) and produced mineralized matrix in vitro in osteogenic conditions.

CONCLUSIONS

We have established a new CRPC cell line as a useful system for modeling human metastatic prostate cancer which presents the mixed phenotype of bone metastases that is commonly observed in prostate cancer patients with advanced disease. This model will help to understand androgen-independent mechanisms involved in the progression of prostate cancer in bone and provides a preclinical model for testing the effects of new treatments for bone metastases.

The paracrine signaling peptide endothelin-1 (ET1) is involved in cardiovascular diseases, cancer and chronic pain. It acts on class A G-protein-coupled receptors (GPCRs) but displays atypical pharmacology. It binds tightly to ET receptor type A (ET(A)) and causes long-lasting effects. In resistance arteries, the long-lasting contractile effects can only be partly and reversibly relaxed by low-molecular-weight ET(A) antagonists (ERAs). However, the neuropeptide calcitonin-gene-related peptide selectively terminates binding of ET1 to ET(A). We propose that ET1 binds polyvalently to ET(A) and that ERAs and the physiological antagonist allosterically reduce ET(A) functions. Combining the two-state model and the two-domain model of GPCR function and considering receptor activation beyond agonist binding might lead to better anti-endothelinergic drugs. Future studies could lead to compounds that discriminate between ET(A)-mediated effects of the endogenous isopeptides ET1, ET2 and ET3 and that become more effective when the activity of the endogenous endothelin system is elevated.

Inflammation and oxidative stress play important roles in the progression of renal damage. The natural polyphenol naringenin is known to exert potent antioxidant and anti-inflammatory effects. In this study, we have investigated the effect of naringenin on kidney dysfunction, fibrosis, endoplasmic reticulum (ER) stress, angiotensin II type I receptor (AT1R) expression and inflammation in daunorubicin (DNR) induced nephrotoxicity model. Nephrotoxicity was induced in rats by intravenous injection of DNR at a cumulative dose of 9 mg/kg. After 1 week, naringenin (20mg/kg/day. p.o) was administered daily for 6 weeks. Biochemical studies were performed to evaluate renal function. Western blotting was performed to measure the protein levels of AT1R, endothelin (ET)1, ET receptor type A (ETAR), extracellular signal-regulated kinase (ERK)1/2, nuclear factor (NF)κB p65, peroxisome proliferator activated receptor (PPAR)γ, oxidative/ER stress, apoptosis, and inflammatory markers in the kidney of DNR treated rats. Histopathological analysis was done using hemotoxylin eosin and Masson trichrome stained renal sections to investigate the structural abnormalities and fibrosis. DNR treated rats suffered from nephrotoxicity as evidenced by worsened renal function, increased blood urea nitrogen, serum creatinine levels in renal tissues and histopathogical abnormalities. Treatment with naringenin mitigated these changes. Furthermore, naringenin up regulated PPARγ and down regulated AT1R, ET1, ETAR, p-ERK1/2, p-NFκB p65, ER stress, apoptosis, and inflammatory markers. Our results suggest that naringenin has an ability to improve renal function and attenuates AT1R, ERK1/2-NFκB p65 signaling pathway in DNR induced nephrotoxicity in rats.

Non-alcoholic fatty liver disease is associated with hepatic microangiopathy and liver inflammation caused by type 2 diabetes mellitus. Oxidised LDL (oxLDL) is involved in proinflammatory and cytotoxic events in various microcirculatory systems. The lectin-like oxLDL receptor 1 (LOX1) plays a crucial role in oxLDL-induced pathological transformation. However, the underlying mechanism of oxLDL's effects on liver microcirculation disturbances remains unclear. In this study, we investigated the effects of oxLDL on LOX1 (OLR1) expression and function, as well as on the fenestration features of human liver sinusoidal endothelial cells (HLSECs) in vitro. Primary HLSECs were obtained and cultured. The cells were treated with various concentrations of oxLDL (25, 50, 100 and 200 μg/ml), and the cytotoxicity and expression of LOX1 were examined. Furthermore, LOX1 knockdown was performed using siRNA technology, and the changes in intracellular reactive oxygen species (ROS), NFκB, p65, (p65), endothelin 1 (ET1 (EDN1)), eNOS (NOS3) and caveolin 1 (CAV1) levels were measured. Cells were treated with 100 μg/ml oxLDL, and the fenestra morphology was visualised using scanning electron microscopy. oxLDL significantly increased LOX1 expression at both the mRNA and protein levels in HLSECs in a dose- and time-dependent manner. oxLDL stimulation increased ROS generation and NFκB activation, upregulated ET1 and caveolin 1 expression, downregulated eNOS expression and reduced the fenestra diameter and porosity. All of these oxLDL-mediated effects were inhibited after LOX1 knockdown. These results reveal a mechanism by which oxLDL stimulates the production of LOX1 through the ROS/NFκB signalling pathway and by which LOX1 mediates oxLDL-induced endothelial injury and the defenestration of HLSECs.

