ET-1 Synthesis Research Articles

Circulating Extracellular Vesicles Are Elevated with Prediabetes and Adversely Affect Cerebral Endothelial Nitric Oxide and Endothelin-1 Production

Experimental and clinical studies indicate that a proatherogenic endothelial phenotype develops in the prediabetic, hyperglycemic state providing the platform for accelerated vascular disease with prediabetes and, in turn, type 2 diabetes. Prediabetes (fasting plasma glucose concentrations: 100-125 mg/dL) is associated with an increased risk of stroke. Circulating endothelial cell-derived microvesicles (EMVs) are now recognized as both a biomarker and mediator of vascular health and disease. In fact, EMVs have been shown to be involved in the pathogenesis of cerebrovascular dysfunction leading to increased stroke risk. Circulating EMVs are elevated in adults with prediabetes; whether EMVs associated with the prediabetic state promote endothelial abnormalities known to increase stroke risk is unknown. The experimental aim of this study was to determine the effect of EMVs isolated from adults with prediabetes on brain endothelial cell nitric oxide (NO) and endothelin (ET)-1 production. Circulating EMVs (CD144-PE) were isolated (flow cytometry) from 20 mid-life adults (age:44-69 yr): 10 normoglycemic (NG; 8M/2F; age: 55±2 yr; BMI: 25.7±0.8 kg/m2; fasting plasma glucose: 87±2 mg/dL) and 10 prediabetic (PreD: 8M/2F; 55±2 yr; 27.1±1.5 kg/m2; 107±2 mg/dL). Human cerebral microvascular endothelial cells (hCMECs) were cultured and separately treated with EMVs from each subject. Expression of intracellular proteins of interest was determined by capillary electrophoresis immunoassay. Circulating EMV concentrations were significantly higher (~65%) in PreD vs NG (180±25 vs 110±13 EMV/μL) and significantly associated with plasma glucose concentrations (r=0.48; P=0.03). Phosphorylation is the primary posttranslational modification regulating eNOS enzyme activity. Phosphorylation of Ser1177 confers the greatest activation of eNOS; whereas, phosphorylation at Thr495 reduces eNOS activation. Expression of p-eNOS (Ser1177) was lower (47.6±2.6 vs 58.6±3.5 AU; P=0.02) and p-eNOS (Thr495) higher (82.6±4.4 vs 31.1±2.5 AU; p<0.001) in cells treated with EMVs from PreD vs NG. Concordantly, NO production was ~20% lower (6.0±0.2 vs 7.2±0.4 μmol/L; p<0.05) in cells treated with EMVs from PreD adults. PreD-associated EMVs also significantly increased (~25-70%) the expression of Big ET-1 (804.4±47.7 vs 656.7±30.7 AU) and endothelin converting enzyme (290.0±24.6 vs 169.6±19.0 AU) as well as ET-1 production (30.2±4.3 vs 24.2±2.0 pg/mL) in hCMECs. In summary, EMVs harvested from adults with PreD reduced endothelial nitric oxide synthase (eNOS) activation and NO production and increased ET-1 synthesis and release in brain endothelial cells in vitro. Increased risk and incidence of stroke associated with prediabetes may be mediated by circulating EMVs. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of Circulating Extracellular Vesicles from Adults with Spinal Cord Injury on Pulmonary Artery Endothelial Cell Function

