Endothelin Receptors Expressed by Immune Cells Are Involved in Modulation of Inflammation and in Fibrosis: Relevance to the Pathogenesis of Systemic Sclerosis.

Endothelin-1 (ET-1) plays a pivotal role in vasoconstriction, fibrosis, and inflammation, the key features of systemic sclerosis (SSc). ET-1 receptors (ETA and ET(B)) are expressed on endothelial cells, smooth muscle cells, and fibroblasts, but their presence on immune cells has not been deeply investigated so far. Endothelin receptors antagonists such as bosentan have beneficial effects on vasoconstriction and fibrosis, but less is known about their potential anti-inflammatory effects. We studied the expression of ET-1 receptors on immune cells (T and B lymphocytes, monocytes, and neutrophils) and the link between ET-1 and inflammation in patients with SSc. We show here that ET-1 exerts a proinflammatory effect in CD4+ T cells, since it induces an increased IFN-γ production; preincubation with antagonists of both receptors reduces IFN-γ production. Moreover, following ET-1 stimulation, neutrophils produce proinflammatory mediators, thus amplifying the effects of activated CD4+ T cells. Our data indicate that ET-1 system is involved in the pathogenesis of inflammation and fibrosis typical of SSc, through the activation of T lymphocytes and neutrophils and the consequent release of proinflammatory and profibrotic cytokines. These findings suggest that dual ET-1 receptors antagonist therapy, besides its effect on vasculopathy, has a profound impact on the immune system favouring antiinflammatory and antifibrogenic effects.

Humoral immunity in hand transplantation: anti-HLA and non-HLA response.

UNLABELLED: Antibodies against donor's HLA antigens and B cell activity are recognized modulators of immune response to allograft. The role of both anti-HLA and non-HLA antibodies is understood in solid organ transplantation, but has not been addressed in composite tissue allografts. AIM: We decided to evaluate the presence and role of anti-HLA and non-HLA antibodies after hand transplantation. METHODS: We assayed anti-HLA and non-HLA antibodies in 5 consecutive hand transplant patients. The presence of anti-HLA antibodies was tested by flow-PRA method (One Lambda). Non-HLA antibodies were defined as anti-endothelial cell (AECA), anti-angiotensin II type 1 receptor (anti-AT1R), anti-endothelin receptor antibodies (anti-ETAR). RESULTS: Anti-HLA antibodies were present in 1 patient in class I and in another one in class II. Both patients developed one episode of acute rejection. AECA were present in only one recipient with borderline activity. Both anti-AT1R and Anti-ETAR were found strongly positive in one patient who repeatedly developed acute rejection episodes. CONCLUSION: The presence of non-HLA antibodies (anti-AT1R and anti-ETAR) and the occurrence of multiple rejection episodes found in one patient here require further investigation into a possible association and role of humoral immunity in composite tissue rejection.

Autoantibodies to angiotensin and endothelin receptors in systemic sclerosis induce cellular and systemic events associated with disease pathogenesis.

