Acute Pressor Response to Psychosocial Stress Is Dependent on Endothelium-Derived Endothelin-1.

BACKGROUND: Acute psychosocial stress provokes increases in circulating endothelin-1 (ET-1) levels in humans and animal models. However, key questions about the physiological function and cellular source of stress-induced ET-1 remain unanswered. We hypothesized that endothelium-derived ET-1 contributes to the acute pressor response to stress via activation of the endothelin A receptor. METHODS AND RESULTS: Adult male vascular endothelium-specific ET-1 knockout mice and control mice that were homozygous for the floxed allele were exposed to acute psychosocial stress in the form of cage switch stress (CSS), with blood pressure measured by telemetry. An acute pressor response was elicited by CSS in both genotypes; however, this response was significantly blunted in vascular endothelium-specific ET-1 knockout mice compared with control mice that were homozygous for the floxed allele. In mice pretreated for 3 days with the endothelin A antagonist, ABT-627, or the dual endothelin A/B receptor antagonist, A-182086, the pressor response to CSS was similar between genotypes. CSS significantly increased plasma ET-1 levels in control mice that were homozygous for the floxed allele. CSS failed to elicit an increase in plasma ET-1 in vascular endothelium-specific ET-1 knockout mice. Telemetry frequency domain analyses suggested similar autonomic responses to stress between genotypes, and isolated resistance arteries demonstrated similar sensitivity to α1-adrenergic receptor-mediated vasoconstriction. CONCLUSIONS: These findings specify that acute stress-induced activation of endothelium-derived ET-1 and subsequent endothelin A receptor activation is a novel mediator of the blood pressure response to acute psychosocial stress.

ET-1 increases reactive oxygen species following hypoxia and high-salt diet in the mouse glomerulus.

AIM: This study was designed to determine whether ET-1 derived from endothelial cells contributes to oxidative stress in the glomerulus of mice subjected to a high-salt diet and/or hypoxia. METHODS: C57BL6/J control mice or vascular endothelial cell ET-1 knockout (VEET KO) mice were subjected to 3-h exposure to hypoxia (8% O2) and/or 2 weeks of high-salt diet (4% NaCl) prior to metabolic cage assessment of renal function and isolation of glomeruli for the determination of reactive oxygen species (ROS). RESULTS: In control mice, hypoxia significantly increased urinary protein excretion during the initial 24 h, but only in animals on a high-salt diet. Hypoxia increased glomerular ET-1 mRNA expression in control, but not in vascular endothelial cell ET-1 knockout (VEET KO) mice. Under normoxic conditions, mice on a high-salt diet had approx. 150% higher glomerular ET-1 mRNA expression compared with a normal-salt diet (P < 0.05). High-salt diet administration significantly increased glomerular ROS production in flox control, but not in glomeruli isolated from VEET KO mice. In C57BL6/J mice, the ETA receptor-selective antagonist, ABT-627, significantly attenuated the increase in glomerular ROS production produced by high-salt diet. In addition, chronic infusion of C57BL6/J mice with a subpressor dose of ET-1 (osmotic pumps) significantly increased the levels of glomerular ROS that were prevented by ETA antagonist treatment. CONCLUSION: These data suggest that both hypoxia and a high-salt diet increase glomerular ROS production via endothelial-derived ET-1-ETA receptor activation and provide a potential mechanism for ET-1-induced nephropathy.

Endothelin-1 is a transcriptional target of p53 in epidermal keratinocytes and regulates ultraviolet-induced melanocyte homeostasis.

Keratinocytes contribute to melanocyte activity by influencing their microenvironment, in part, through secretion of paracrine factors. Here, we discovered that p53 directly regulates Edn1 expression in epidermal keratinocytes and controls UV-induced melanocyte homeostasis. Selective ablation of endothelin-1 (EDN1) in murine epidermis (EDN1(ep-/-) ) does not alter melanocyte homeostasis in newborn skin but decreases dermal melanocytes in adult skin. Results showed that keratinocytic EDN1 in a non-cell autonomous manner controls melanocyte proliferation, migration, DNA damage, and apoptosis after ultraviolet B (UVB) irradiation. Expression of other keratinocyte-derived paracrine factors did not compensate for the loss of EDN1. Topical treatment with EDN1 receptor (EDNRB) antagonist BQ788 abrogated UV-induced melanocyte activation and recapitulated the phenotype seen in EDN1(ep-/-) mice. Altogether, the present studies establish an essential role of EDN1 in epidermal keratinocytes to mediate UV-induced melanocyte homeostasis in vivo.

