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Decreased cerebral blood flow (CBF) may contribute to the pathology of multiple sclerosis (MS), but the underlying mechanism is unknown. We investigated whether the potent vasoconstrictor endothelin-1 (ET-1) is involved. We found that, compared with controls, plasma ET-1 levels in patients with MS were significantly elevated in blood drawn from the internal jugular vein and a peripheral vein. The jugular vein/peripheral vein ratio was 1.4 in patients with MS vs. 1.1 in control subjects, suggesting that, in MS, ET-1 is released from the brain to the cerebral circulation. Next, we performed ET-1 immunohistochemistry on postmortem white matter brain samples and found that the likely source of ET-1 release are reactive astrocytes in MS plaques. We then used arterial spin-labeling MRI to noninvasively measure CBF and assess the effect of the administration of the ET-1 antagonist bosentan. CBF was significantly lower in patients with MS than in control subjects and increased to control values after bosentan administration. These data demonstrate that reduced CBF in MS is mediated by ET-1, which is likely released in the cerebral circulation from reactive astrocytes in plaques. Restoring CBF by interfering with the ET-1 system warrants further investigation as a potential new therapeutic target for MS.

Substance P (SP) is a well-known neuropeptide implicated in the wound-healing process. The wound occasionally causes a pigmented scar. In the present study, we examined whether increased levels of SP affected melanogenesis. When human melanocytes were treated with SP, the melanin content increased and the pigmentation process accelerated in a dose-dependent manner. In addition to melanogenesis-related genes, the expression of neurokinin 1 receptor, endothelin 1 (EDN1), and EDN receptor type B (EDNRB) also increased at both the messenger RNA and protein levels. Interestingly, secreted EDN1 was observed in the melanocyte culture medium, and this phenomenon was significantly enhanced by SP treatment. Through knockdown experiments using small interfering RNAs (siRNAs), we confirmed that endothelin-converting enzyme 1 (ECE1), EDN1, and EDNRB were involved in SP-induced pigmentation and found that EDN1 secretion was affected by ECE1 and EDN1 siRNAs, but not by EDNRB siRNA. These findings indicate that ECE1 is essential for EDN1 secretion in melanocytes and that EDNRB functions downstream of secreted EDN1 to increase the cAMP levels and activate the melanogenesis-related phosphorylation cascade. This study provides in vitro evidence for a melanogenic function of SP in the skin and suggests that the SP-related signal is a potent target for regulating stress- or wound-induced pigmentation.

The molecular mechanism linking aldosterone and endothelin-1 in the development of diabetic nephropathy has not been completely elucidated. Here, we provide evidence showing that streptozotocin-induced diabetic rats have significantly increased aldosterone and endothelin-1 in the kidney tissue and markedly decreased expression of Dot1a and Af9. Blocking aldosterone with spironolactone significantly reduced proteinuria, glomerulosclerosis, tubulointerstitial injury and endothelin-1 expression, and significantly increased Dot1a and Af9 expression. Increasing Dot1a and Af9 expression by spironolactone or by stable transfection led to impaired endothelin-1 expression in NRK-52 cells. In contrast, downregulation of Dot1a and Af9 by aldosterone in NRK-52E cells caused upregulation of endothelin-1. Genetic inactivation of Dot1l, which encodes Dot1a, in Aqp2-expressing principal cells of mouse kidney impaired association of Dot1a and H3 dimethyl K79 with the specific subregions of endothelin-1 promoter, and upregulates endothelin-1 mRNA and protein expression. Our data suggest that Dot1a and Af9 repress endothelin-1 in vitro and in vivo. Excessive aldosterone induces kidney injury, in part possibly by downregulating Dot1a and Af9, and thus relieving Dot1a-Af9-mediated repression to increase endothelin-1 transcription. Spironolactone ameliorates kidney injury in Streptozotocin-induced diabetic rats, possibly by restoring Dot1a-Af9-mediated repression to reduce endothelin-1 expression. Therefore, Dot1a and Af9 as aldosterone-downregulated targets are negative regulators of endothelin-1 transcription in vitro and in vivo, and may be considered as new potential therapeutic targets of kidney injury in diabetes.

Endothelialization of a vascular stent is a critical step to prevent late stent thrombosis. In this study, electron beam deposition was utilized to create different scales of roughness on titanium stents, including flat features (F-Ti), a mixture of nanometer and submicron features (S-Ti), and nanometer features (N-Ti). The role of stent surface roughness on initial protein adsorption, platelet adhesion, rat aortic endothelial cell adhesion, migration, and nitric acid/endothelin-1 secretion was investigated in vitro. Results revealed the highest endothelial cell attachment on S-Ti after 4 h. Moreover, under flow conditions, the endothelial cell layer remained the most intact on S-Ti. These results were positively correlated with improved vitronectin adsorption on S-Ti. Endothelial cells also showed the fastest migration on S-Ti of all the samples over a 96 h time period. Endothelial cells on S-Ti exhibited the highest nitric acid/endothelin-1 ratio of all the samples, indicating potentially the best antithrombic endothelial cellular phenotype. This study also revealed the lowest platelet adhesion on S-Ti of all the samples. In summary, without using pharmaceutical agents, significantly less platelet and greater endothelial responses on nanometer to submicron rough titanium were observed in this study compared to flat titanium and, thus, nanometer to submicron surface features on titanium should be further studied for vascular stent applications.