INTRODUCTION: Vasculopathy, inflammatory fibrosis and functional autoantibodies (Abs) are major manifestations of systemic sclerosis (SSc). Abs directed against the angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR) are associated with characteristic disease features including vascular, inflammatory, and fibrotic complications indicating their role in SSc pathogenesis. Therefore, the impact of anti-AT1R and anti-ETAR Abs on initiation of inflammation and fibrosis was analyzed. METHODS: Anti-AT1R and anti-ETAR Ab-positive immunoglobulin G (IgG) from SSc patients (SSc-IgG) was used for experiments. Healthy donor IgG served as a normal control, and AT1R and ETAR activation was inhibited by antagonists. Protein expression was measured with ELISA, mRNA expression with real time-PCR, endothelial repair with a scratch assay, and collagen expression with immunocytochemistry. Transendothelial neutrophil migration was measured with a culture insert system, and neutrophil ROS activation with immunofluorescence. Neutrophils in bronchoalveolar lavage fluids (BALFs) were analyzed microscopically after passive transfer of SSc-IgG or NC-IgG into naïve C57BL/6J mice. KC plasma levels were quantified by a suspension array system. Histologic analyses were performed by using light microscopy. RESULTS: Anti-AT1R and anti-ETAR Ab-positive SSc-IgG induced activation of human microvascular endothelial cells (HMEC-1). Elevated protein and mRNA levels of the proinflammatory chemokine interleukin-8 (IL-8, CXCL8) and elevated mRNA levels of the vascular cell adhesion molecule-1 (VCAM-1) were induced in HMEC-1. Furthermore, activation of HMEC-1 with SSc-IgG increased neutrophil migration through an endothelial cell layer and activation of reactive oxygen species (ROS). SSc-IgG decreased HMEC-1 wound repair and induced type I collagen production in healthy donor skin fibroblasts. Effects of migration, wound repair, and collagen expression were dependent on the Ab-levels. Passive transfer of anti-AT1R and anti-ETAR Ab-positive SSc-IgG into naïve C57BL/6J mice increased neutrophil BALF counts. In parallel, increased levels of the murine functional IL-8 homologue, chemokine KC, were found in the plasma of SSc-IgG-treated mice as well as structural alterations of the lungs. CONCLUSIONS: We conclude that angiotensin and endothelin-receptor activation via anti-AT1R and anti-ETAR Abs mediate pathogenic effects, indicating their contribution to pathogenesis of SSc. Therefore, anti-AT1R and anti-ETAR Abs could provide novel targets for therapeutic intervention in the treatment of SSc.

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Pulmonary hypertension associated with sarcoidosis.

Pulmonary involvement is common in sarcoidosis, an immune-mediated inflammatory disorder that is characterized by non-caseating granulomas in tissue. Sarcoid patients with advanced pulmonary disease, especially end-stage pulmonary fibrosis, risk developing pulmonary hypertension (World Health Organization group III pulmonary hypertension secondary to hypoxic lung disease). Increased levels of endothelin (ET)-1 in plasma and bronchoalveolar lavage of some sarcoid patients suggest that ET-1 may be driving pulmonary fibrosis and sarcoidosis-associated pulmonary hypertension. Although a relationship between raised levels of ET-1 and clinical phenotype is yet to be identified, early evidence from studies of ET-1 blockade with drugs such as bosentan is encouraging. Such therapy possibly could be combined with standard anti-inflammatory agents to improve outcome.

Increased gene expression and production of murine endothelin receptors after birth.

We developed the real-time PCR quantification of endothelin-A (ET-A) and endothelin-B (ET-B) receptor genes and present their relative expression levels in various adult tissues and during development in mouse using the 2(-Delta Delta C(T)) method. ET-A and ET-B receptors were detected in all tissues examined. Gene expression of ET-A and ET-B receptors increases during the later stages of embryonic development in lung, heart, liver, kidney, and skin and reaches a maximum on the first one or two days after birth. The results, in agreement with our data on endothelin (ET) ligands, suggest that the ET system may be involved in the emergence and maintenance of functions vital after birth in these organs. These findings were corroborated through observation of the correlation between the gene expression and (poly)peptide production of the ET system in normal skin before and after parturition.

Expression and cellular localisation of renal endothelin-1 and endothelin receptor subtypes in autosomal-dominant polycystic kidney disease.

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.

Endothelin B receptor-like immunoreactivity in podocytes of the rat kidney.