Spinal cord injury (SCI) is associated with an increased risk and prevalence of cardiopulmonary and cerebrovascular disease-related morbidity and mortality. Despite an improved understanding of the neurological and physical consequences associated with SCI, factors that initiate, promote and accelerate cardiopulmonary disease are poorly understood. It has become apparent that the increased incidence of pulmonary disorders in adults with chronic SCI is not solely due to worsening of traditional risk factors, but also involves ill-defined factors. Several studies have implicated circulating endothelial cell-derived microvesicles (EMVs) in the etiology of cardiopulmonary diseases. We have previously reported that circulating EMVs are elevated in adults with chronic SCI and the expression of their microRNA cargo promotes disease. Reduction in pulmonary nitric oxide (NO) bioavailability and increased endothelin (ET)-1 production underly many cardiopulmonary disorders associated with SCI. The experimental aim of this study was to determine the effect of EMVs isolated from adults with chronic tetraplegic SCI on pulmonary endothelial cell NO and ET-1 production. As part of an ongoing study, circulating EMVs (CD144-PE) were isolated (flow cytometry) from 7 non-injured adults (all male; age: 38±4 yr) and 7 motor complete SCI adults with tetraplegic SCI (male; 44±5 yr). All subjects were free of overt cardiometabolic disease. Human pulmonary artery endothelial cells (HPAECs) were cultured and separately treated with EMVs from each subject for 24 hr. Expression of intracellular NO and ET-1 proteins of interest was determined by capillary electrophoresis immunoassay. Circulating EMV concentrations were significantly higher (~200%) in SCI vs non-injured (154±36 vs 54±7 EMV/μL). Expression of p-eNOS (Ser1177), the primary activation site for eNOS, was ~30% lower (96.0±2.3 vs 140.7±8.9 AU; p<0.01) and p-eNOS (Thr495), primary inhibitory site, ~40% higher (40.7±1.7 vs 28.8±3.1 AU; p<0.01) in cells treated with EMVs from SCI vs non-injured. As a result, NO production was ~20% lower (6.5±0.3 vs 8.3±0.7 μmol/L; P=0.03) in HPAECs treated with EMVs from adults with SCI. SCI-associated EMVs also significantly increased (~70%) the expression of Big ET-1 (60.6±3.6 vs 36.1±2.7 AU) resulting in enhanced endothelial ET-1 production (884.0±22.9 vs 778.7±18.0 pg/mL). In conclusion, EMVs harvested from adults with SCI markedly reduced eNOS activation and NO production and increased ET-1 synthesis and release in pulmonary endothelial cells in vitro. Circulating EMVs represent a potential mechanistic factor underlying pulmonary disorders and increased disease risk with SCI. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Characterisation of pomegranate juice polyphenols identifies oenothein B as an endothelium-dependent vasodilator and regulator of endothelial function that enhances actions of oligomeric procyanidins

Abstract Background Cardiovascular disease mortality is reduced in individuals with high daily consumption of dietary polyphenols. Optimal daily intakes, choice of polyphenol, and mechanism of action have yet to be fully defined. Pomegranate juice (PJ) reduces blood pressure and improves endothelial function. These effects are attributed to the high levels of polyphenols, primarily ellagitannins. Purpose The aims of these investigations were (1) to identify PJ polyphenol(s) that altered endothelial function and could potentially lower blood pressure, and (2) to compare these endothelial actions to oligomeric procyanidins (OPC) purified from apple polyphenol extracts. Methods PJ polyphenols were purified by Sephadex LH-20 chromatography and RP-HPLC, and identified by LC-MS/MS and NMR. Fractions were screened for biological activity using cultured bovine aortic endothelial cells by measuring the degree of inhibition of endothelin-1 synthesis. Endothelial actions were further assessed by measuring changes in intracellular Ca2+ using FLIPR Ca2+-6 assay, qRT-PCR (EDN1, eNOS and KLF2 mRNAs), and NO synthesis (nitrite). Results Six PJ polyphenols were isolated. These were identified as punicalin, four punicalagin isomers, and oenothein B (OTB, a cyclic dimeric ellagitannin; LC-MS [M-H]-= 1567.1). At concentrations ≤5 µg/ml only OTB inhibited ET-1 synthesis (IC50 1.5 µg/ml). The identity of OTB was confirmed by NMR, and by comparison with OTB purified from Epilobium species. Further characterisation of the actions of OTB on endothelial cells showed pre-incubation with 1.25, 2.5 and 5 µg/ml OTB increased binding of biotinylated-OPC by 1.7, 2.3 and 3.8 fold. Subsequent studies of the interactions between OTB and OPC showed synergistic effects for decreases in ET-1 synthesis, EDN1 mRNA, and increases in KLF2 mRNA, eNOS mRNA and NO release. The effect of combining OTB (1 µg/ml) and OPC (1 µg/ml) lasted for over 24 h (relative to control: ET-1 -57%, EDN1 mRNA -63%, eNOS mRNA +69%, KLF2 mRNA +356%), when individually these polyphenols no longer had any significant effect. Intracellular Ca2+ responses also showed synergistic interactions, however the profile of [Ca2+] changes for OTB and OPC alone differed, suggesting separate signalling pathways initiating these endothelial responses. Endothelium-dependent vasodilator effects of OTB were confirmed on isolated mouse aorta (EC50 0.27 µg/ml). Conclusions These effects of OTB may underlie previously reported decreases in blood pressure and improvements in endothelial function after pomegranate juice consumption. Detailed understanding of the mechanism of action of OTB, and its interactions with OPC could lead to new therapeutic modalities for treating endothelial dysfunction in a broad spectrum of people at risk of cardiovascular disease.

