OBJECTIVE

Smooth muscle alpha-actin (SMA) is a cytoskeletal protein characteristic to vascular smooth muscle cells (VSMC), and it serves to facilitate cell contraction and migration. Bacterial lipopolysaccharide (LPS), a major mediator of septic shock secondary to infection, is known to directly affect VSMC. The objective of this study was to investigate the effect of LPS on the expression levels of SMA in VSMC.

METHODS

This study was performed on cultured VSMC derived from human aorta, human coronary artery, or rat aorta.

RESULTS

We show that SMA expression in VSMC, induced by endothelin-1 (ET1) or transforming growth factor-beta (TGF-beta), is potently inhibited by a LPS. This parallels a decreased migration of VSMC after LPS treatment. Downregulation of SMA by LPS is not a result of altered signaling of ET1 or TGF-beta receptors, and it is not mediated by canonical (for LPS) mechanisms, such as production of prostaglandins or nitric oxide, or secretion of other endocrine factors. On a molecular level, downregulation of SMA expression by LPS occurs at the level of transcription, as both SMA mRNA levels and SMA promoter activity are inhibited by LPS. The SMA promoter is controlled largely by two major regulatory elements-CArG boxes activated by serum response factor (SRF), and TGF-beta control elements (TCE). LPS does not affect the activity of SRF, but it potently inhibits both basal and inducible TCE activation.

CONCLUSIONS

We show for the first time that LPS attenuates SMA transcription and protein expression in VSMC likely through inhibition of a TCE element on the SMA promoter.

Serum response factor (SRF) is activated by contractile and hypertrophic agonists, such as endothelin-1 (ET1) to stimulate expression of cytoskeletal proteins in vascular smooth muscle cells (VSMCs). While studying the regulation of smooth muscle alpha-actin (SMA) expression at the level of protein stability, we discovered that inhibition of proteasome-dependent protein degradation by N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) or lactacystin (LC) did not enhance the levels of SMA, but, unexpectedly, attenuated SMA expression in response to ET1, without affecting the viability of VSMCs. Down-regulation of SMA protein by MG132 or LC occurred at the level of SMA transcription and via the inhibition of SRF activity. By contrast, MG132 and LC potentiated the activity of activator protein-1 transcription factor. Regulation of SRF by MG132 was not related to inhibition of nuclear factor-kappaB, an established target of proteasome inhibitors, and was not mediated by protein kinase A, a powerful regulator of SRF activity. Signaling studies indicate that inhibition of ET1-induced SRF activity by MG132 occurs at the level downstream of heterotrimeric G proteins Gq/11 and G13, of small GTPase RhoA, and of actin dynamics but at the level of SRF-DNA binding. MG132 treatment did not result in ubiquitination or accumulation of SRF. By contrast, the levels of c-Jun were rapidly increased upon incubation of cells with MG132, and ectopic overexpression of c-Jun mimicked the effect of MG132 on SRF activity. Together, these data suggest that inhibition of proteasome results in down-regulation of SMA expression via up-regulation of c-Jun and repression of SRF activity at the level of DNA binding.

Wound healing evaluation is important in forensic pathology, in which angiogenesis plays an important role. We have already shown that vascular endothelial growth factor A (VEGF) is produced in the rat skin incision wounds by neutrophils, endothelial cells, and fibroblasts. In this study, we assessed the changes in the mRNA expressions of various factors possibly involved in angiogenesis including angiopoietin (ANGPT) 1 and 2, cadherin 5 (CDH5), granulocyte-macrophage colony stimulating factor (CSF2/GM-CSF), granulocyte colony stimulating factor (CSF3/G-CSF), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand12 (CXCL12/SDF1), endothelin 1 (ET1), fibroblast growth factor 1 (FGF 1), hepatocyte growth factor (HGF), hypoxia inducible factor 1 alpha (HIF1a), leptin, matrix metallopepitidase 9 (MMP9), serpine/plasminogen activator inhibitor1 (PAI1), platelet-derived growth factor-A (PDGF-A), transforming growth factor alpha and beta 1 (TGFa and b1), tenomodulin (TNMD), and troponin I type 2 (TNNI2) in the early stage of the rat skin incision wounds by real time RT-PCR. Factors reported to be involved in lymphangiogenesis such as fibroblast growth factor 2 (FGF 2), c-fos induced growth factor (FIGF/VEGF-D), forkhead box C2 (FOXC2), and prospero homeobox 1 (PROX1) were also studied. One and 3 days after the dorsal skin incisions, wounds on male Sprague-Dawley rats showed the statistically significant increases in the mRNA expressions for CXCL2, CSF3, MMP9, PAI1, and CSF2, whereas TGFa, TNNI2, FGF1, TNMD, leptin, and CXCL12 showed the statistically significant decreases. Interestingly, lymphgangiogenic factors FOXC2, PROX1, and FGF2 also showed the statistically significant decreases. In situ hybridization and immunohistochemistry showed the mRNA and protein positivity in endothelial cells, fibroblasts, and some leukocytes at the bottom of the wound tissue for PAI1, CSF3, and MMP9, 1 day after the skin incisions. Our novel findings show the possible involvement of several factors involved in angiogenesis and lymphangiogenesis in the early stage of wound healing process, which may be useful for forensic wound evaluations.