The distribution of endothelin B receptor (ETBR)-like immunoreactivity in the rat renal glomerulus was investigated using an affinity-purified antibody against a synthetic peptide corresponding to the amino acid residues 425-439 of the rat ETBR. Light microscopy showed ETBR-like immunoreactivity to be localized predominantly near the glomerular blood capillaries. By immunoelectron microscopy using the pre-embedding method, intense immunodeposits indicating ETBR were detected in podocytes, particularly in their foot processes, in contrast with the weak immunoreaction in endothelial cells of the glomerular blood capillaries and in the mesangial cells. In sections stained with the post-embedding method using immunogold particles, positive signals were also found on the plasma membrane of podocyte foot processes as well as the cytoplasm just beneath the cell membrane. These findings suggest that endothelin stimulates ETBR mainly on podocytes, thus resulting in a decrease of the glomerular blood flow and glomerular filtration rates.

Quantitative analysis of endothelin and nitric oxide synthase in rat model of postobstructive pulmonary vasculopathy.

There is increasing evidence that endothelin (ET) and endothelial nitric oxide synthase (eNOS) may contribute various kinds of pulmonary vascular remodeling, including postobstructive pulmonary vasculopathy (POPV), which resulted from chronic ligation of unilateral pulmonary artery. The aim of this study was to investigate the expression of ET-1, ET-A receptor, ET-B receptor, and eNOS quantitatively in POPV rats. One month after a left thoracotomy with either left main pulmonary artery ligation (ligated group) or no ligation (control group), rat pulmonary arteries and lungs were used for Western blot analysis using specific antibodies against ET-1, ET-A receptor, ET-B receptor, and eNOS. ET-A receptor was more highly expressed in the pulmonary arteries of ligated rats compared to the control. The expression of ET-1, ET-B receptor,and eNOS was not different between ligated and control rats. These findings suggest that ET-A receptor overexpression would play a main role for pulmonary arterial remodeling in POPV rats, whereas eNOS may serve as a compensatory mediator.

Distinct expression of endothelin receptor subtypes A and B in luteinized human granulosa cells.

Endothelins (ET) are potent vasoconstrictive peptides originally isolated from vascular endothelial cells. Their biological effects are mediated through two different receptors, the endothelin-1 (ET-1)-selective endothelin receptor subtype ETA and the non-selective receptor subtype ETB. ET-1 protein has been found in human ovarian follicular fluid and ET-1 mRNA expression has been demonstrated in ovarian tissue. These findings indicate that the endothelin-system participates in the modulation of ovarian function, probably acting in an autocrine/paracrine manner. In the current study we used freshly aspirated, luteinized human granulosa cells (hGC) representing an in vitro model of the early corpus luteum. By means of RT-PCR and immunocytochemistry we investigated whether luteinized human granulosa cells express ETA and ETB receptors. Specific amplification products of ETA transcripts were detected in all samples investigated. In contrast, only after using a three-fold amount of ETB reverse transcripts we were able to demonstrate specific, but weak amplification products. In addition, immunocytochemical staining for ETA but not for ETB was found in granulosa cell preparations. The present study provides clear evidence that human granulosa cells predominantly express ETA receptor subtype mRNA and protein hinting to its possible role in follicle maturation and corpus luteum formation.

Limitation of infarct size and attenuation of myeloperoxidase activity by an endothelin A receptor antagonist following ischaemia and reperfusion.