Postinjury Inhibition of Endothelin‐1 Dependent Renal Vasoregulation Mitigates Rhabdomyolysis‐Induced Acute Kidney Injury

Rhabdomyolysis is a life‐threatening condition resulting from the breakdown of skeletal muscle fibers leading to the release of myoglobin into the blood. Increased circulating myoglobin can cause kidney damage, and acute kidney injury (AKI) occurs in 33‐50% of patients with myoglobinuria. Persistent constriction of the renal microvessels, which reduces renal blood flow (RBF) and glomerular filtration rate (GFR), contributes to rhabdomyolysis‐induced AKI. Since activation of G‐protein‐coupled receptors (GPCR) and ion channels regulate renal vascular resistance (RVR), rhabdomyolysis may likely modulate RVR via GPCR and vascular ion channel mechanisms. Pretreatment of rats with an endothelin receptor antagonist has been shown to alleviate rhabdomyolysis‐induced AKI. However, the downstream and upstream links between rhabdomyolysis and increased ET‐1 synthesis are unknown. The downstream effectors of ET‐1‐induced vasoconstriction are smooth muscle cell (SMC) Ca2+‐permeable ion channels. Yet, the ion channel mechanisms that trigger renal vasoconstriction in rhabdomyolysis‐induced AKI remain unknown. Also, most cases of rhabdomyolysis‐induced AKI cannot be predicted. Whether post‐injury inhibition of the vascular mechanisms of ET‐1 ameliorates, rhabdomyolysis‐induced AKI is unknown. This study investigates post‐injury renal production and vascular regulation of the ET system in rhabdomyolysis‐induced AKI. Six h of rhabdomyolysis increased peptidase endothelin converting enzyme (ECE‐1)‐dependent biosynthesis of ET‐1 in the kidneys. Rhabdomyolysis‐induced AKI was not sex‐dependent. At 24 h, rhabdomyolysis also increased renal reactive oxygen species (ROS), ECE‐1, and ET‐1 levels. Furthermore, ET‐1 activated rat renal vascular smooth muscle cell TRPC3 leading to a reduction in RBF and increased RVR. Protein expression levels of the ET receptors (ETA and ETB) and TRPC3 channels in renal microvessels were unaltered. However, postinjury inhibition of ECE1, ET Receptors, and TRPC3 mitigated rhabdomyolysis‐induced GFR impairment, renal hypoperfusion, AKI biomarker elevation, and morphological kidney damage. To further explore the role of TRPC3 ion channels in rhabdomyolysis‐induced AKI, we subjected TRPC3 knock‐out (KO) rats to rhabdomyolysis. Compared to their wild‐type counterparts, KO rats show protection against rhabdomyolysis‐induced AKI. Our findings suggest that ECE‐driven proteolytic ET‐1 production contributes to rhabdomyolysis‐induced AKI. Downstream, ET‐derived DAG activates renal vascular smooth muscle cell TRPC3 channels leading to extracellular calcium entry, vasoconstriction, increased RVR, and reduced GFR.

MO596HYPERPHOSPHATEMIA INDUCES ENDOTHELIN-1 SYNTHESIS AND INFLAMMATION IN LUNG FIBROBLASTS

Abstract Background and Aims Hyperphosphatemia is an aging-related condition associated to chronic diseases such as chronic kidney disease (CKD). In both cases, aging and CKD, it has been described a decline lung function, however, relationship between high serum levels of phosphate and lung damage has not been described yet. The aim of this work was to evaluate if phosphate could induce inflammation in lung, studying the mechanisms implied. Method In vitro studies were performed on a cell line from human lung fibroblasts (Wi-38) which were treated with a phosphate donor termed β-glycerophosphate (BGP, 10 mM) and also with endothelin 1 (ET-1, 10 nM) at different times. To assess inflammation, the expression of several cytokines such as TNF-α,IL-1β, IL-6 and MCP-1 were measured by real time PCR. mRNA expressions of prepro-ET-1 and endothelin-converting enzyme-1 (ECE-1) were also analyzed by real time PCR. Reactive oxygen species (ROS) production was evaluated by confocal microscopy in live cells using the fluorogenic probe CellROX as oxidative stress reagent. Results Treatment with BGP induced a significant rise of the pro-inflammatory cytokines expression, TNF-α, IL-1β, IL-6 and MCP-1, on lung fibroblasts. Moreover, BGP was able to regulate ET-1 system, as we found a significant increase of mRNA expressions not only of prepro-ET-1 but also of ECE-1, reaching a peak around 2 and 6 hours, respectively. Later, it was checked whether ET-1 could induce inflammation itself in lung fibroblasts. Cells incubated with ET-1 show similar results as BGP, increasing expressions of all cytokines, TNF-α, IL-1β, IL-6 and MCP-1.ET-1 response was earlier than BGP, around at 2 hours with ET-1 instead of at 8 hours with BGP. In order to elucidate a possible mechanism, ROS production was assessed after BGP treatment. BGP induced ROS production at 15 min in lung fibroblasts. Conclusion BGP induces the synthesis of pro-inflammatory cytokines as well as the system of synthesis of ET-1, besides to rise ROS production.ET-1 itself also increases pro-inflammatory cytokines expression. ET-1 could contribute to the inflammation observed in lung fibroblast after BGP treatment. We propose oxidative stress as a potential mechanism implied, as it is well known that ROS can mediate in inflammation and in the ET system. However, more studies are necessary to confirm this role. All in all, these results relate hyperphosphatemia with a higher inflammation in lung fibroblasts which could decline lung function in chronic diseases associated to aging as CKD.