The major factors influencing the rate of progression of chronic renal disease in autosomal-dominant polycystic kidney disease (ADPKD) are unknown and there are currently no effective treatments for slowing the progression of chronic renal failure in ADPKD patients. As a first step in investigating the potential role of endothelin-1 (ET1) and its receptors (ETA and ETB) in the pathophysiology of progression in ADPKD, we have studied their expression and cellular localisation in ADPKD kidney. Immunoreactive ET1 was detected in cyst epithelia, mesangial cells and vascular smooth muscle cells suggesting continuing ET1 synthesis in the cystic kidney. Compared to healthy controls, ETA mRNA was 5-10-fold higher in ADPKD cystic kidney. In cystic kidney, neo-expression of ETA receptors was found overlying glomeruli and cysts and markedly increased in medium-sized renal arteries by microautoradiography. This is the first study to demonstrate a specific upregulation of ETA receptors in human renal disease. Future studies should address whether ETA selective antagonists may be effective in slowing renal disease progression in ADPKD.

BACKGROUND

Diagnosis of canine idiopathic pulmonary fibrosis (IPF) is challenging. Endothelin-1 (ET1) is a biomarker of IPF in humans, but whether ET1 can detect and differentiate IPF from other canine respiratory diseases is unknown.

OBJECTIVE

To evaluate whether measurement of the concentration of ET1 in serum and bronchoalveolar lavage fluid (BALF) can be used to distinguish canine IPF from chronic bronchitis (CB) and eosinophilic bronchopneumopathy (EBP).

METHODS

Twelve dogs with IPF, 10 dogs with CB, 6 dogs with EBP, 13 privately owned healthy West Highland White Terriers (WHWT), and 9 healthy Beagle dogs.

METHODS

Prospective, case control study. ET1 concentration was determined by ELISA in serum and in BALF.

RESULTS

No significant difference in serum ET1 concentration was detected between healthy Beagle dogs and WHWT. Serum ET1 concentration was higher in dogs with IPF (median interquartile range; 2.32 pg/mL, 2.05-3.38) than healthy Beagle dogs (1.28, 1.07-1.53; P < .001), healthy WHWT (1.56, 1.25-1.85; P < .001), dogs with EBP (0.94 0.68-1.01; P = .001), and dogs with CB (1.54 0.74-1.82; P = .005). BALF ET1 concentration was below the detection limit in healthy WHWT and in dogs with CB, whereas it was measurable in all dogs with IPF. A cut-off serum concentration of 1.8 pg/mL had a sensitivity of 100% and a specificity of 81.2% for detection of IPF, with an area under the receiver operating characteristic curve of 0.818.

CONCLUSIONS

Serum ET1 can differentiate dogs with IPF from dogs with EBP or CB. ET1 can be detected in BALF of dogs with IPF.

The powerful vasoconstrictor peptide endothelin-1 (ET1) has been shown to reduce local cerebral blood flow in brain areas supplied by the middle cerebral artery (MCA) to a pathologically low level upon intracerebral injection adjacent to the MCA. This reduction manifests itself as an ischemic infarct, that is fully developed within 3 days after ET1 injection. The aim of the present study is to examine the effect of ET1 on electroencephalographic (EEG) activity. ET1 was microinjected unilaterally at a dose of 60 pmol in 3 microl of saline to the MCA in conscious rats. EEG signals were recorded from the frontoparietal cortical area, supplied by MCA, from the first up to the fourteenth day after ET1 injection. EEG activity was analyzed by the fast Fourier transformation. A significant shift to a lower EEG frequency, i.e., augmentation of slow waves and a reduction of alpha-like and faster EEG waves was found post-ET1. This effect was maximal after 3-7 days when the most severe destruction of neurons in this cortical area occurs, as has been previously demonstrated. The results suggest that the quantitative EEG analysis may provide useful additional information about the functional disturbances associated with focal cerebral ischemia.

