Endothelin. Immunohistologic localization in aorta and biosynthesis by cultured human aortic endothelial cells.

BACKGROUND: Endothelin-1 (ET-1) has been shown to exist in many organs and to have various biologic functions including vasoconstriction. However, an exact location of ET gene expression of the tissues is not fully investigated. Human aortic tissue was examined to elucidate the exact location of ET gene expression. EXPERIMENTAL DESIGN: Human aortas were obtained at autopsy and fixed in either conventional 10% formalin or 3% paraformaldehyde. The aortic thin sections were subjected to examinations of an immunohistochemistry and in situ hybridization of ET-1. Human aortic endothelial cells were cultured by a previously reported method. ET-1 released in the supernatant from the cultured endothelial cells was radioimmunoassayed. RESULTS: Immunohistologic study of ET-1 revealed a linear staining of the endothelial monolayer and diffuse staining in the intimal and medial smooth muscle cells on human aorta except for fetal aorta. In situ hybridization signals were intense in the endothelial cells from the elderly as well as younger subjects as examined with 35S-labeled anti-sense probe RNA. Fetal aortic endothelial cells revealed the least signals that meant developing but still immature gene translation. Smooth muscle cells showed positive but weak in situ hybridization signals. Control immunohistologic and hybridization studies were negative. ET-1 biosynthesis by cultured human aortic endothelial cells was invariably low in the subjects under the age of 50, ranging from 0.23 to 0.40 pmol/1 x 10(5) cells for 3 days. On the other hand, endothelial cells from the elderly subjects generally synthesized a greater amount of endothelin in vitro. CONCLUSIONS: These findings indicate that ET-1 is most highly expressed in endothelial cells, although not as highly but certainly, expressed in intimal and medial smooth muscle cells. This fact gives a new insight into the biophysiologic and pathologic roles of ET. In addition, these methods are applicable to investigate the gene expression of ET-1 in all organs and tissues.     

The endothelin system in human glioblastoma

Endothelin-1 (ET-1) is a powerful mitogenic and/or anti-apoptotic peptide produced by many cancer cells. To evaluate the potential role of the endothelin system in glioblastoma we first determined the cellular distribution of the mRNA and proteins of the components of the endothelin system, preproendothelin-1 (PPET-1), endothelin-converting enzyme-1 (ECE-1), and ET(A) and ET(B) receptors in human glioblastoma tissue and glioblastoma cell lines. PPET-1, ECE-1, and ET(A) receptor were highly expressed in glioblastoma vessels and in some scattered glioblastoma areas whereas ET(B) receptor was mainly found in cancer cells. This suggests that glioblastoma vessels constitute an important source of ET-1 that acts on cancer cells via the ET(B) receptor. Four human glioblastoma cell lines expressed mRNA for all of the components of the ET-1 pathway. Bosentan, a mixed ET(A) and ET(B) receptor antagonist, induced apoptosis in these cell lines in a dose-dependent manner. Apoptosis was potentiated by Fas Ligand (APO-1L, CD95L), a pro-apoptotic peptide, only in LNZ308 cells, corresponding to the known functional Fas expression in these cell lines. LNZ308 cells also expressed the long and short forms of the cellular FLICE/caspase-8 inhibitory protein (FLIP). Bosentan and a protein kinase C inhibitor down-regulated short FLIP in these cells. ET-1 induced transient phosphorylation of extracellular signal-regulated kinase but did not induce long-term thymidine incorporation in LNZ308 glioblastoma cells. These results suggest that, in glioblastoma cells, ET-1, mainly acting via the ET(B) receptor, is a survival/antiapoptotic factor produced by tumor vasculature, but not a proliferation factor, involving protein kinase C and extracellular signal-regulated kinase pathways, and stabilization of the short form of FLIP.     