Endothelin-1 Regulation of Exercise-Induced Changes in Flow: Dynamic Regulation of Vascular Tone.

Although endothelin (ET)-1 is a highly potent vasoconstrictor with considerable efficacy in numerous vascular beds, the role of endogenous ET-1 in the regulation of vascular tone remains unclear. The perspective that ET-1 plays little role in the on-going regulation of vascular tone at least under physiologic conditions is supported by findings that potential ET-1 constriction is minimized by the release of the vasodilator and ET-1 synthesis inhibitor, nitric oxide (NO). Indeed, ET-1 release and constriction is self-limited by ET-1-induced, endothelial ETB receptor-mediated release of NO. Moreover, even if the balance between ET-1 and NO were reversed as the result of lowered NO activity, as occurs in a number of pathophysiologies associated with endothelial dysfunction, the well-known resistance of ET-1 constriction to reversal (as determined with exogenous ET-1) precludes ET-1 in the dynamic, i.e., moment-to-moment, regulation of vascular tone. On the other hand, and as presently reviewed, findings of ET-1-dependent modulation of organ blood flow with exercise under physiologic conditions demonstrate the dynamic regulation of vascular tone by ET-1. We speculate that this regulation is mediated at least in part through changes in ET-1 synthesis/release caused by pulsatile flow-induced shear stress and NO.

TRAF3IP2 mediates high glucose-induced endothelin-1 production as well as endothelin-1-induced inflammation in endothelial cells.

Hyperglycemia-induced production of endothelin (ET)-1 is a hallmark of endothelial dysfunction in diabetes. Although the detrimental vascular effects of increased ET-1 are well known, the molecular mechanisms regulating endothelial synthesis of ET-1 in the setting of diabetes remain largely unidentified. Here, we show that adapter molecule TRAF3 interacting protein 2 (TRAF3IP2) mediates high glucose-induced ET-1 production in endothelial cells and ET-1-mediated endothelial cell inflammation. Specifically, we found that high glucose upregulated TRAF3IP2 in human aortic endothelial cells, which subsequently led to activation of JNK and IKKβ. shRNA-mediated silencing of TRAF3IP2, JNK1, or IKKβ abrogated high-glucose-induced ET-converting enzyme 1 expression and ET-1 production. Likewise, overexpression of TRAF3IP2, in the absence of high glucose, led to activation of JNK and IKKβ as well as increased ET-1 production. Furthermore, ET-1 transcriptionally upregulated TRAF3IP2, and this upregulation was prevented by pharmacological inhibition of ET-1 receptor B using BQ-788, or inhibition of NADPH oxidase-derived reactive oxygen species using gp91ds-tat and GKT137831. Notably, we found that knockdown of TRAF3IP2 abolished ET-1-induced proinflammatory and adhesion molecule (IL-1β, TNF-α, monocyte chemoattractant protein 1, ICAM-1, VCAM-1, and E-selectin) expression and monocyte adhesion to endothelial cells. Finally, we report that TRAF3IP2 is upregulated and colocalized with CD31, an endothelial marker, in the aorta of diabetic mice. Collectively, findings from the present study identify endothelial TRAF3IP2 as a potential new therapeutic target to suppress ET-1 production and associated vascular complications in diabetes. NEW & NOTEWORTHY This study provides the first evidence that the adapter molecule TRAF3 interacting protein 2 mediates high glucose-induced production of endothelin-1 by endothelial cells as well as endothelin-1-mediated endothelial cell inflammation. The findings presented herein suggest that TRAF3 interacting protein 2 may be an important therapeutic target in diabetic vasculopathy characterized by excess endothelin-1 production.