OBJECTIVE

To investigate the effects of prolonged inhibition of phosphodiesterase type-5, using once-daily long-acting phosphodiesterase type-5 inhibitor (tadalafil) on erectile function and biomarkers of endothelial function in male patients with systemic sclerosis (SSc) and erectile dysfunction (ED).

METHODS

In an open-label study, 14 nonconsecutive male patients with SSc with different degrees of ED were enrolled into the study irrespective of their clinical response to tadalafil, and received once-daily tadalafil 10 mg for 12 weeks. Primary endpoints were variations from baseline of penile arterial inflow [peak systolic velocity (PSV, cm/s); measured with dynamic color duplex ultrasound] and the erectile function domain score (measured with the International Index of Erectile Function questionnaire). Secondary endpoints were variations from baseline of morning erections (determined by modified question 13 of the Structured Interview on Erectile Dysfunction questionnaire) and plasma concentrations of endothelin-1 (ET1).

RESULTS

The PSV and the erectile function domain score were significantly improved by once-daily tadalafil (from 21.3 +/- 6.4 to 30.0 +/- 7.0 cm/s and from 13.0 +/- 6.8 to 17.0 +/- 9.0 vs baseline, respectively; p <0.05). Question 13 scores decreased dramatically after treatment compared with baseline (from 2.2 +/- 0.2 to 0.8 +/- 0.5 arbitrary units; p < 0.001), and plasma ET1 levels decreased (from 24 +/- 15 to 9.8 +/- 7.4 pg/ml; p < 0.05).

CONCLUSIONS

In men with SSc-related ED, once-daily tadalafil improved both erectile function and vascular measures of cavernous arteries. Increases in morning erections and decreases in plasma ET1 levels were found, which may play a potential role in preventing progression of penile fibrosis and erectile dysfunction.

Urotensin II (UII) is a potent vasoactive cyclic peptide thought to play a role in myocardial hypertrophy and remodelling. We therefore determined UII plasma levels in congestive heart failure (CHF) patients and its relationship with the severity of the disease and well-established markers of left ventricular function. UII was significantly higher in CHF patients (n = 57) than in controls (n = 48) [geometric mean (pg/ml), 95% PI: 1.32 (0.67-2.59) versus 0.84 (0.31-1.61), p < 0.0001], was related to the functional class of the disease and correlated negatively with left ventricular ejection fraction (r = -0.316, P = 0.016). Furthermore, UII correlated significantly with Big-ET1 (r = 0.32, p = 0.03), BNP (r = 0.42, p = 0.005) but poorly with Nt-proANP (r = 0.28, p = 0.07). Our results suggest that UII could play a role in worsening the course of congestive heart failure and is associated with established markers of cardiovascular dysfunction.

The purpose of this study was to characterize the magnitude and duration of cerebral blood flow (CBF) reduction in the somatosensory cortical region in a rat model of middle cerebral artery occlusion (MCAO) induced by endothelin-1 (ET1) microinjection under isoflurane anesthesia. MCAO was induced by microinjection of ET1 proximal to the MCA in 41 isoflurane-anesthetized male Sprague-Dawley rats. Three doses of ET1 were studied, 60 pmol (Group 1), 150 pmol (Group 2), and 300 pmol (Group 3). CBF was monitored for 4h following injection using a laser Doppler probe stereotaxically inserted into the left somatosensory cortical region. Computed tomography perfusion imaging was used to verify the extent and duration of blood flow reduction in a subset of 12 animals. The magnitude and duration of blood flow reduction was variable (60-92% of baseline). The 300 pmol dose provided the greatest sustained decrease in blood flow. Evidence of tissue damage was obtained in cases where CBF decreased to <40% of baseline. At the doses studied, ET1-induced ischemia in the presence of isoflurane anesthesia can be used as a minimally invasive but variable model of MCAO. The model is well suited for acute imaging studies of ischemia.