Knockout of endothelin-1 in vascular endothelial cells protects against insulin resistance induced by high-salt diet in mice.

The increased plasma Endothelin-1 (ET-1) level has been associated with development of insulin resistance in obese and hypertensive patients. However, the underlying mechanism remains elusive. Here we investigate the potential role of endothelial cell-derived ET-1 in mediating insulin resistance induced by high-salt diet. To address this issue, we used vascular endothelial cell-specific ET-1 knockout (VEETKO) mice and its littermates fed with a high-salt diet containing 8% NaCl for 3 weeks, and evaluated the metabolic parameters. High-salt diet increased systolic blood pressure similarly in both genotypes. We observed impairment of glucose tolerance in control mice despite comparable increase of serum insulin concentration with VEETKO mice. We further found that VEETKO mice showed preservation of circulating adiponectin level - an adipokine with insulin-sensitizing property - and prevention of the upregulation of the pro-inflammatory adipokine TNF-α, which lead towards better insulin sensitivity. These results provide evidence that blockade of endothelin signaling may be proven beneficial in preventing high-salt induced insulin resistance.

Endothelial cell-derived endothelin-1 promotes cardiac fibrosis in diabetic hearts through stimulation of endothelial-to-mesenchymal transition.

BACKGROUND: Persistently high plasma endothelin-1 (ET-1) levels in diabetic patients have been associated with the development of cardiac fibrosis, which results from the deposition of extracellular matrix and fibroblast recruitment from an as-yet unknown source. The underlying mechanism, however, remains elusive. Here, we hypothesize that ET-1 might contribute to the accumulation of cardiac fibroblasts through an endothelial-to-mesenchymal transition in diabetic hearts. METHODS AND RESULTS: We induced diabetes mellitus in vascular endothelial cell-specific ET-1 knockout [ET-1(f/f);Tie2-Cre (+)] mice and their wild-type littermates using the toxin streptozotocin. Gene expression and histological and functional parameters were examined at 8, 24, and 36 weeks after the induction of diabetes mellitus. Diabetes mellitus increased cardiac ET-1 expression in wild-type mice, leading to mitochondrial disruption and myofibril disarray through the generation of superoxide. Diabetic mice also showed impairment of cardiac microvascularization and a decrease in cardiac vascular endothelial growth factor expression. ET-1 further promotes cardiac fibrosis and heart failure through the accumulation of fibroblasts via endothelial-to-mesenchymal transition. All of these features were abolished in ET-1(f/f);Tie2-Cre (+) hearts. Targeted ET-1 gene silencing by small interfering RNA in cultured human endothelial cells ameliorated high glucose-induced phenotypic transition and acquisition of a fibroblast marker through the inhibition of transforming growth factor-beta signaling activation and preservation of the endothelial cell-to-cell contact regulator VE-cadherin. CONCLUSIONS: These results provide new insights suggesting that diabetes mellitus-induced cardiac fibrosis is associated with the emergence of fibroblasts from endothelial cells and that this endothelial-to-mesenchymal transition process is stimulated by ET-1. Targeting endothelial cell-derived ET-1 might be beneficial in the prevention of diabetic cardiomyopathy.

Vascular endothelial cell-derived endothelin-1 mediates vascular inflammation and neointima formation following blood flow cessation.