Osmolar regulation of endothelin-1 production by the inner medullary collecting duct

AimsEndothelin-1 (ET-1) is an autocrine inhibitor of collecting duct (CD) Na+ and water reabsorption. CD ET-1 production is increased by a high salt diet and is important in promoting a natriuretic response. The mechanisms by which a high salt diet enhances CD ET-1 are being uncovered. In particular, elevated tubule fluid flow, as occurs in salt loading, enhances CD ET-1 synthesis. Tubule fluid solute content and interstitial osmolality can also be altered by a high salt diet, however their effect on CD ET-1 alone, or in combination with flow, is poorly understood. Main methodsET-1 mRNA production by a mouse inner medullary CD cell line (mIMCD3) in response to changing flow and/or osmolality was assessed. Key findingsFlow or hyperosmolality (using NaCl, mannitol or urea) individually caused an ~2-fold increase in ET-1 mRNA, while flow and hyperosmolality together increased ET-1 mRNA by ~14 fold. The hyperosmolality effect alone and the synergistic effect of flow + hyperosmolality was inhibited by chelation of intracellular Ca2+, however were not altered by blockade of downstream Ca2+-signaling pathways (calcineurin or NFATc), inhibition of cellular Ca2+ entry channels (purinergic receptors or polycystin-2), or blockade of the epithelial Na+ channel. Inhibition of NFAT5 with rottlerin or NFAT5 siRNA greatly reduced the stimulatory effect of osmolality alone and osmolality + flow on mIMCD3 ET-1 mRNA levels. SignificanceBoth flow and osmolality individually and synergistically stimulate mIMCD3 ET-1 mRNA content. These findings may be relevant to explaining high salt diet induction of CD ET-1 production.

Flow regulation of endothelin-1 production in the inner medullary collecting duct.

Collecting duct-derived endothelin (ET)-1 is an autocrine inhibitor of Na(+) and water reabsorption; its deficiency causes hypertension and water retention. Extracellular fluid volume expansion increases collecting duct ET-1, thereby promoting natriuresis and diuresis; however, how this coupling between volume expansion and collecting duct ET-1 occurs is incompletely understood. One possibility is that volume expansion increases tubular fluid flow. To investigate this, cultured IMCD3 cells were subjected to static or flow conditions. Exposure to a shear stress of 2 dyn/cm(2) for 2 h increased ET-1 mRNA content by ∼2.3-fold. Absence of perfusate Ca(2+), chelation of intracellular Ca(2+), or inhibition of Ca(2+) signaling (calmodulin, Ca(2+)/calmodulin-dependent kinase, calcineurin, PKC, or phospholipase C) prevented the flow response. Evaluation of possible flow-activated Ca(2+) entry pathways revealed no role for transient receptor potential (TRP)C3, TRPC6, and TRPV4; however, cells with TRPP2 (polycystin-2) knockdown had no ET-1 flow response. Flow increased intracellular Ca(2+) was blunted in TRPP2 knockdown cells. Nonspecific blockade of P2 receptors, as well as specific inhibition of P2X7 and P2Y2 receptors, prevented the ET-1 flow response. The ET-1 flow response was not affected by inhibition of either epithelial Na(+) channels or the mitochondrial Na(+)/Ca(2+) exchanger. Taken together, these findings provide evidence that in IMCD3 cells, flow, via polycystin-2 and P2 receptors, engages Ca(2+)-dependent signaling pathways that stimulate ET-1 synthesis.

Endothelial MRTF-A mediates angiotensin II induced cardiac hypertrophy

Angiotensin II (Ang II) stimulates endothelin (ET-1) transcription, which contributes to cardiac hypertrophy and fibrosis. We have previously reported that myocardin related transcription factor A (MRTF-A) is indispensable for ET-1 transcription in vascular endothelial cells under hypoxic conditions, indicating that MRTF-A might mediate Ang II-induced pathological hypertrophy. Here we report that Ang II augmented the expression of MRTF-A in cultured endothelial cells and in the lungs of mice with cardiac hypertrophy. Over-expression of MRTF-A enhanced, whereas depletion of MRTF-A attenuated, transcriptional activation of ET-1 gene by Ang II. MRTF-A deficiency ameliorated Ang II induced cardiac hypertrophy and fibrosis in mice paralleling diminished synthesis and release of ET-1. Mechanistically, MRTF-A was recruited to the ET-1 promoter by c-Jun/c-Fos (AP-1) in response to Ang II treatment. Once bound, MRTF-A altered the chromatin structure by modulating histone acetylation and H3K4 methylation on the ET-1 promoter. More importantly, mice with endothelial-specific MRTF-A silencing by lentiviral particles phenocopied mice with systemic MRTF-A deletion in terms of Ang II-induced pathological hypertrophy. In conclusion, we data have unveiled a MRTF-A-containing complex that links ET-1 transactivation in endothelial cells to cardiac hypertrophy and fibrosis by Ang II.