Inheritance analysis of congenital left ventricular outflow tract obstruction malformations: Segregation, multiplex relative risk, and heritability

The left ventricular outflow tract (LVOTO) malformations, aortic valve stenosis (AVS), coarctation of the aorta (COA), and hypoplastic left heart (HLH) constitute a mechanistically defined subgroup of congenital heart defects that have substantial evidence for a genetic component. Evidence from echocardiography studies has shown that bicuspid aortic valve (BAV) is found frequently in relatives of children with LVOTO defects. However, formal inheritance analysis has not been performed. We ascertained 124 families by an index case with AVS, COA, or HLH. A total of 413 relatives were enrolled in the study, of which 351 had detailed echocardiography exams for structural heart defects and measurements of a variety of aortic arch, left ventricle, and valve structures. LVOTO malformations were noted in 30 relatives (18 BAV, 5 HLH, 3 COA, and 3 AVS), along with significant congenital heart defects (CHD) in 2 others (32/413; 7.7%). Relative risk for first-degree relatives in this group was 36.9, with a heritability of 0.71-0.90. Formal segregation analysis suggests that one or more minor loci with rare dominant alleles may be operative in a subset of families. Multiplex relative risk analysis, which estimates number of loci, had the highest maximum likelihood score in a model with 2 loci (range of 1-6 in the lod-1 support interval). Heritability of several aortic arch measurements and aortic valve was significant. These data support a complex but most likely oligogenic pattern of inheritance. A combination of linkage and association study designs is likely to enable LVOTO risk gene identification. This data can also provide families with important information for screening asymptomatic relatives for potentially harmful cardiac defects.     

BMP4 is required in the anterior heart field and its derivatives for endocardial cushion remodeling, outflow tract septation, and semilunar valve development

The endocardial cushions play a critical role in septation of the four-chambered mammalian heart and in the formation of the valve leaflets that control blood flow through the heart. Within the outflow tract (OFT), both cardiac neural crest and endocardial-derived mesenchymal cells contribute to the endocardial cushions. Bone morphogenetic protein 4 (BMP4) is required for endocardial cushion development and for normal septation of the OFT. In the present study, we show that anterior heart field (AHF)-derived myocardium is an essential source of BMP4 required for normal endocardial cushion expansion and remodeling. Loss of BMP4 from the AHF in mice results in an insufficient number of cells in the developing OFT endocardial cushions, defective cushion remodeling, ventricular septal defects, persistent truncus arteriosus, and abnormal semilunar valve formation.     

Endothelin and endothelin receptors A and B in the human testis

 Human testicular capillaries interconnect Leydig cells and seminiferous tubules. Microcirculation and blood flow are therefore essential for the maintenance of spermatogenesis. The expression and the localisation of ET (endothelin) and its receptors in testicular tissue, in seminiferous tubules and in human testicular capillaries were studied. ET-1 mRNA was detected in whole testicular tissue and in seminiferous tubules whereas isolated testicular capillaries were negative. Big ET-1 (Big endothelin 1) and ET peptides were localised in Leydig and Sertoli cells whereas interstitial and intramural capillaries (within the lamina propria) remained unstained. ET was also found in mature spermatids. ET-A (endothelin receptor A) mRNA was detected in seminiferous tubules and whole testicular tissue whereas testicular blood vessels were negative. ET-A immunostaining was displayed in Leydig and Sertoli cells and in spermatids. ET-B (endothelin receptor B) mRNA was detected in whole testicular tissue, seminiferous tubules and in testicular capillaries. ET-B peptide was prominent in Leydig cells, peritubular cells, endothelial cells and pericytes of interstitial and intramural capillaries as well as in vascular endothelial and smooth muscle cells. From these results we conclude that ET produced in Leydig and Sertoli cells can act in a paracrine manner via ET-B on the human testicular microvasculature and the peritubular cells. The presence of both ET-A and ET-B in Leydig cells and of ET-A in Sertoli cells leads to the assumption that ET could influence these cells as an autocrine factor. Accepted: 9 October 1998     