Using the canine chronic cerebral vasospasm model, we studied the effects of a potent new nonpeptidic endothelin-1 (ET1) receptor antagonist, bosentan (Ro 47-0203, 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2'-bipyr imidin-4 - yl]-benzenesulfonamide). Endothelin (ET) receptors are composed of the ETA receptors and the ETB receptors; ET1 acts on both of these receptors. Although it has been previously thought that the ETA receptor mediates vasoconstriction, whereas the ETB receptor mediates vasodilation, recent evidence suggests that ETB receptor also contributes to vasoconstriction. Because bosentan is a mixed antagonist that acts on both receptors, its use might indicate whether or not ET is involved in the pathogenesis of cerebral vasospasm. In this study, beagle dogs received a double injection of autologous arterial blood into the cisterna magna at 2-day intervals (i.e., on Days 0 and 2). The diameter of the basilar artery (BA) was angiographically examined up to Day 7. A total of 24 dogs were randomly allocated to either the treatment group or the no-treatment group. Eight dogs were treated with 10 mg/kg bosentan by a one-dose injection into a central venous catheter. Bosentan was given twice a day starting immediately after the first subarachnoid hemorrhage for 6 days until Day 5. Sixteen dogs served as controls, with untreated subarachnoid hemorrhage. After the injection of bosetan, blood pressure decreased by about 25 mm Hg for a few minutes and then returned to normal. In the dogs treated with bosentan, the BA spasm on Day 7 was significantly ameliorated compared with the BA spasm in the untreated dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelins (ETs), potent endothelium-derived mediators, stimulate formation of nitric oxide, which, in turn, protects against suicidal erythrocyte death or eryptosis, characterized by phosphatidylserine exposure at the erythrocyte surface and triggered by increase in cytosolic Ca(2+) ([Ca(2+)](i)). The present study explored whether the ET1-receptor ETB influences suicidal erythrocyte death. To this end, [Ca(2+)](i) (Fluo3-fluorescence) and phosphatidylserine exposure (annexin V-binding) were determined utilizing FACS analysis. Energy depletion increased [Ca(2+)](i) and phosphatidylserine-exposure, effects significantly blunted by ET1 (IC(50) approximately 100 nM) and the ETB receptor-agonist sarafotoxin 6c (IC(50) approximately 10 nM) but not by ET2 and ET3. ET1 and sarafotoxin significantly delayed the kinetics of suicidal erythrocyte death following energy depletion. ETB stimulation did not blunt the effect of Ca(2+)-ionophore ionomycin (1 microM) on phosphatidylserine exposure. The in vivo significance was tested using rescued ETB-knockout (etb(-/-)) and wild-type (etb(+/+)) mice. The number of phosphatidylserine-exposing erythrocytes, of reticulocytes and spleen size were significantly larger in etb(-/-) mice than in etb(+/+)-mice. The etb(-/-) erythrocytes were more susceptible to the eryptotic effect of oxidative stress and more rapidly cleared from circulating blood than etb(+/+) erythrocytes. Finally, the spleens from etb(-/-) mice were enlarged and contained markedly more phosphatidylserine-exposing erythrocytes than spleens from etb(+/+) mice. The observations disclose a novel function of ET1, i.e., protection from suicidal erythrocyte death.

Endothelin-1 (ET1) is an endogenous vasoconstrictor released by the vascular system to regulate the contractility of vascular smooth muscle cells (VSMC). It is implicated in the pathogenesis of hypertension and diabetic vasculopathy. In rabbit inferior vena cava (IVC), 10 nM ET1 induces tonic contraction mainly via type A endothelin receptor activation. Using confocal imaging of Fluo-3 loaded in thein situ VSMC within the intact IVC, we found that ET1 elicited [Ca2+]i oscillations with an average frequency of 0.31 +/- 0.01 Hz. These [Ca2+]i oscillations occurred as repetitive Ca2+ waves traveling along the longitudinal axis of the cells with an average velocity of 29 +/- 3 microm/s. The Ca2+ waves were not synchronized between neighboring VSMC nor were they propagated between them. Nifedipine (10 microM) inhibited the tonic contraction by 27.0 +/- 5.0% while SKF96365 (50 microM) abolished the remaining contraction. In a parallel Ca2+ study, nifedipine reduced the frequency of the oscillations to 0.22 +/- 0.01 Hz while SKF96365 abolished the remaining [Ca2+]i oscillations. Subsequent application of 25 mM caffeine elicited no further Ca2+ signal. Thus, we conclude that ET1 stimulates tonic contraction in the rabbit IVC by inducing [Ca2+]i oscillations and that stimulated Ca2+ entry through both the L-type voltage-gated Ca2+ channels and a nifedipine-resistant and SKF96365-sensitive pathway is crucial for the maintenance of [Ca2+]i oscillations and tonic contraction.