Asthme sévère : nouvelles cibles thérapeutiques

Severe asthma is a highly incapacitating disease with no effective preventive or curative treatment. The 10% of patients with "refractory" or "difficult" asthma have chronic symptoms, episodic exacerbations and persistent airway obstruction, despite chronic? 2-agonist and steroid therapy. A major objective of current asthma research is to identify the underlying cellular and molecular mechanisms and thus to develop new treatments. Persistent airway eosinophilia is a hallmark of severe asthma. IL-5 is essential for terminal differentiation of committed eosinophil precursors and is also involved in eosinophily degranulation and priming. By releasing cytokines and cationic proteins, eosinophils contribute to airway inflammation and damage the bronchial mucosa. A monoclonal antibody against IL-5 has been shown to reduce exacerbations of refractory asthma. Interventions targeting eosinophil cationic proteins might have therapeutic potential. Structural changes in the bronchial wall, collectively known as airway remodeling, are believed to play a prominent role in the persistent airflow obstruction associated with severe asthma. In this setting the airway epithelium shows major abnormalities, including loss of barrier function, phenotypic changes, and functional disorders. The abnormal respiratory epithelium is believed to orchestrate airway remodeling through aberrant production of extracellular matrix components, fibrogenic cytokines and chemokines, and growth factors responsible for the proliferation, migration and activation of smooth muscle cells and fibroblasts. Recently, increased ET-1 synthesis by the bronchial epithelium was observed in severe refractory asthma, and was found to correlate with airway remodeling and airway obstruction. ET-1 might represent a novel therapeutic target in severe steroid-refractory asthma.

Cerebral Malaria: A Vasculopathy

Cerebral Malaria: A Vasculopathy

Contribution of oxidative stress to pulmonary arterial hypertension

Recent data implicate oxidative stress as a mediator of pulmonary hypertension (PH) and of the associated pathological changes to the pulmonary vasculature and right ventricle (RV). Increases in reactive oxygen species (ROS), altered redox state, and elevated oxidant stress have been demonstrated in the lungs and RV of several animal models of PH, including chronic hypoxia, monocrotaline toxicity, caveolin-1 knock-out mouse, and the transgenic Ren2 rat which overexpresses the mouse renin gene. Generation of ROS in these models is derived mostly from the activities of the nicotinamide adenine dinucleotide phosphate oxidases, xanthine oxidase, and uncoupled endothelial nitric oxide synthase. As disease progresses circulating monocytes and bone marrow-derived monocytic progenitor cells are attracted to and accumulate in the pulmonary vasculature. Once established, these inflammatory cells generate ROS and secrete mitogenic and fibrogenic cytokines that induce cell proliferation and fibrosis in the vascular wall resulting in progressive vascular remodeling. Deficiencies in antioxidant enzymes also contribute to pulmonary hypertensive states. Current therapies were developed to improve endothelial function, reduce pulmonary artery pressure, and slow the progression of vascular remodeling in the pulmonary vasculature by targeting deficiencies in either NO (PDE-type 5 inhibition) or PGI(2) (prostacyclin analogs), or excessive synthesis of ET-1 (ET receptor blockers) with the intent to improve patient clinical status and survival. New therapies may slow disease progression to some extent, but long term management has not been achieved and mortality is still high. Although little is known concerning the effects of current pulmonary arterial hypertension treatments on RV structure and function, interest in this area is increasing. Development of therapeutic strategies that simultaneously target pathology in the pulmonary vasculature and RV may be beneficial in reducing mortality associated with RV failure.

Abstract 4872: Beta Blockers and Acute Release of ET-1 in Response to Anger Recall Stress in Patients With CAD

Acute anger is associated with triggered AMI and transient myocardial ischemia. Anger provoked ischemia differs from demand related ischemia with regard to differential epicardial vs. microvascular processes. Use of beta-blockers is standard of care for patients with CAD. Endothelin-1 plays a central role in vascular regulation and homeostasis, particularly in diseased coronary segments, and has been linked to endothelial dysfunction during laboratory emotional stress. Beta-blockade has been reported to reduce plasma levels of ET-1 in patients with marked LV dysfunction/CHF and to inhibit the synthesis and release of ET-1 in cellular preparations. We studied the relationship of beta-blockade use on laboratory anger stress-provoked levels of ET-1 in patients with CAD without marked LV dysfunction/CHF. Methods and results: 104 patients with history of stable CAD underwent sequential 10-min resting baseline (BL) and 6 minute anger-recall stress (AR). Blood samples drawn at the end of rest and stress were assayed for ET-1 by ELISA (fmo/mL) and change from BL to AR calculated. As expected, the hemodynamic response to anger stress was significantly lower (p=0.015) among patients taking beta-blockers (n=81) than those not taking these agents (n=23). These two groups did not differ in ET-1 at BL (2.27±3.77 vs. 1.24±1.89, p = 0.98). Those not taking beta-blockers however, showed a significantly greater % increase in ET-1 from BL to AR (16.12%±29.09% vs. 2.14%±23.85%, p <0.036); this relationship persisted after adjusting for rate pressure product, age, diabetes, HTN, and statin use (p=0.03). Comments: Beta-blocker use was associated with a significantly less anger-stress provoked increase in ET-1. This may be one of the mechanisms behind beneficial effects longitudinally of beta-blocker use on incident coronary events in patients with CAD.