Endothelin (ET)-1 stimulates nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and increases superoxide production in some cells such as vascular smooth muscle cells. Here, we reported that ET1 inhibited NADPH oxidase activity, superoxide generation, and cell proliferation in human abdominal aortic endothelial cells (HAAECs) via the ETB1-Pyk2-Rac1-Nox1 pathway. Superoxide production was determined by assessing ethidium fluorescence using flow cytometry in HAAECs exposed to ET1 (10-30 nm) at different time intervals. ET1 significantly decreased superoxide production in HAAECs in the presence of NG-nitro-L-arginine methyl ester, indicating that ET1 suppressed superoxide generation independent of nitric oxide synthase. ET1 significantly attenuated NADPH oxidase activity and cell proliferation, which could be abolished by silence of Nox1 gene, suggesting that ET1-induced inhibition of NADPH oxidase activity was mediated by Nox1. Furthermore, RNA interference silence of ETB1 receptors significantly increased NADPH oxidase activity, and blocked the inhibitory effect of ET1 on NADPH oxidase activity. Activation of ETB1 receptors by ET1 suppressed protein phosphorylation of pyk2 (Y402) and Rac1, suggesting that ET1 inhibited NADPH oxidase activity via ETB1-Pyk2-Rac1 pathway. Indeed, inhibition of Pyk2 by AG-17 abolished ET1-induced suppression of NADPH oxidase activity. ET1 also attenuated angiotensin II-induced activation of NADPH oxidase and cell proliferation. This study demonstrated, for the first time, that ET1, via ETB1, inhibited NADPH oxidase activity in HAAECs by suppressing the Pyk2-Rac1-Nox1 pathway. This finding reveals a novel function of ETB1 receptors in regulating endothelial NADPH oxidase activity, superoxide production, and cell proliferation, opening a new avenue for understanding the role of ETB1 receptors in protecting endothelial cells.

OBJECTIVE

Ligand activation of peroxisome proliferator-activated receptors (PPARs) prevents cardiomyocyte hypertrophy, but the underlying signalling mechanisms remain unknown. We previously reported that the anti-hypertrophic effect of the dietary polyunsaturated fatty acid, conjugated linoleic acid (CLA), was associated with the upregulation of diacylglycerol (DAG) kinase (DGK). DGK catalyses phosphorylative conversion/attenuation of DAG, thereby modulating protein kinase C (PKC) and G-protein signalling. As the anti-hypertrophic effects of CLA were attenuated by inhibitors of PPARs, the present aim was to investigate the involvement of DGK in the anti-hypertrophic actions of bona fide selective PPAR agonists.

RESULTS

Endothelin-1 (ET1)-induced hypertrophy of neonatal, and then adult, Sprague-Dawley rat cardiomyocytes served as experimental paradigms. Expression of DGKζ, the predominant DGK isoform in myocytes, was stimulated by ligands of PPARγ (troglitazone) or PPARα (fenofibrate) and was accompanied by increased DGK activity. Troglitazone or fenofibrate prevented hypertrophic indicators elicited by ET1, including myocyte size augmentation, de novo protein synthesis, hypertrophic gene expression, and activation of the pro-hypertrophic signal, PKCε. shRNA knockdown of DGKζ abolished the growth-inhibitory effects of PPARs and restored all ET1-induced aspects of hypertrophy. Importantly, the involvement of DGK in the ability of troglitazone and fenofibrate to block ET1-induced hypertrophy and PKCε signalling was verified in adult rat myocytes.

CONCLUSIONS

Collectively, these findings show that the anti-hypertrophic actions of PPARs require DGKζ. Thus, within the cardiomyocyte, there exists a PPAR-DGK signalling axis that underpins the ability of PPAR ligands to inhibit ET1-dependent hypertrophy.