Conscious Wistar rats with stereotaxically and unilaterally implanted cannula just above the middle cerebral artery (MCA) were injected with the powerful vasoconstrictor peptide endothelin-1 (ET1, 60 pmol in 3 microl). The purpose was to examine the long-term (from the 1st to the 14th day) changes in neuronal bioelectrical activity together with sensorimotor deficits after ET1-induced MCA occlusion (MCAO). Extracellular multi-unit activity (MUA) recorded from the ipsilateral fronto-parietal cortical area (supplied by MCA) and sensorimotor behavior (one postural reflex test and six limb placing tests) were examined. A significant suppression of the multi-unit activity was observed until the 14th day post-ET1. The rats exhibited significant unilateral sensorimotor deficits with a maximum at the 3-7 days after ET1 and a spontaneous partial recovery by days 11-14. A significant correlation was found between the suppression of the multi-unit activity and the sensorimotor deficits between the 3rd and the 10th day post-ET1. The results suggest that studying the bioelectrical activity in combination with the behavioral sensorimotor functions may be of use to assess the functional disturbances associated with focal cerebral ischemia and would help to examine the therapeutic benefits of various cerebroprotective treatments before initiating human clinical trials.

It is known that UV modulates the expression of paracrine factors that regulate melanocyte function in the skin. We investigated the consequences of repetitive UV exposure of human skin in biopsies of 10 subjects with phototypes 2-3.5 taken 1-4 years later. The expression of melanogenic factors (TYR, MART1, MITF), growth factors/receptors (SCF/KIT, bFGF/FGFR1, ET1/EDNRB, HGF, GM-CSF), adhesion molecules (beta-catenin, E-cadherin, N-cadherin), cell cycle proteins (PCNA, cyclins D1, E2) as well as Bcl-2, DKK1, and DKK3, were analyzed by immunohistochemistry. Most of those markers showed no detectable changes at>> or = 1 year after the repetitive UV irradiation. Although increased expression of EDNRB protein was detected in 3 of 10 UV-irradiated subjects, there was no detectable change in the expression of ET1 protein or in EDNRB mRNA levels. In summary, only the expression of TYR, MART1, and/or EDNRB, and only in some subjects, was elevated at>> or = 1 year after UV irradiation. Thus the long-term effects of repetitive UV irradiation on human skin did not lead to significant changes in skin morphology and there is considerable subject-to-subject variation in responses. The possibility that changes in the expression and function of EDNRB triggers downstream activation of abnormal melanocyte proliferation and differentiation deserves further investigation.

Genetic responses that characterize experimental autoimmune myocarditis (EAM) have not yet been determined. To investigate gene expression in the myocardium of EAM, absolute copy numbers of 44 mRNA species [calcium-handling proteins, contractile proteins, natriuretic peptides (NPs), cytokines, chemokines, growth factors, renin-angiotensin-aldosterone (RAA) system, endothelins (ETs) and extracellular matrix] in synthesized cDNA from a fixed quantity of total heart RNA were assessed using real-time reverse-transcriptase PCR at days 0, 14, 21 and 28 after immunization. alpha-Cardiac myosin showed a 26.3-fold decrease and beta-cardiac myosin a 3.75-fold increase at day 14. Atrial NP and brain NP increased 47.7- and 6.35-fold at days 21 and 14 respectively. Angiotensin II type 1 receptor, angiotensin-converting enzyme and ET1 increased 22.3-fold at day 21, 6.30-fold at day 21 and 16.8-fold at day 14 respectively. Aldosterone receptor decreased 2.15-fold at day 14, but aldosterone synthetase was detected only at days 14 and 21. Interleukin (IL)-2, IL-10, interferon-gamma and monocyte chemo-attractant protein-1 increased 9.08-fold at day 14, 398-fold at day 21, 43.1-fold at day 14 and 142-fold at day 14 respectively. Collagen type 3, collagen type 1 and fibronectin increased 34.6-, 1.74- and 44.4-fold respectively at day 21. Interestingly, osteopontin showed a 4540-fold increase and it was the highest mRNA of all at day 14. An isoform of cardiac myosin and NP are dramatically changed in EAM. RAA system and ET expressions are changed differently during the EAM time course. Cytokine, chemokine and extracellular matrix greatly increase and, in particular, large numbers of osteopontin mRNA are expressed in early EAM.

BACKGROUND

Classic sulfonyloureas (SUs) are known to attenuate ischaemic preconditioning. Gliclazide is an SU agent believed to be more protective. We assessed the effects of diet, glibenclamide, or gliclazide on the warm-up effect in type 2 diabetic patients with stable angina.

METHODS

The study group consisted of 64 men, aged 54+/-5 years: 17 patients without diabetes (G I) and 47 diabetic patients: 16 patients treated with glibenclamide (G II), 16 with gliclazide (G III) and 15 patients treated with diet (G IV). After the baseline positive exercise test (ET1), all patients reexercised after 30-min rest (ET2). We analysed exercise duration (ED, s), time to 1 mm ST depression (T-STD, s), max STD (mm), heart rate-systolic blood pressure product at 1 mm STD, or ischaemic threshold (mmHg/min x 100) and the total ischaemic time (s).