Endothelin-1 and Transforming Growth Factor-β1 Independently Induce Fibroblast Resistance to Apoptosis via AKT Activation

Myofibroblast apoptosis is critical for the normal resolution of wound repair responses, and impaired myofibroblast apoptosis is associated with tissue fibrosis. Lung expression of endothelin (ET)-1, a soluble peptide implicated in fibrogenesis, is increased in murine models of pulmonary fibrosis and in the lungs of humans with pulmonary fibrosis. Mechanistically, ET-1 has been shown to induce fibroblast proliferation, differentiation, contraction, and collagen synthesis. In this study, we examined the role ET-1 in the regulation of lung fibroblast survival and apoptosis. ET-1 rapidly activates the prosurvival phosphatidylinositol 3'-OH kinase (PI3K)/AKT signaling pathway in normal and fibrotic human lung fibroblasts. ET-1-induced activation of PI3K/AKT is dependent on p38 mitogen-activated protein kinase (MAPK), but not extracellular signal-regulated kinase (ERK) 1/2, JNK, or transforming growth factor (TGF)-beta1. Activation of the PI3K/AKT pathway by ET-1 inhibits fibroblast apoptosis, and this inhibition is reversed by blockade of p38 MAPK or PI3K. TGF-beta1 has been shown to attenuate myofibroblast apoptosis through the p38 MAPK-dependent secretion of a soluble factor, which activates PI3K/AKT. In this study, we show that, although TGF-beta1 induces fibroblast synthesis and secretion of ET-1, TGF-beta1 activation of PI3K/AKT is not dependent on ET-1. We conclude that ET-1 and TGF-beta1 independently promote fibroblast resistance to apoptosis through signaling pathways involving p38 MAPK and PI3K/AKT. These findings suggest the potential for novel therapies targeting the convergence of prosurvival signaling pathways activated by these two profibrotic mediators.

Treatment with TNF-α or bacterial lipopolysaccharide attenuates endocardial endothelial cell-mediated stimulation of cardiac fibroblasts

BackgroundThe endocardial endothelium that lines the inner cavity of the heart is distinct from the microvascular endothelial cells and modulates cardiac muscle performance in a manner similar to the vascular endothelial modulation of vascular structure and vasomotor tone. Although the modulatory effects of endocardial endothelium (EE) on cardiomyocytes are firmly established, the regulatory effects of endocardial endothelium on the cardiac interstitium and its cellular components remain ill defined.Methods and ResultsWe investigated whether the stimulatory effect of EE on cardiac fibroblasts would be altered when EECs are activated by the cytokine tumor necrosis factor-α (TNF-α) or the endotoxin bacterial lipopolysaccharide (LPS). Both TNF-α and LPS were found to independently attenuate the stimulatory effect of EE on cardiac fibroblasts. These agents lowered the synthesis or release of ET-1 and increased the secretion of TGF-β and NO.ConclusionThe findings of this study using endocardial endothelial cells (EECs) and neonatal cardiac fibroblasts demonstrate that pro-inflammatory cytokines cause altered secretion of paracrine factors by EECs and inhibit proliferation and lower collagen synthesis in fibroblasts. These changes may influence fibroblast response and extra cellular matrix remodeling in pathological conditions of the heart.