AIMS: Although endothelin-1 (ET-1) has been suggested to contribute to the pathogenesis of neointima formation and atherosclerosis, the individual roles of ET-1 derived from certain cell types in this disease remain unclear. In this study, we determined the role of vascular endothelial ET-1 on vascular inflammation and neointima formation using vascular endothelial ET-1-knockout [ET-1(f/f); Tie2-Cre (+)] mice. METHODS AND RESULTS: Intimal hyperplasia was induced by complete ligation of the left carotid artery in 12-week-old male ET-1(f/f);Tie2-Cre (+) mice (n = 35) and the wild-type (WT) littermates (n = 34). Following this intervention, neointima formation was reduced in ET-1(f/f);Tie2-Cre (+) mice compared with the WT mice, independent of the difference in blood pressure. This reduction was associated with a decrease in inflammatory cell recruitment to the vessel wall, which was accompanied by reduced expression levels of endothelial adhesion molecules as well as chemokines and a decrease in vascular smooth muscle cell proliferation. CONCLUSION: The results of our study provide direct evidence for the role of vascular endothelial ET-1 in mediating vascular inflammation and neointima formation following vascular injury in addition to promoting vasoconstriction and cell proliferation. Furthermore, this study suggests a strategy for the efficient design of ET receptor antagonists with targeted inhibition of ET-1 signalling in vascular endothelial cells.

Role of the renin-angiotensin-aldosterone system in collecting duct-derived endothelin-1 regulation of blood pressure.

Renal collecting duct (CD)-specific knockout of endothelin-1 (ET-1) causes hypertension and impaired Na excretion. A previous study noted failure to suppress the renin-angiotensin-aldosterone axis in these knockout (KO) mice, hence the current investigation was undertaken to examine the role of this system in CD ET-1 KO. Renal renin content was similar in kidneys from CD ET-1 KO and control mice during normal Na intake; high-Na intake suppressed renal renin content to a similar degree in KO and control. Plasma renin concentrations paralleled changes in renal renin content. Valsartan, an angiotensin receptor blocker (ARB), abolished the hypertension in CD ET-1 KO mice during normal Na intake. High-Na intake + ARB treatment increased blood pressure in CD ET-1 KO, but not in controls. High-Na intake was associated with reduced Na excretion in CD ET-1 KO animals, but no changes in water excretion or creatinine clearance were noted. Spironolactone, an aldosterone antagonist, also normalized blood pressure in CD ET-1 KO mice during normal Na intake, whereas high-Na intake + spironolactone raised blood pressure only in CD ET-1 KO animals. In summary, hypertension in CD ET-1 KO is partly due to angiotensin II and aldosterone. We speculate that CD-derived ET-1 may regulate, via a novel pathway, renal renin production.

Altered collecting duct adenylyl cyclase content in collecting duct endothelin-1 knockout mice.

BACKGROUND: Endothelin-1 (ET-1) inhibition of vasopressin (AVP)-stimulated water reabsorption by the inner medullary collecting duct (IMCD) is associated with reduced cAMP accumulation. To determine the effect of ET-1 deficiency, AVP-stimulated cAMP responsiveness was assessed in IMCD from mice with collecting duct-specific deletion of ET-1 (CD ET-1 KO) and from control animals. METHODS: Cyclic AMP production, adenylyl cyclase (AC) mRNA, and AC protein were measured in acutely isolated IMCD. RESULTS: CD ET-1 KO IMCD had enhanced AVP-stimulated cAMP accumulation. Inhibition of calcium-stimulated AC using BAPTA did not prevent enhanced AVP responsiveness in CD ET-1 KO IMCD. Factors known to be modified by ET-1, including nitric oxide, cyclooxygenase metabolites, and superoxide did not affect the increased AVP responsiveness of CD ET-1 KO IMCD. Differential V2 receptor or G-protein activity was not involved since CD ET-1 KO IMCD had increased cAMP accumulation in response to forskolin and/or cholera toxin. CD ET-1 KO did not affect mRNA or protein levels of AC3, one of the major known collecting duct AC isoforms. However, the other known major collecting duct AC isoform (AC5/6) did have increased protein levels in CD ET-1 KO IMCD, although AC5 (weak signal) and 6 mRNA levels were unchanged. CONCLUSION: ET-1 deficiency increases IMCD AC5/6 content, an effect that may synergize with acute ET-1 inhibition of AVP-stimulated cAMP accumulation.

Endogenous endothelin-1 is required for cardiomyocyte survival in vivo.