It has previously been shown that endothelin (ET) receptor antagonists limit myocardial ischaemia/reperfusion (I/R) injury. The mechanism behind this effect is still unclear. The aim of this study was to elucidate the possible relationship between cardioprotection by an ET(A) receptor antagonist and inhibition of neutrophil accumulation or activation in the myocardium determined as myeloperoxidase (MPO) activity during I/R. Anaesthetised pigs were subjected to 45 min ischaemia by ligation of the left anterior descending coronary artery (LAD) followed by 4 h of reperfusion. Infiltration of MPO-containing cells, presumably neutrophils, into the ischaemic area was confirmed with an immunohistochemical technique using antibodies against porcine MPO. Vehicle (n = 7) or the selective ET(A) receptor antagonist LU 135252 (LU; n = 7) were given into the LAD during the last 10 min of ischaemia and the first 5 min of reperfusion. There were no significant differences in LAD flow, mean arterial pressure, heart rate, or rate pressure product between the groups during I/R. The area at risk was similar in the two groups. LU reduced the final infarct size to 40+/-6% of the area at risk compared to 80+/-6% in the vehicle group (P < 0.001). Endothelin-like immunoreactivity increased 2-fold in the ischaemic area in the vehicle group (P < 0.01), but not in the group given LU. MPO activity was higher (2.5x) in the ischaemic than in the non-ischaemic myocardium of the vehicle group. The MPO activity in the ischaemic myocardium was significantly lower in the group given LU (7.0+/-1.2 units g(-1)) than in the vehicle group (14.2+/-1.9 units g(-1); P < 0.01). There was a significant correlation between the infarct size and MPO activity (P < 0.01, r = 0.68). In conclusion, local administration of the selective ET(A) receptor antagonist LU during the last period of ischaemia and early reperfusion reduces the extent of myocardial necrosis and MPO activity. This suggests that LU may exert its cardioprotective effect by inhibiting neutrophil-mediated injury.

Localization of endothelin-A and -B receptors during the postnatal development of rat cerebellum.

Intense expression of mRNA of endothelin-B receptor (ETBR) has been detected in the Bergmann glia of cerebellum by in situ hybridization, but the intracellular localization has not been reported because of the absence of a useful antibody for immunohistochemical investigations. We made polyclonal antibodies against the carboxyl terminus of human ETBR (420-442) and ETAR (403-427), and performed light- and electron-microscopic immunohistochemistry of the wild-type and ETBR-deficient (sl/sl) rat cerebella. Localization of ETBR during postnatal development was examined by double-staining immunofluorescence using antibodies against ETBR and S-100 beta. In the wild-type rats, ETBR immunoreactivity appeared from postnatal day 5 (P5) and was distributed diffusely in the processes and cell bodies of S-100 beta-positive glial cells. By P14, ETBR immunoreactivity was concentrated in the Golgi apparatus of Bergmann glial cell soma and the plasma membrane of its processes. The ETBR-positive astrocytes in the granular layer decreased in number during P7-14 and had disappeared by week 3. At 3 weeks, ETBR immunoreactivity was restricted to the Golgi apparatus of Bergmann glia. In the sl/sl rats, ETBR immunoreactivity was not observed at all. In contrast to ETBR, ETAR immunoreactivity appeared transiently in the cytoplasm of all astrocytes (Bergmann glia and astrocytes in the granular layer) in the 9- to 14-day-old wild rats and 7- to 14-day-old sl/sl rats, and disappeared within 3 weeks in both. Granule cells did not express immunoreactivity for ETBR and ETAR from the neonatal stage to adulthood. Changes in the intracellular localization of ETBR and transient expression of ETAR may be correlated with the changes of glial functions and proliferation during postnatal development of rat cerebellum.

Enhanced expression of endothelin receptor subtypes in cirrhotic rat liver.

BACKGROUND/AIMS: A number of vasoactive substances have been implicated as potential mediators of intrahepatic portal hypertension. Endothelin (ET)-1 has been suggested to be involved in the regulation of hepatic microcirculation and development of portal hypertension. The aim of this study was to clarify the localization of two subtypes of ET receptors, ET A (ETAR) and B receptors (ETBR), in normal rat liver, and how the receptor expressions are altered in CCl4-induced cirrhotic rat liver. METHODS: Liver specimens were examined immunohistochemically after reacting with anti-ETAR and anti-ETBR rabbit polyclonal antibodies. Immunogold staining was also performed using the same antibodies, and examined under light and electron microscopy. RESULTS: In normal rat liver, immunohistochemistry revealed expression of ETAR and ETBR on the hepatic sinusoidal lining cells. By immunogold electron microscopy, electron-dense gold particles indicating the presence of ETARs were localized mainly on hepatic stellate cells (HSCs) and to a lesser extent on sinusoidal endothelial cells (SECs), while ETBRs were expressed equally intensely on HSCs and SECs. In cirrhotic animals, both ETAR and ETBR increased significantly on HSCs, while there were no significant increases in either receptor on SECs. CONCLUSIONS: In the normal state, HSCs possess both ETARs and ETBRs, while SECs mainly possess ETBRs. In cirrhosis, endothelins may exert more intense effects on HSCs via the enhanced ETARs and ETBRs, causing an increase in hepatic sinusoidal microvascular tone.