RESULTS

In G I, all analysed variables improved significantly during ET2 relative to ET1. Glibenclamide (G II) completely abolished the protective effect of exercise-induced ischaemia because only ED increased during ET2 (431 vs. 451, P<0.05). In G III, however, ED (486 vs. 537, P<0.001), T-STD (364 vs. 388, P<0.05) and max STD (2.5 vs. 2.0, P<0.05) improved significantly during ET2, whereas ischaemic threshold and total ischaemic time did not (PNS). In G IV, similar to G I, all variables improved significantly during ET2 relative to ET1.

CONCLUSIONS

Warm-up effect is preserved in diabetic patients with stable angina treated with diet, partially preserved in gliclazide-treated and abolished in glibenclamide-treated patients.

3-Phenoxybenzoate is a transient metabolite from the breakdown of a number of pyrethroid insecticides in soil. In this study, we identified and characterized a bacterium which could grow on 3-phenoxybenzoate, converting it to phenol. On the basis of morphological and biochemical features, the 3-phenoxybenzoatedegrading isolate was determined to be a Pseudomonas species, probably a strain of Pseudomonas delafieldii, now designated Pseudomonas strain ET1. Pseudomonas strain ET1 grew on 3-phenoxybenzoate with a generation time of 3 h and a specific rate of metabolism of (2.6 +/- 0.9) x 10 g of 3-phenoxybenzoate consumed cell h. The K(m) for 3-phenoxybenzoate metabolism was 1.4 +/- 0.8 muM. The metabolism of 3-phenoxybenzoate was constitutive and not subject to catabolite repression. The metabolism of a variety of substituted diaryl ether compounds was examined. 3- and 4-Phenoxybenzoates were metabolized, but 2-phenoxybenzoate was not. Phenoxy-substituted benzyl aldehyde was metabolized, but phenoxy-substituted benzyl alcohol, benzene, phenol, and aniline were not. Derivatives of 3-phenoxybenzoate substituted in the 4' position with hydroxyl, methyl, or chlorine were metabolized, yielding the corresponding 4-substituted phenol. 3-(2-Hydroxyphenoxy)benzoate was not metabolized, but 3-phenoxy-4-fluorobenzoate was. These results indicate that the metabolism of the tested diaryl ether compounds was restricted to 4-phenoxybenzoate, 3-phenoxybenzyl aldehyde, and 3-phenoxybenzoate derivatives without a substitution in the 2' position.

Endothelin-1 (ET-1) is a potent vasoconstrictor while nitric oxide (NO) has strong vasodilatory effects. Recent studies have indicated that vasoconstrictors and NO may mutually modulate their production and/or activity, thus regulating each other in the context of microcirculatory maintenance. We examined the question whether ET-1 may affect NO formation by controlling the expression of the inducible isoform of the NO synthase (iNOS) in cultured rat glomerular mesangial cells (MCs), as induced by the inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) plus interleukin-1 beta (IL-1 beta). We found that ET-1 in MCs markedly reduced cytokine-induced NO production (measured as stable NO2-) and inhibited the expression of iNOS mRNA (Northern blot analysis) and of iNOS protein (Western blotting). Inhibition of cytokine-stimulated iNOS mRNA expression by ET-1 was almost complete at the level of gene transcription while post-transcriptional effects were not detected. The ETA receptor antagonist BQ-123 blocked the inhibitory effect of ET1. The ETA agonist sarafotoxin 6b (S6b) inhibited, while the ETB agonist-sarafotoxin 6c (S6c) did not inhibit cytokine-initiated iNOS transcription in MCs. The results demonstrate that ET-1 can strongly inhibit cytokine induction of iNOS and formation of NO in cultured MCs, and that this action is mediated via the ETA receptor. While the precise mechanism(s) and biological relevance of this ET-1 effect are presently unclear, it is conceivable that down-regulation of iNOS by the vasopressor ET-1 may serve in vivo to prevent massive NO build-up and subsequent vasomotor collapse in the glomerular capillary tuft. This could help to maintain glomerular ultrafiltration in states of endotoxin excess as well as during glomerular formation and action of TNF-alpha and IL-1 beta causing iNOS induction and subsequent overproduction of NO.