Chromosome abnormalities in congenital heart disease

Refinements in cytogenetic techniques have promoted progress in understanding the role that chromosome abnormalities play in the cause of congenital heart disease. To determine if mutations at specific loci cause congenital heart disease, irrespective of the presence of other defects, and to estimate the prevalence of chromosome abnormalities in selected conotruncal cardiac defects, we reviewed retrospectively cytogenetic and clinical databases at St. Louis Children's Hospital. Patients with known 7q11.23 deletion (Williams syndrome), Ullrich-Turner syndrome (UTS), and most autosomal trisomies were excluded from this analysis. Two groups of patients were studied. Over a 6.5-year period, 57 patients with chromosomal abnormalities and congenital heart disease were identified. Of these, 37 had 22q11 deletions; 5 had abnormalities of 8p; and 15 had several other chromosome abnormalities. The prevalence of chromosome abnormalities in selected conotruncal or aortic arch defects was estimated by analysis of a subgroup of patients from a recent 22-month period. Chromosome abnormalities were present in 12% of patients with tetralogy of Fallot, 26% in tetralogy of Fallot/pulmonary atresia, 44% in interrupted aortic arch, 12% in truncus arteriosus, 5% in double outlet right ventricle, and 60% in absent pulmonary valve. We conclude that chromosome analysis should be considered in patients with certain cardiac defects. Specifically, fluorescent in situ hybridization (FISH) analysis of 22q11 is indicated in patients with conotruncal defects or interrupted aortic arch. High resolution analysis should include careful evaluation of the 8p region in patients with either conotruncal or endocardial cushion defects. Am. J. Med. Genet. 70:292–298, 1997. © 1997 Wiley-Liss, Inc.     

Cellular distribution of the endothelin system in the human brain

The vasoconstrictor endothelin-1 (ET-1) may also act as a neuropeptide. ET-1 is formed by the catalytic action of endothelin-converting enzyme-1 (ECE-1) on big ET-1 and its cellular actions are mediated via ET(A) and ET(B) receptors. Although localisation of these components in rodent brain has been extensively investigated, no single study has mapped their distribution in human brain. Here we describe the localisation of ET-1 mRNA, ET-1, ECE-1, ET(A) and ET(B) receptors within 24 human brain regions. In situ RT-PCR has previously detected ET-1 mRNA in 22 areas (excluding the post-central gyrus and pineal gland), and ET-1 immunoreactivity was visualised in cells of all regions. Using specific antibodies we have immunolocalised ECE-1 and ET(B) receptors in cells of 24 areas, and ET(A) receptors in nine regions (choroidal epithelial cells, neurones in the diencephalon, hippocampus, amygdaloid, dentate nucleus, Purkinje cells of the cerebellum, flocculo-nodular lobe and vermis). ET-1 mRNA, ET-1, ECE-1 and ET(B) receptors were observed in cortical pyramidal cells, neurones (brainstem, basal nuclei, thalamus, insula and claustrum, limbic region), cells in the anterior pituitary gland; nerve cell processes in the pars nervosa; pinealocytes and choroidal epithelial cells. Only ET-1 mRNA, ET-1, ECE-1, and ET(B) receptors were visualised in cerebral capillary endothelial cells. The presence of ET-1 mRNA, ECE-1 and ET-1 in 22 brain regions confirms ET expression and processing in human brain. The localisation of ET-1 and ET(B) receptors suggests receptor-mediated action akin to a neurotransmitter role for ET-1.     