Transforming growth factor-β (TGF-β) and endothelin (ET) are found in elevated amounts in the airways of individuals with asthma. The cellular source of these peptides and their role in mediating the airway fibrosis of chronic asthma are unknown. In response to mechanical stresses similar to those occurring in vivo during airway constriction, bronchial epithelial cells increase the steady-state level of mRNA for both ET-1 and ET-2, followed by increased release of ET protein. Mechanical stress also enhances release of TGF-β2 from a preformed cell-associated pool. TGF-β2 and ET act individually and, more importantly, synergistically to promote fibrotic protein synthesis in reporter fibroblasts. To confirm the role of these intermediates in stress-induced fibrosis, conditioned medium from mechanically stressed bronchial epithelial cells was shown to elicit fibrotic protein synthesis in reporter fibroblasts; this effect was significantly inhibited by combined treatment with ET receptor antagonists and a neutralizing antibody to TGF-β2. These data are consistent with a primary pathogenic role for mechanical stress–induced release of both TGF-β2 and ET in the subepithelial fibrosis that characterizes chronic asthma.

Vascular Peptide Endothelin-1 Links Fat Accumulation With Alterations of Visceral Adipocyte Lipolysis Vanessa van Harmelen 1 , Anna Eriksson 1 , Gaby Åström 1 , Kerstin Wåhlén 1 , Erik Näslund 2 , Fredrik Karpe 3 , Keith Frayn 3 , Tommy Olsson 4 , Jonas Andersson 4 , Mikel Rydén 1 and Peter Arner 1 1 Department of Medicine, Karolinska Institutet at the Karolinska University Hospital, Stockholm, Sweden 2 Department of Clinical Sciences, Danderyds Hospital, Karolinska Institutet, Stockholm, Sweden 3 Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital Oxford, U.K 4 Department of Medicine, Umeå University Hospital, Umeå, Sweden Address correspondence and reprint requests to Peter Arner, MD, PhD, Karolinska Institutet, Department of Medicine, M63, Karolinska University Hospital, Huddinge, SE-141 86 Stockholm, Sweden. E-mail: peter.arner{at}ki.se Abstract OBJECTIVE— Visceral obesity increases risk of insulin resistance and type 2 diabetes. This may partly be due to a region-specific resistance to insulin's antilipolytic effect in visceral adipocytes. We investigated whether adipose tissue releases the vascular peptide endothelin-1 (ET-1) and whether ET-1 could account for regional differences in lipolysis. RESEARCH DESIGN AND METHODS— One group consisted of eleven obese and eleven nonobese subjects in whom ET-1 levels were compared between abdominal subcutaneous and arterialized blood samples. A second group included subjects undergoing anti-obesity surgery. Abdominal subcutaneous and visceral adipose tissues were obtained to study the effect of ET-1 on differentiated adipocytes regarding lipolysis and gene and protein expression. RESULTS— Adipose tissue had a marked net release of ET-1 in vivo, which was 2.5-fold increased in obesity. In adipocytes treated with ET-1, the antilipolytic effect of insulin was attenuated in visceral but not in subcutaneous adipocytes, which could not be explained by effects of ET-1 on adipocyte differentiation. ET-1 decreased the expression of insulin receptor, insulin receptor substrate-1 and phosphodiesterase-3B and increased the expression of endothelin receptor-B (ET B R) in visceral but not in subcutaneous adipocytes. These effects were mediated via ET B R with signals through protein kinase C and calmodulin pathways. The effect of ET-1 could be mimicked by knockdown of IRS-1. CONCLUSIONS— ET-1 is released from human adipose tissue and links fat accumulation to insulin resistance. It selectively counteracts insulin inhibition of visceral adipocyte lipolysis via ET B R signaling pathways, which affect multiple steps in insulin signaling. ERK, extracellular signal–related kinase ET-1, endothelin-1 ETAR, endothelin receptor-A ETBR, endothelin receptor-B FFA, free fatty acid GPDH, glycerol-3-phosphate dehydrogenase IRS, insulin receptor substrate MEK, mitogen-activated protein kinase kinase OM, omental PI3K, phosphatidylinositol 3-kinase PKC, protein kinase C SC, subcutaneous TNF, tumor necrosis factor Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 19 November 2007. DOI: 10.2337/db07-0893. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted November 13, 2007. Received July 2, 2007. DIABETES