Glomerular expression of chemotactic protein-1/chemokine (C-C motif) ligand-2 (MCP-1/CCL2) correlates with the degree of renal damage, suggesting a role of this chemokine in the pathogenesis of renal diseases. Bindarit is an original indazolic derivative able to inhibit MCPs synthesis and to significantly decrease MCP-1/CCL2 urinary excretion in patients with Lupus Nephritis, in correlation with reduction in albuminuria. Aim of the present work was to elucidate the effect of MCP-1/CCL2 synthesis inhibition on in vitro models of mesangial cell dysfunction. ET1 (10nM) and AngII (10nM) significantly stimulated MCP-1/CCL2 release by human renal mesangial cells (HRMCs) after 3-12h stimulation. Bindarit (10-300 μM) significantly inhibited MCP-1/CCL2 release in response to both stimuli within 12h. Bindarit also inhibited mRNA MCP-1/CCL2 expression, confirming an effect of the drug at transcriptional level. Bindarit significantly and concentration-dependently inhibited HRMC proliferation, measured as either cell duplication or total DNA/well, and impaired mRNA collagen IV expression, collagen deposition and fibronectin expression induced by AngII and ET1. Exposure of HRMCs to bindarit also impaired MMP2 activation in response to both stimuli, measured by means of gelatin zymography. These data confirm the important role of MCP-1/CCL2 synthesis in mesangial cell dysfunction and support the potential of therapeutic intervention targeting this chemokine in kidney disease.

BACKGROUND

Endothelin (ET1) promotes the growth of osteoblastic breast and prostate cancer metastases. Conversion of big ET1 to mature ET1, catalyzed primarily by endothelin converting enzyme 1 (ECE1), is necessary for ET1's biological activity. We previously identified the Ece1, locus as a positional candidate gene for a pleiotropic quantitative trait locus affecting femoral size, shape, mineralization, and biomechanical performance.

METHODS

We exposed TMOb osteoblasts continuously to 25 ng/ml big ET1. Cells were grown for 6 days in growth medium and then switched to mineralization medium for an additional 15 days with or without big ET1, by which time the TMOb cells form mineralized nodules. We quantified mineralization by alizarin red staining and analyzed levels of miRNAs known to affect osteogenesis. Micro RNA 126-3p was identified by search as a potential regulator of sclerostin (SOST) translation.

RESULTS

TMOb cells exposed to big ET1 showed greater mineralization than control cells. Big ET1 repressed miRNAs targeting transcripts of osteogenic proteins. Big ET1 increased expression of miRNAs that target transcripts of proteins that inhibit osteogenesis. Big ET1 increased expression of 126-3p 121-fold versus control. To begin to assess the effect of big ET1 on SOST production we analyzed both SOST transcription and protein production with and without the presence of big ET1 demonstrating that transcription and translation were uncoupled.

CONCLUSIONS

Our data show that big ET1 signaling promotes mineralization. Moreover, the results suggest that big ET1's osteogenic effects are potentially mediated through changes in miRNA expression, a previously unrecognized big ET1 osteogenic mechanism.

Alteration of cochlear blood flow may be involved in the etiology of inner ear disorders like sudden hearing loss, fluctuating hearing loss and tinnitus. The aim of the present study was to localize the vasodilator calcitonin gene-related peptide (CGRP) and to identify CGRP receptors and their signaling pathways in the gerbil spiral modiolar artery (SMA) that provides the main blood supply of the cochlea. CGRP was localized in perivascular nerves by immunocytochemistry. The vascular diameter and cytosolic Ca2+ concentration [Ca2+]i in the smooth muscle cells were measured simultaneously with videomicroscopy and fluo-4-microfluorometry. Calcitonin receptor-like receptor (CRLR) mRNA was identified by RT-PCR as a specific 288 bp fragment in total RNA isolated from the vascular wall. The SMA was preconstricted by a 2-min application of 1 nM endothelin-1 (ET1). CGRP, forskolin, and dibutyryl-cAMP caused a vasodilation (EC50 = 0.1 nM, 0.3 mM, and 20 mM). CGRP and forskolin caused an increase in cAMP production and a transient decrease in the [Ca2+]i. The CGRP-induced vasodilation was antagonized by CGRP8-37 (KDB = 2 mM). The K+-channel blockers iberiotoxin and glibenclamide partially prevented the CGRP- or forskolin-induced vasodilations but failed to reverse these vasodilations. These results demonstrate that CGRP is present in perivascular nerves and causes a vasodilation of the ET1-preconstricted SMA. The data suggest that this vasodilation is mediated by an increase in the cytosolic cAMP concentration, a transient activation of iberiotoxin-sensitive BK and glibenclamide-sensitive KATP K+ channels, a transient decrease in the [Ca2+]i and a long-lasting Ca2+ desensitization.

OBJECTIVE

Metabolic syndrome (MetS) is considered as a proinflammatory and prothrombotic state with atherogenic risk factors including dyslipidemia, obesity and glucose intolerance. Oxidative stress is a unifying basis of several disorders including diabetes mellitus (DM) and MetS. We therefore designed this cross-sectional study to investigate the potential interaction among iron metabolism, inflammation and endothelial plexus in MetS and DM patients.