BACKGROUND: Endothelin-1 (ET-1) has potent vasoconstrictor and hypertrophic actions. Pharmacological antagonists of endothelin receptors attenuate cardiac hypertrophy, have been approved for treatment of pulmonary hypertension, and are under investigation for treatment of heart failure. To investigate the role of ET-1 in the heart, we created mice with cardiomyocyte deletion of ET-1. METHODS AND RESULTS: Mice with cardiomyocyte-specific deletion of ET-1 are phenotypically normal when young. Remarkably, as the mice age or when young animals are subjected to aortic banding, they develop an unexpected phenotype of progressive systolic dysfunction and cardiac dilation. Echocardiography, necropsy, histology, and molecular phenotype confirm a dilated cardiomyopathy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling analysis reveals greater abundance of apoptotic nuclei in the ET-1-deficient hearts. Transcriptional and Western analyses suggest enhanced tumor necrosis factor (TNF)-mediated apoptosis with increases in caspase-8 activity. These ET-1-deficient hearts also have diminished nuclear factor (NF)-kappaB activity, resulting in diminution of downstream inhibitors of TNF signaling. CONCLUSIONS: Local ET-1 gene expression is necessary to maintain normal cardiac function and cardiomyocyte survival in mice with both age and hemodynamic stress. This cardiac-protective effect is mediated by paracrine ET-1 modulation of TNF-related apoptosis, in part through upregulation of NF-kappaB signaling.

Increased sensitivity to acute and persistent pain in neuron-specific endothelin-1 knockout mice.

Endothelin-1 (ET-1) exists in endothelial cells as well as a variety of other cell types. The presence of ET-1 and its receptors in neurons suggests its possible role as a neurotransmitter and/or neuromodulator. Studies utilizing exogenous ET-1 have suggested that ET-1 affects pain transmission. This study was designed to examine the possible role(s) of neuronal ET-1 in pain processing. We produced neuron-specific ET-1 knockout mice using the Cre/loxP system with a synapsin I promoter and examined the effects produced by the lack of neuronal ET-1 on pain behavior using common pain models and a model of stress-induced analgesia. In acute nociceptive pain models, paw withdrawal thresholds to radiant heat and mechanical stimuli applied with von Frey hairs were significantly lower in the knockout mice compared with control. This indicated that the absence of neuronal ET-1 leads to greater sensitivity to acute nociceptive stimuli. After inflammation was produced by intraplantar injection of carrageenan, there was a significantly greater degree of thermal hyperalgesia and mechanical allodynia in the knockout mice even after the difference in baseline values was compensated. Furthermore, in a neuropathic pain model produced by spinal nerve ligation, there was also a greater degree of mechanical allodynia in the knockout mice. Finally, in a swim-stress model, the magnitude of stress-induced analgesia was less in the knockout mice, indicating the involvement of neuronal ET-1 in stress-induced analgesia. The results suggest that there is a basal release of ET-1 from neurons that affect baseline pain thresholds as well as an additional release during persistent pain states that acts to suppress the pain. The involvement of neuronal ET-1 in stress-induced analgesia further suggests its role in endogenous pain inhibitory systems. To confirm that ET-1 is released in persistent pain states and to determine which part of the CNS is involved, we measured the concentrations of ET-1 before and after inducing peripheral inflammation in different parts of the CNS involved in endogenous pain inhibitory systems in normal mice. We found that ET-1 was increased in the hypothalamus while no significant increase was observed in the midbrain, medulla and spinal cord. The results of the present study suggest that neuronal ET-1 is involved in endogenous pain inhibitory control likely via pathways through the hypothalamus.

Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.