Self-regulation of the endothelin receptor system in choriocarcinoma cells.

The human trophoblast secretes endothelin-1 (ET-1) and expresses ET receptors. The present study tested whether the transformed BeWo, JAR and JEG-3 choriocarcinoma cells: (1) secrete endothelin-1 (ET-1); (2) express both ET-A and ET-B receptor subtypes; and (3) have the potential to allow for autologous regulation of ET-receptor proteins. The cells were cultured for 24/48 h with or without 10% FCS and, in experiments on receptor regulation, with ET-1 (5-20 nM and 10 microM). ET-1 secretion was measured by RIA and receptor levels by immunoblotting. All cell types secreted ET-1 albeit at different levels and sensitivity to FCS. All cell lines expressed both ET-A (JEG-3>BeWo=JAR) and ET-B (JEG-3=JAR>BeWo) receptor subtypes, which could be up- and downregulated depending on ET-1 concentration, culture time and FCS presence. It is concluded that BeWo, JAR and JEG-3 choriocarcinoma cells secrete ET-1 and express both ET-A and ET-B receptor subtypes. The receptor levels can be regulated by ET-1. This provides the molecular basis for an autocrine system with the potential of autologous regulation of yet unidentified ET-1-induced functions.

Pulmonary vascular remodeling distal to pulmonary artery ligation is accompanied by upregulation of endothelin receptors and nitric oxide synthase.

There is increasing evidence that the pathogenesis and progression of many forms of pulmonary vasculopathy are related to abnormalities in endothelial mediators, including endothelin-1 (ET-1) and nitric oxide (NO). Using a rat model of chronic unilateral pulmonary artery ligation, we investigated the role of ET-1 and NO in postobstructive pulmonary vasculopathy (POPV). Eight months after a left thoracotomy with either left main pulmonary artery ligation (ligated group) or no ligation (sham group), rat lungs, including those contralateral to the ligation (hyperperfused group), were fixed and mounted for histologic sectioning. Morphometric measurements were carried out by computer-assisted image analysis and immunohistochemical staining was performed using specific antibodies against ET-1, ETA, and EBB receptors, and endothelial NO synthase (eNOS). Compared to sham lungs, the ligated lungs showed (1) an increase in muscular, adventitial, and intimal thickness of pulmonary artery; (2) increase in external diameter of the bronchial artery (39.8 +/- 2.2 microns vs. 16.8 +/- 0.9 microns in sham group; P < .005) and number of bronchial arteries per bronchiole (3.21 +/- mu 0.26 vs. 1.86 +/- mu 0.21 in sham group; P < .001); and (3) increase in the intensity of eNOS and ETA, B receptor immunoreactivity. No morphometric or immunohistochemical differences were observed between the hyperperfused and sham lungs. These findings suggest that increased synthesis of endothelial NO may serve as a compensatory mediator in ET-1-mediated vascular remodeling.

Subtype-specific desensitization of human endothelin ETA and ETB receptors reflects differential receptor phosphorylation.