METHODS

A total of 62 patients [median age 54 (23-76) years; male/female 16/46] and 18 healthy controls [median age 38 (30-64) years; male/female 6/12] were included in the study. Patient population was classified as MetS (n = 30) and DM (n = 32).

RESULTS

Leukocyte count (p = 0.002) and osteopontin (OPN) levels (p = 0.008) were significantly higher, while C-reactive protein (CRP) (p = 0.056) and IL-6 (p = 0.059) represented a relative increase in the patient group. Leptin, endothelin 1 (ET1), hepcidin, nitric oxide synthase (NOS), erythrocyte sedimentation rate (ESR), iron, transferrin saturation (TS) and ferritin levels were not significantly different between the patient and control groups. Endothelin 1 was found to be higher in the DM group compared to MetS group (p = 0.15, p = 0.049). Leukocyte count, leptin, hepcidin, OPN, NOS, IL-6, ESR, CRP, iron, TS and ferritin levels were not different between DM and MetS groups. A positive correlation was demonstrated between leptin and OPN (p = 0.001, r = 0.360), ferritin and hepcidin (p < 0.01, r = 0.633), IL-6 and CRP (p = 0.023, r = 0.319), leptin and NOS (p = 0.005, r = 0.309) and OPN and NOS (p < 0.001, r = 0.803). There was a negative correlation between hepcidin and NOS (p = 0.009, r = -0.289). When the study cohort was divided into two particular groups based on median ferritin and hepcidin levels, hepcidin (p = 0.002), ALT (p = 0.001) and LDL (p = 0.049) levels were higher in the high-ferritin group. Nitric oxide synthase levels (p = 0.033) were lower, whereas ferritin levels (p = 0.004) were higher in the high-hepcidin group.

CONCLUSIONS

Mechanisms involved in the vicious circle of MetS including inflammation, endothelial vasculature and iron metabolism remain to be elucidated. The role of iron metabolism in this complex interaction should be confirmed with further studies.

BACKGROUND

The envelope (E) protein of dengue virus (DENV) is the major immunogen for dengue vaccine development. At the C-terminus are two α-helices (EH1 and EH2) and two transmembrane domains (ET1 and ET2). After synthesis, E protein forms a heterodimer with the precursor membrane (prM) protein, which has been shown as a chaperone for E protein and could prevent premature fusion of E protein during maturation. Recent reports of enhancement of DENV infectivity by anti-prM monoclonal antibodies (mAbs) suggest the presence of prM protein in dengue vaccine is potentially harmful. A better understanding of prM-E interaction and its effect on recognition of E and prM proteins by different antibodies would provide important information for future design of safe and effective subunit dengue vaccines.

RESULTS

In this study, we examined a series of C-terminal truncation constructs of DENV4 prME, E and prM. In the absence of E protein, prM protein expressed poorly. In the presence of E protein, the expression of prM protein increased in a dose-dependent manner. Radioimmunoprecipitation, sucrose gradient sedimentation and pulse-chase experiments revealed ET1 and EH2 were involved in prM-E interaction and EH2 in maintaining the stability of prM protein. Dot blot assay revealed E protein affected the recognition of prM protein by an anti-prM mAb; truncation of EH2 or EH1 affected the recognition of E protein by several anti-E mAbs, which was further verified by capture ELISA. The E protein ectodomain alone can be recognized well by all anti-E mAbs tested.

CONCLUSIONS

A C-terminal domain (EH2) of DENV E protein can affect the expression and stability of its chaperone prM protein. These findings not only add to our understanding of the interaction between prM and E proteins, but also suggest the ectodomain of E protein alone could be a potential subunit immunogen without inducing anti-prM response.

Blood vessels composed of endothelial cells (ECs) contact with mesenchymal stem cells (MSCs) in different tissues, suggesting possible interaction between these 2 types of cells. We hypothesized that endothelin-1 (ET1), a secreted paracrine factor of ECs, can differentially direct the lineages of adipose-derived stem cells (ASCs) and bone marrow-derived MSCs (BMSCs). Predifferentiated ASCs and BMSCs were treated with ET1 for 2 cell passages and then induced for multilineage differentiation. Our results showed that adipogenesis of ET1-pretreated ASCs and osteogenesis of ET1-pretreated BMSCs were increased compared to those of control cells. The effect of ET1 on enhancing adipogenesis of ASCs and osteogenesis of BMSCs was attenuated by blocking endothelin receptor type A (ETAR) and/or endothelin receptor type B (ETBR). Western blot analysis indicated that regulation by ET1 was mediated through activation of the protein kinase B and ERK1/2 signaling pathways. We analyzed subpopulations of ASCs and BMSCs with or without ETAR and/or ETBR, and we found that ETAR+/ETBR- and ETAR-/ETBR+ subpopulations of ASCs and those of BMSCs pretreated with ET1 were prone to turning into adipocytes and osteoblasts, respectively, after differentiation induction. Our findings provide insight into the differential regulation of MSC specification by ET1, which may help develop viable approaches for tissue regeneration.-Lee, M.-S., Wang, J., Yuan, H., Jiao, H., Tsai, T.-L., Squire, M. W., Li, W.-J. Endothelin-1 differentially directs lineage specification of adipose- and bone marrow-derived mesenchymal stem cells.