Endothelin-1 (ET-1) is a 21-amino-acid peptide, derived from vascular endothelial cells, with potent vasoconstrictor activity. ET-1 has been implicated in diverse physiological or pathological processes, including the vascular changes associated with sepsis. However, the factors that regulate ET-1-associated toxicity during bacterial infections, or in other settings, are not fully understood. Both the pathology associated with certain allergic and autoimmune disorders, and optimal host defence against bacterial and parasitic infections are mediated by mast cells. In vitro, mast cells can produce ET-1 (ref. 11), undergo ET-1-dependent and endothelin-A receptor (ET(A))-dependent activation, and release proteases that degrade ET-1 (ref. 14). Although the potential relationships between mast cells and the ET-1 system thus may be complex, the importance of interactions between ET-1 and mast cells in vivo is obscure. Here we show that ET(A)-dependent mast-cell activation can diminish both ET-1 levels and ET-1-induced pathology in vivo, and also can contribute to optimal survival during acute bacterial peritonitis. These findings identify a new biological function for mast cells: promotion of homeostasis by limiting the toxicity associated with an endogenous mediator.

Temporal requirement of signaling cascade involving endothelin-1/endothelin receptor type A in branchial arch development.

Intercellular interactions within the branchial arch (BA) system is essential for craniofacial development. Endothelin-1 (ET-1), produced by the branchial epithelium and core mesenchyme, acts on cranial neural crest-derived ectomesenchymcal cells expressing endothelin A receptor (ETAR) and regulates expression of crucial genes such as Dlx6, a member of distalless homeobox gene family, and its downstream target dHAND, a basic helix-loop-helix transcription factor. To investigate the role of ET-1 and subsequent signaling cascades in BA development, we examined when and how they activate dHAND and Dlx6 expression. ETAR blockade by BQ123 in mouse embryo culture has revealed that ET-1/ETAR signaling is critical for dHAND and Dlx6 expression in the mandibular arch mesenchyme around embryonic day (E)8.75-E9.0 and becomes dispensable by E9.5. dHAND and Dlx6 expression after E9.5 was dependent on the presence of the epithelium, which was partly mediated by FGF-like signals. These findings indicate that ET-1/ETAR and subsequent epithelial signals are sequentially involved in BA development by maintaining dHAND and Dlx6 expression. Furthermore, discordance of dHAND and Dlx6 expression domains and heterogeneity with respect to dependency on ET-1 and FGF-like signals suggest that genetic hierarchy involving Dlx6 and dHAND is differently controlled among subdomains within the mandibular arch.

Endothelin-1 regulates cardiac sympathetic innervation in the rodent heart by controlling nerve growth factor expression.

The cardiac sympathetic nerve plays an important role in regulating cardiac function, and nerve growth factor (NGF) contributes to its development and maintenance. However, little is known about the molecular mechanisms that regulate NGF expression and sympathetic innervation of the heart. In an effort to identify regulators of NGF in cardiomyocytes, we found that endothelin-1 specifically upregulated NGF expression in primary cultured cardiomyocytes. Endothelin-1-induced NGF augmentation was mediated by the endothelin-A receptor, Gibetagamma, PKC, the Src family, EGFR, extracellular signal-regulated kinase, p38MAPK, activator protein-1, and the CCAAT/enhancer-binding protein delta element. Either conditioned medium or coculture with endothelin-1-stimulated cardiomyocytes caused NGF-mediated PC12 cell differentiation. NGF expression, cardiac sympathetic innervation, and norepinephrine concentration were specifically reduced in endothelin-1-deficient mouse hearts, but not in angiotensinogen-deficient mice. In endothelin-1-deficient mice the sympathetic stellate ganglia exhibited excess apoptosis and displayed loss of neurons at the late embryonic stage. Furthermore, cardiac-specific overexpression of NGF in endothelin-1-deficient mice overcame the reduced sympathetic innervation and loss of stellate ganglia neurons. These findings indicate that endothelin-1 regulates NGF expression in cardiomyocytes and plays a critical role in sympathetic innervation of the heart.

Mice with cardiomyocyte-specific disruption of the endothelin-1 gene are resistant to hyperthyroid cardiac hypertrophy.