Endothelins regulate blood pressure in mammals through G protein-coupled receptors. Two receptor subtypes, ETA and ETB, exist which differ by their agonist profiles. Here we show subtype-specific differences in the inactivation of these endothelin receptors. Using a modified inositol phosphate accumulation assay, we found that stimulation of ETA by endothelin-1 results in sustained activation of the subtype, retaining >30% of its initial activity even 20 min after agonist administration, whereas the ETB rapidly deactivated after agonist stimulation, losing >80% of its initial activity within 5 min after endothelin application. The discrepancy in receptor inactivation is reflected by subtype-specific differences in receptor phosphorylation. Whereas ETA failed to undergo ligand-induced phosphorylation, the ETB was rapidly phosphorylated in response to agonist stimulation. By contrast, the kinetics of ligand-induced internalization were essentially identical for the receptor subtypes, suggesting endothelin receptor internalization being independent of ligand-induced receptor phosphorylation. Interestingly, a strong correlation was observed between the time course of ETA inactivation and ETA internalization. Therefore, our data suggest a subtype-specific inactivation of human endothelin receptors: fast receptor phosphorylation in the case of ETB and slow receptor internalization in the case of ETA. Subtype-specific modulation of endothelin receptors may account for the short-term hypotensive effects of endothelins via rapidly down-regulating ETB receptors and the long-lasting hypertensive effects due to sustained ETA activation.

Identification of a 65 kDa endothelin receptor in bovine cardiac sarcolemmal vesicles.

Endothelin-1, an endothelial cell-derived vasoconstrictor peptide, also exerts a potent positive inotropic effect on cardiac tissue. Characterization of specific binding of endothelin-1 to bovine cardiac sarcolemmal vesicles is reported. In the presence of 1 mM CaCl2, the observed binding for 125I-endothelin-1 had a Kd of 6.2 nM with an observed Bmax of 14 pmol/mg sarcolemmal protein. In the presence of 1 mM EDTA (and no added Ca2+) Bmax was reduced to 9 pmol/mg sarcolemmal protein while the Kd remained unchanged. Binding affinity for sarafotoxin S6b was at least one order of magnitude less than for endothelin-1. 125I-Endothelin-1 covalently cross-linked to a sarcolemmal protein with an apparent molecular weight of 65 kDa. Site-directed polyclonal antibodies to a sequence located on the third extramembranal segment of a previously cloned endothelin ETA receptor from bovine lung were produced. Using Western blot analysis, the site-directed polyclonal antibody recognized a sarcolemmal protein at 65 kDa. We conclude that sarcolemmal membranes from bovine ventricular myocardium contain an endothelin binding site and that it is a protein with an apparent molecular weight of 65 kDa.

Immunochemical characterization and localization of endothelin ETB receptor.

A highly specific antiserum was raised against purified bovine endothelin ETB receptor and used to determine the tissue distribution of the receptor subtype ETB and to localize the receptor immunohistochemically in the kidney, adrenal gland, lung, cerebellum, and pituitary gland whose functions are known to be under strong influence of endothelin. The antiserum raised in a rabbit specifically recognized the receptor band on Western blot analysis of membrane proteins. Furthermore, it immunoprecipitated only ETB, establishing its ETB specificity. By determination of the percentage of the total number of the endothelin receptors that is immunoprecipitable with the antiserum, the amounts of the ETB relative to those of the ET receptors were found to vary from tissue to tissue: lung (70%), cerebellum (55%), pituitary gland (50%), kidney (25%), adrenal gland (10%), and testis (< 2%). This means that, in the lung, ET is the major form, whereas in the testis, ETA is predominant, comprising >95% of the receptors. Immunohistochemical examination of tissue sections revealed endothelium localization of the ETB endothelin receptor.

Endothelium localization of ETB receptor revealed by immunohistochemistry.

The ETB receptor has been found to be predominantly expressed in the vascular endothelium of various bovine tissues by immunostaining with a highly specific antiserum that does not cross-react with ETA. The tissues examined include the lung, trachea, kidney, adrenal gland, pituitary gland, and cerebellum. The wide-spread localization of ETB in the endothelial lining suggests that there is an ETB-mediated autocrine system of endothelin, which plays an important role in regulation of the functions of endothelial cells.