小鼠胚胎细胞视黄酸结合蛋白1阳性神经嵴细胞的时空分布与功能

目的 探讨小鼠胚胎心神经嵴细胞的形成、分布模式及其在心血管系统发育过程中的作用。方法 选用抗细胞视黄酸结合蛋白1（CRABP1）、抗α-平滑肌肌动蛋白（α-SMA）、抗心肌肌球蛋白重链（MHC）、抗胰岛因子1（Isl-1）抗体,对45只胚龄8～12d小鼠胚胎连续切片进行免疫
组织化学染色。结果 胚龄8d,CRABP1在神经褶的外胚层未见阳性表达。胚龄8.5～9d,在心管与鳃弓水平,神经褶开始出现CRABP1阳性细胞,且有部分细胞从神经褶背侧分离进入邻近间充质。胚龄10d,神经管两侧间充质内的CRABP1阳性细胞迁移至鳃弓、弓动脉壁内皮周围以及流出道
心胶质内。胚龄11～12d,弓动脉内皮周围、流出道心内膜垫内CRABP1表达明显下降,但弓动脉管壁α-SMA阳性平滑肌细胞数量增加。主肺动脉隔及其分隔形成的升主动脉和肺动脉干管壁内均可见Isl-1阳性细胞,但未见CRABP1表达。结论 小鼠胚胎CRABP1阳性神经嵴细胞形成的时间窗
限定在胚龄8.5～9d。胚龄10d后,CRABP1阳性神经嵴细胞经过迁移,参与弓动脉中膜平滑肌和流出道心内膜垫的形成。CRABP1不能用于标记迁移后的神经嵴细胞。     

Inactivation of Bmp4 from the Tbx1 expression domain causes abnormal pharyngeal arch artery and cardiac outflow tract remodeling

Maldevelopment of outflow tract and aortic arch arteries is among the most common forms of human congenital heart diseases. Both Bmp4 and Tbx1 are known to play critical roles during cardiovascular development. Expression of these two genes partially overlaps in pharyngeal arch areas in mouse embryos. In this study, we applied a conditional gene inactivation approach to test the hypothesis that Bmp4 expressed from the Tbx1 expression domain plays a critical role for normal development of outflow tract and pharyngeal arch arteries. We showed that inactivation of Bmp4 from Tbx1-expressing cells leads to the spectrum of deformities resembling the cardiovascular defects observed in human DiGeorge syndrome patients. Inactivation of Bmp4 from the Tbx1 expression domain did not cause patterning defects, but affected remodeling of outflow tract and pharyngeal arch arteries. Our further examination revealed that Bmp4 is required for normal recruitment/differentiation of smooth muscle cells surrounding the PAA4 and survival of outflow tract cushion mesenchymal cells.     

Endothelins-1/3 and endothelin-A/B receptors expressing glial cells with special reference to activated microglia in experimentally induced cerebral ischemia in the adult rats

We reported previously that amoeboid microglial cells (AMC) in the developing brain exhibited endothelins (ETs) expression which diminished with advancing age and was undetected in microglia in the more mature brain. This study sought to explore if microglia in the adult would be induced to express ETs in altered conditions. By immunofluorescence microscopy, ETs and endothelin (ET)-B receptor were undetected in microglial cells in sham-operated and normal control rats. However, in adult rats subjected to middle cerebral artery occlusion (MCAO), lectin labeled activated microglia which occurred in large numbers in the marginal zones in the ischemic cortex at 3 days and 1 week intensely expressed ETs specifically endothelin (ET)-1 and ET-B receptor; ET-3 and ET-A receptor were absent in these cells. By RT-PCR and ELISA, ET-1 and -3 mRNA and protein expression level was progressively increased in the ischemic cerebral cortex after MCAO compared with the controls. ET-A and ET-B receptor mRNA and protein levels were concomitantly up-regulated. It is suggested that increased release of ET-1 following MCAO by massive activated microglia can exert an immediate constriction of local blood vessels bearing ET-A receptor. ET-1 may also interact with activated microglia endowed with ET-B receptor via an autocrine manner that may be linked to chemokines/cytokines production. ET-1, ET-3 and ET-B receptor were also localized in reactive astrocytes along with some oligodendrocytes. We conclude that activated microglia together with other glial cells in the marginal zone after MCAO are the main cellular source of ETs that may be involved in regulation of vascular constriction and glial chemokines/cytokines production. However, dissecting the role of individual component of the endothelin system in the various glial cells, notably activated microglia, would be vital in designing of an effective therapeutic strategy for clinical treatment of stroke in which microglial cells have been implicated.     