Endothelin 1 (ET-1), a potent vasoconstrictor peptide expressed by endothelium, is also produced in the heart in response to a variety of stresses. It induces hypertrophy in cultured cardiac myocytes but only at concentrations far greater than those found in plasma. We tested whether ET-1 generated by cardiac myocytes in vivo is a local signal for cardiac hypertrophy. To avoid the perinatal lethality seen in systemic ET-1-null mice, we used the Cre/loxP system to generate mice with cardiac myocyte-specific disruption of the ET-1 gene. We used the alpha-myosin heavy chain promoter to drive expression of Cre and were able to obtain 75% reduction in ET-1 mRNA in cardiac myocytes isolated from these mice at baseline and after stimulation, in vivo, for 24 h with tri-iodothyronine (T3). Necropsy measurements of cardiac mass indexed for body weight showed a 57% reduction in cardiac hypertrophy in response to 16 days of exogenous T3 in mice homozygous for the disrupted ET-1 allele compared to siblings with an intact ET-1 gene. Moreover, in vivo MRI showed only a 3% increase in left ventricular mass indexed for body weight in mice with the disrupted allele after 3 weeks of T3 treatment versus a 27% increase in mice with an intact ET-1 gene. A reduced hypertrophic response was confirmed by planimetry of cardiac myocytes. We conclude that ET-1, produced locally by cardiac myocytes, and acting in a paracrine/autocrine manner, is an important signal for myocardial hypertrophy that facilitates the response to thyroid hormone.

Molecular determinants of neural crest migration.

Normal septation of the cardiac outflow tract requires migration of neural crest cells from the posterior rhombencephalon to the branchial arches and developing conotruncal endocardial cushions. Proper migration of these cells is mediated by a variety of molecular cues. Adhesion molecules, such as integrins, are involved in the interaction of neural crest cells with the extracellular matrix, while cadherins allow neural crest cells to interact with each other during their migration. Pax3 appears to be important for proliferation of neural crest precursors, and connexin-43-mediated gap junction communication influences the rate of migration. Endothelin and its receptors are required for normal postmigratory differentiation. Platelet-derived growth factor and retinoic acid have roles in neural crest migration and differentiation as well. Finally, the similarity between the cardiovascular malformations seen in the DiGeorge and 22q11 deletion syndromes and animal models of neural crest deficiency has led to the examination of the role of genes located near or within the DiGeorge critical region in neural crest migration.

Gene expression of bone matrix proteins and endothelin receptors in endothelin-1-deficient mice revealed by in situ hybridization.

Endothelin-1 (ET-1) was first found as a vasoconstrictor protein excreted by vascular endothelial cells, but recently ET-1 has been considered to have widespread functions that include regulation of osteochondrogenic metabolism. We analyzed sections of head regions in ET-1 knockout mice that are known to have abnormalities in pharyngeal arch-derived tissues and found that there was severe hypoplasia in facial bones. The hypoplasia suggests that the matrix mineralization system of facial bones is disrupted in ET-1-/- homozygous mice. To elucidate whether osteogenic cells in facial bones are the targets for ET-1 and whether expression of bone matrix genes are modulated by ET-1, we examined gene expression of ET-1 receptors, ETA and ETB, and that of the bone matrix proteins, osteonectin (ON) and osteopontin (OP), both in the head regions of ET-1+/- heterozygous and ET-1-/- homozygous mice by means of in situ hybridization. Different patterns of expression between ETA and ETB mRNAs were observed in both groups. In 18.5 days post coitus fetuses, ETA mRNA was most strongly expressed in osteogenic cells along craniofacial bones, but ETB mRNA was most strongly expressed in trunks of trigeminal nerve. This finding suggests that ET-1 may modulate osteogenic cells through ETA receptor but not through ETB receptor. The expression patterns of ETA, OP, and ON mRNAs were distinct between the two groups. In the lower jaw of ET-1+/- heterozygous mice, the ETA, ON, and OP mRNA positive cells were scattered in the inner and outer regions of the thick bone matrix, but in ET-1-/- homozygous mice, cells containing those mRNAs were located close to each other at the surface of thin bone matrix. However, cellular expression of ON and OP mRNAs in osteogenic cells of ET-1-/- homozygous mice was not suppressed as compared with ET-1+/- heterozygous mice. We conclude that ET-1 may regulate proliferation and migration of osteogenic cells in the maxillofacial region, rather than modulating the expression level of ON and OP mRNAs.