Abstract Interleukin (IL)-4 and IL-13 are key proinflammatory cytokines in asthma. Studies in transgenic mice show that both cytokines cause inflammation, but only IL-13 causes subepithelial fibrosis, a characteristic feature of asthma. We compared the in vitro profibrogenic effects of IL-4 and IL-13 using bronchial fibroblasts from asthmatic subjects. In the presence of transforming growth factor (TGF)- β the cells transformed into contractile myofibroblasts and expressed α -smooth muscle actin and procollagen I. IL-4 and IL-13 also stimulated proliferation, but were relatively ineffective in promoting myofibroblast transformation. TGF- β was more potent than the cytokines in stimulating release of endothelin-1 and vascular endothelial growth factor, whereas IL-4 and IL-13 were more potent stimuli for eotaxin release. Although neither IL-4 nor IL-13 induced profibrotic responses, both cytokines caused a corticosteroid-insensitive stimulation of TGF- β 2 release from primary bronchial epithelial cells. These data indicate that epithelial activation by IL-13 or IL-4 plays a critical role in initiating remodeling through release of TGF- β 2. TGF- β 2 then activates the underlying myofibroblasts to secrete matrix proteins and smooth muscle and vascular mitogens to propagate remodeling changes into the submucosa. In contrast, direct activation of submucosal fibroblasts by IL-4 and IL-13 has a proinflammatory effect via eotaxin release and recruitment of eosinophils into the airways.

Background Adrenomedullin (ADM), a novel vasorelaxant peptide, was found in human/rat ovaries. The present study investigated the interaction of ADM and endothelin-1 (ET-1) in follicles and newly formed corpora lutea (CL) and the actions of ADM on progesterone production in CL during pregnancy. Methods The peptide and gene expression level of adrenomedullin in small antral follicles, large antral follicles and CL was studied by real-time RT-PCR and EIA. The effect of ADM treatment on oestradiol production in 5-day follicular culture and on progesterone production from CL of different pregnant stages was measured by EIA. The interaction of ADM and ET-1 in follicles and CL at their gene expression level was studied by real-time RT-PCR. Results In the rat ovary, the gene expression of Adm increased during development from small antral follicles to large antral follicles and CL. In vitro treatment of preantral follicular culture for 5 days with ADM increased oestradiol production but did not affect follicular growth or ovulation rate. The regulation of progesterone production by ADM in CL in culture was pregnancy-stage dependent, inhibitory at early and late pregnancy but stimulatory at mid-pregnancy, which might contribute to the high progesterone production rate of the CL at mid-pregnancy. Moreover, the interaction between ADM and ET-1 at both the production and functional levels indicates that these two vasoactive peptides may form an important local, fine-tuning regulatory system together with LH and prolactin for progesterone production in rat CL. Conclusions As the CL is the major source of progesterone production even after the formation of placenta in rats, ADM may be an important regulator in progesterone production to meet the requirement of pregnancy.

Early infections and wound healing disorders after implantation of a total knee replacement occur regardless of the intraoperative use of a tourniquet. The biochemical regulatory processes responsible for the disturbances in microcirculation and thus the potential therapeutic options have yet to be elucidated. The hypothesis of the present paper was that endothelin-1 (ET-1), a mediator of microcirculation disturbances in parenchymatous organs, also is released after major operations on peripheral joints. The concentration of ET-1 in the plasma was determined preoperatively and at 10 postoperative time points (5 min–48 h) with (group A, n = 10) and without the use of a tourniquet (group B, n = 10). The ET-1 concentration achieved its maximum 6 h after opening the tourniquet, which corresponded to 3.3 times the preoperative value. Without a tourniquet, the concentration maximum (2.9 times the baseline value) was achieved already 1.5 h after the end of the operation. However, the total amount of ET-1 secreted over 24 h was identical in both groups ( p > 0.5). We conclude that the tissue hypoxia resulting from the use of a tourniquet modulates ET-1 secretion, but that traumatization during the operation has a much stronger influence on the total amount secreted. ET-1 antagonists thus should be discussed for the drug prophylaxis of wound healing disorders, regardless of the use of a tourniquet.

Endogenous direct current electric fields (dcEFs) play a significant role in major biological processes such as embryogenesis, wound healing, and tissue regeneration. In this study, the galvanotaxis of human umbilical vein endothelial cell line EA.Hy926 was investigated by using a novel designed bioreactor. The physical features of the bioreactor were discussed and analyzed by both numerical simulation method and equivalent circuit model method. EA.Hy926 cells were cultured in the bioreactor for 10–24 h under 50–250 mV/mm dcEFs. Cell migration direction, distance, and velocity were recorded under an online time-lapse microscope. The effects of serum and growth factor on cell galvanotatic migration were investigated. To further explore the role of dcEFs in regulating endothelial cells, we analyzed the endothelial cell proliferation and secretion of nitric oxide (NO), endothelin-1 (ET-1) in response to dcEFs of physiological strength. Our results showed that EA.Hy926 cells had an obvious directional migration to the cathode, and the EF-directed migration was voltage dependent. The results also showed dcEFs did not affect cell proliferation, but affected the productions of NO and ET-1. Our study also showed the novel bioreactor, with a compact and planar style, makes it more convenient and more reasonable for EF stimulation experiments than earlier chamber designs.