The cardiac endothelin system in established pressure overload left ventricular hypertrophy

In normal hearts, endothelin-1 (ET-1) has been shown to initiate myocyte growth and to modulate cardiac function. However, regulation of the various components of the system and the functional effects of ET-1 in established left ventricular hypertrophy (LVH) are less clear. We thus studied ET-1, ET(A) receptor, and endothelin converting enzyme (ECE-1) mRNA regulation as well as the effects of ET-1 on coronary resistance, LV contractility and relaxation in hypertrophied rat hearts. Cardiac pressure overload, secondary to banding of the ascending aorta, resulted in a transient increase of cardiac ET-1 and ET(A) receptor mRNAs that reached a maximum at 2 days (+75% and +40%, respectively, P<0.05, each). ET-1 mRNA levels reached a second peak at 84 days of pressure overload (+60%, P<0.05), at the later time point in conjunction with elevated ECE-1 mRNA levels (+20%, P<0.05). The functional implications of ET-1 were examined in a study of isolated perfused hearts. Both hearts with established LVH and sham control hearts responded to ET-1 perfusion (10(-1)] to 10(-9) M) with an increase of coronary perfusion pressure (CPP; +85+/-15 and +75+/-8 mm Hg; P<0.001 each) and a slight decrease of LV systolic pressure (LVP; -12+/-9 and -9+/-7 mm Hg; P = NS). In contrast, ET-1 increased LV end-diastolic pressure (LVEDP) only in LVH hearts (+22+/-7 mm Hg, P<0.05 versus baseline and +20+/-7 mm Hg, P<0.05 versus sham). Direct stimulation of protein kinase C mimicked the effects of ET-1, whereas inhibition of this kinase or the Na+ -H+ exchanger blunted the effects of ET-1 on CPP, LVP, and LVEDP. Interestingly, coadministration of the vasodilator and the nitric oxide (NO) donor nitroglycerin not only prevented the increase of CPP and LVEDP, but also uncovered a slight positive inotropic effect of ET-1 in LVH hearts. Thus, the cardiac expression of ET-1, ET(A), and ECE-1 mRNAs displays a distinct pattern during early and advanced cardiac pressure overload. Furthermore, ET-1 mediates a slight depression of systolic, and a profound depression of diastolic, functional parameters in hearts with established LVH, effects that appear to be secondary to ET-1-related coronary vasoconstriction. The data suggest a functional role of the endothelin system in hearts with established pressure overload hypertrophy.     

Differential adrenomedullin release and endothelin receptor expression in distinct subpopulations of human airway smooth-muscle cells

Although adrenomedullin (ADM) is implicated in the control of airway tone, regulation of ADM release from airway smooth-muscle cells (ASMCs) has not been explored. Preliminary experiments have indicated that human ASMC populations were heterogeneous in their rate of ADM release and expression of endothelin (ET)(A) and ET(B) receptors. We isolated these phenotypically distinct ASMCs from explants derived from the same airway segment. ASMCs possessing exclusively ET(A) receptors appeared smaller and proliferated faster than ET(A)/ET(B) isolates. Macroautoradiographic analysis confirmed the presence of both receptors in human bronchi. ADM release and messenger RNA expression was greater in ET(A)/ET(B) isolates compared with ET(A) isolates. No measurable ET release was detected from ASMCs. Exogenous ET-1 (1 to 100 nM) more potently stimulated the release of ADM from ET(A)/ET(B) compared with ET(A) isolates. In addition, ET-3 (1 to 100 nM) stimulated ADM release only from ET(A)/ET(B) isolates, implicating the ET(B) receptor in this response. Exogenous ET-1 potentiated platelet- derived growth factor-stimulated [3H]thymidine uptake in ET(A)/ ET(B) but not ET(A) isolates. ET-3 did not affect [3H]thymidine uptake in either cell type. Possession of ET(A)/ET(B) receptors is associated with higher rates of ADM release and slower proliferation, but a capacity for ET-1 stimulated DNA synthesis via ET(A) receptors. These results support a paracrine role for the regulation of ADM release predominantly via the ET(B) receptor in human ASMCs.     

Ece1 and Tbx1 define distinct pathways to aortic arch morphogenesis.