BACKGROUND

Primary biliary cirrhosis (PBC) is an organ specific autoimmune disease of still unidentified genetic etiology. We have shown that endothelins (ETs), produced by the liver endothelial cells are increased in PBC and may play a major pathogenetic role.

OBJECTIVE

To study gene polymorphisms related to the endothelial cells (eNOS, EDN-1 genes) and, to investigate whether the previously reported association of CTLA4 gene polymorphisms is replicated in a genetically homogeneous Greek population.

METHODS

Genomic DNA was extracted from 100 PBC patients (83 females, 93% AMA+, 74/100 Ludwig stage I-II) and 158 healthy controls. eNOS, CTLA4 and ET1 polymorphisms were determined by PCR-RFLPs analysis.

RESULTS

Both eNOS intron4 VNTR and eNOS exon7 G894T SNP were significantly associated with increased risk in PBC. EDN-11 rs2071942 "A" and rs5370 "T" alleles appeared a tendency for association with disease progression. No association was found between PBC and the CTLA4 SNPs analyzed.

CONCLUSIONS

We demonstrated that eNOS, a gene related to the liver endothelium function is associated with PBC. Contrarily, the important in adaptive immunity gene CTLA4 was not associated with the disease in the homogeneous population analyzed. These results are compatible partially with our previous hypothesis that defects of the liver endothelial system, leading to endothelin overproduction, may be a fundamental early pathogenetic mechanism in PBC.

Radiation-induced heart disease is a severe side effect of thoracic radiotherapy. Studies suggest that mast cells play a protective role in radiation-induced heart disease and that the endothelin (ET) system mediates protective effects of mast cells in other disorders. This study examined whether mast cells modulate the cardiac ET system and examined the effects of ET receptor inhibition in a rat model of radiation-induced heart disease. Mast cell-deficient (Ws/Ws), mast cell-competent (+/+) and Sprague-Dawley rats received 18 Gy irradiation to the heart. Left ventricular mRNA of ET1 and its receptors (ETA and ETB) was measured in Ws/Ws and +/+ rats at 1 week and 3 months. Sprague-Dawley rats were treated with the ETA/ETB antagonist bosentan, and at 6 months cardiac changes were assessed using the Langendorff perfused rat heart preparation, immunohistochemistry and real-time PCR. Ws/Ws and +/+ rat hearts did not differ in baseline mRNA. In contrast, +/+ rats hearts exhibited up-regulation of ET1 after irradiation, whereas Ws/Ws rats hearts did not, suggesting the possibility of interactions between mast cells and the cardiac ET system. Bosentan induced reductions in left ventricular systolic pressure, developed pressure and +dP/dtmax but did not affect fibrosis. Because of the known opposing effects of ETA and ETB, studies with selective antagonists may clarify the role of each receptor.

BACKGROUND

Human Urotensin II (hU-II) is the most potent vasoconstrictor known to date. HU-II receptors are predominant in the human heart and arterial vessels, suggesting hU-II to be of importance as a cardiovascular mediator.

METHODS

We studied 32 consecutive patients (60+/-12 years) with chronic heart failure (CHF) and 10 control subjects (54+/-12 years, n.s.) with cardiopulmonary exercise testing. Blood samples for the measurement of plasma hU-II and big-endothelin-1 (big-ET1) were obtained at rest and at peak exercise.

RESULTS

Peak VO(2) was significantly higher in controls than in CHF patients (19.8+/-3.8 vs. 14.7+/-3.6 ml min(-1) kg(-1), P<0.001). Big-ET1 levels were increased in CHF compared to controls at rest (2.8+/-1.8 vs. 1.7+/-0.1 fmol/ml, P<0.01) and at peak exercise (2.7+/-1.7 vs. 1.6+/-0.2 fmol/ml, P<0.005). HU-II concentrations were comparable in patients with CHF and controls at rest (2990+/-1104 vs. 3290+/-508 pg/ml, n.s.) and peak exercise (3063+/-1185 vs. 3213+/-1188 pg/ml, n.s.). Resting hU-II levels demonstrated no correlation with peak VO(2) in controls or CHF patients.

CONCLUSIONS

The measurement of circulating plasma levels of hU-II does not seem to be very helpful in studying the effects of hU-II in human cardiovascular regulation. A local paracrine or autocrine mediator effect of hU-II in CHF is possible.