Pharyngeal arch artery (PAA) remodeling defects account for several cases of congenital heart disease. Mutations in the Endothelin-1 genetic pathway or Tbx1, a candidate gene for DiGeorge syndrome, cause similar aortic arch defects. Previous research suggests that Tbx1 may trigger diffusible signals from the pharyngeal arches to support the growth of the PAAs that contribute to the mature aortic arch. The demonstration of genetic interaction between Tbx1 and Fgf8 pointed to FGF signaling as a possible candidate. Because Fgf8 interacts with Endothelin-1 signaling and because Endothelin-1 signaling interacts with neural crest-derived cells in the pharyngeal apparatus, we hypothesized that Tbx1 and Endothelin-1 signaling may contribute to the same pathway required for aortic arch morphogenesis. Therefore, we have analyzed mice mutated for the endothelin converting enzyme (Ece1) or Tbx1 genes and compound mutants. Results show that the two genes have different roles in the remodeling of the PAAs and do not interact. We propose that Tbx1 is required for the formation and early growth and remodeling of the PAAs, whereas Ece1 is necessary for regression of the cranial arch arteries and growth of the most caudal arch arteries. The latter function is likely related to the known role of the Endothelin-1 pathway in neural crest function.     

Localization and role of endothelin-1 and endothelin receptors in the human Fallopian tube.

The present experiments were designed to investigate the localization and role of endothelin-1 (ET-1) and endothelin receptors (ET(A) and ET(B)) in human Fallopian tubes obtained from patients in the follicular phase. Immunohistochemical studies revealed the predominant localization of ET-1 and of ET(B) receptors in the tubal epithelium and also within the muscle layer to a lesser degree. ET(A) receptors were dominant within the muscle layer. Scatchard plot analysis of the [(125)I]ET-1 binding also revealed the localization of ET(A) and ET(B) receptors on the Fallopian tubal membrane. A dissociation equilibrium constant of 34.6 +/- 3.3 pmol/l and a maximum binding site concentration of 1137.0 +/- 239.1 fmol/mg protein were obtained from the Scatchard plot analysis. Treatment of Fallopian tubal strips with ET-1 produced a tonic contraction which was inhibited by an ET(A) antagonist but not by an ET(B) antagonist. However, the increase in frequency and decrease in amplitude of rhythmic contractions caused by ET-1 were modulated by the ET(B) antagonist but remained unaffected by the ET(A) antagonist. These results suggest that ET-1 modulates the motility of the Fallopian tube through excitation of ET(A) and/or ET(B) receptors and possibly plays some role in oocyte capture.     

Development-related changes in the expression of shear stress responsive genes KLF-2, ET-1, and NOS-3 in the developing cardiovascular system of chicken embryos.

Blood flow patterns play an important role in cardiovascular development, as changes can cause congenital heart malformations. Shear stress is positively correlated to blood flow. Therefore, it is likely that shear stress is also involved in cardiac development. In this study, we investigated the expression patterns of ET-1, NOS-3, and KLF-2 mRNA in a series of developmental stages of the chicken embryo. These genes are reported to be shear responsive. It has been demonstrated that KLF-2 is confined to areas of high shear stress in the adult human aorta. From in vitro studies, it is known that ET-1 is down-regulated by shear stress, whereas NOS-3 is up-regulated. Therefore, we expect ET-1 to be low or absent and NOS-3 to be high at sites where KLF-2 expression is high. Our study shows that, in the early stages, expression patterns are mostly not shear stress-related, whereas during development, this correlation becomes stronger. We demonstrate overlapping expression patterns of KLF-2 and NOS-3 in the narrow parts of the cardiovascular system, like the cardiac inflow tract, the atrioventricular canal, outflow tract, and in the early stages in the aortic sac and the pharyngeal arch arteries. In these regions, the expression patterns of KLF-2 and NOS-3 exclude that of ET-1. Our results suggest that, in the embryonic cardiovascular system, KLF-2 is expressed in regions of highest shear stress, and that ET-1 and NOS-3 expression, at least in the later stages, is related to shear stress.