Endothelin and Aged Blood Vessels

Age is one of the most important risk factors for cardiovascular disease. For example, a 30-year-old male smoker with untreated hypertension, diabetes, dyslipidemia, and a strong family history of premature coronary heart disease (CHD) would have a 10-year coronary heart disease event rate of only 16% (“intermediate risk”) by Framingham criteria; however, given exactly the same risk profile for a 60-year-old, the risk climbs to >50%. Aging, particularly sedentary aging, leads not only to the development of atherosclerosis but also to ventricular and arterial stiffening.1 This reduces functional capacity and contributes to chronic diseases of the elderly, such as systolic hypertension or heart failure with a preserved ejection fraction. In the search for the underlying mechanism for this effect of sedentary aging, one common feature that has been identified universally in older men is an increased baseline vascular tone,2 which has both hemodynamic and metabolic consequences.3 Although this age-related increase in vascular tone is mediated in part by a chronically elevated sympathetic α-adrenergic vasoconstriction,2 endothelial function also plays a critical role in vascular stability and the regulation of vasomotor function. The endothelium regulates vascular tone through the release of dilator and constrictor substances. Endothelin (ET)-1 is the most potent endothelium-derived constricting factor and influences peripheral vascular tone by interacting with ETA and ETB receptors on smooth muscle cells and the endothelium.4 Several animal studies have examined the profound effects of vascular aging on the ET-1 pathway and support a central role for ET-1 to explain the age-related increase in vascular tone.5–7 In the present issue of Hypertension , Van Guilder et al8 present new and important information regarding the role of aging and physical fitness on the vasomotor effects of ET-1. Using a cross-sectional design in a small but carefully studied …

Differential Role of TGF‐β1/bFGF and ET‐1 in Graft Fibrosis in Heart Failure Patients

Collagen overproduction characteristic for dilated cardiomyopathy (DCM) is coregulated by endothelin (ET)-1, transforming growth factor (TGF)-beta1, basic fibroblast growth factor (bFGF) and matrix metalloproteases (MMPs). Whether these molecules affect grafts transplanted to heart failure patients is unknown. In 67 idiopathic DCM patients, 31 patients with ischemic cardiomyopathy (ICM) and 16 controls, the myocardial bFGF, TGF-beta1, pro-collagen (PrCol) type 1 (PrCol1-alpha1, -alpha2) and MMP expressions were examined using real-time RT-PCR or Western blotting. mRNA expression was measured in grafts for 1 year. TGF-beta1/bFGF stimulation or gene silencing was used to examine their effect on collagen synthesis in cardiac tissue cultures. TGF-beta1 and PrCol1 were upregulated in DCM only, while bFGF was upregulated in both groups versus controls. TGF-beta1 downregulated MMP-1 and upregulated collagen 1, whereas bFGF upregulated MMP-13 in DCM tissue. Post-transplant PrCol1-alpha1, -alpha2 and ET-1 mRNA increased over time in grafts of DCM patients only, while other factors returned to control baseline levels in DCM and ICM. These data indicate that cardiac transplantation corrects the dysregulated TGF/bFGF/MMP-1/MMP-13, but not the excess collagen and ET-1 synthesis in cardiac grafts transplanted to DCM patients. ET-1 might be a major pathologic trigger for graft fibrosis in DCM.

Endothelin mediates superoxide production in angiotensin II-induced hypertension in rats

Angiotensin II and endothelin-1 (ET) are two hormones involved in cardiovascular diseases and well known for their capacity to induce free radical generation in vascular and cardiac tissues. In addition to its prooxidative effect, angiotensin II can increase the synthesis of ET-1 in vascular smooth muscle cells (VSMC). Our objective was to determine whether the ET-1 synthesis in VSMC is involved in angiotensin II-induced superoxide anion production in rats. Our results show that treatments of isolated VSMC with angiotensin II and ET increased superoxide. However, this increase occurred in a bimodal fashion for angiotensin II with a fast transient production (10 min) and a late sustained production (6 h), while ET-1 induced superoxide formation after a delay of 6 h. LU302872 and BQ-123, a nonselective and a selective ETA receptor antagonists, respectively, prevented angiotensin II-induced superoxide anion production only during the late phase. In contrast, BQ-3020, a selective ETB receptor antagonist, had no effect. In vivo, LU302872 reduced the aortic superoxide production induced by angiotensin II administered for 12 days. In conclusion, our results suggest that the superoxide generation induced by chronic angiotensin II infusion may be mediated by ET-1 acting on ETA receptors in VSMC in vitro. Furthermore, this effect appears to contribute to the excess superoxide production during the chronic activation of the renin-angiotensin system in vivo.

A high-salt diet stimulates thick ascending limb eNOS expression by raising medullary osmolality and increasing release of endothelin-1.

A high-salt diet increases renal endothelin (ET) production and thick ascending limb (THAL) endothelial nitric oxide synthase (eNOS) expression. ET stimulates THAL eNOS expression via ET(B) receptors. The tonicity of the renal medulla is highly variable, and hyperosmolality stimulates ET-1 synthesis by endothelial cells. We hypothesized that a high-salt diet raises medullary osmolality, increases ET release by the THAL, and thus enhances eNOS expression. Seven days of high salt (1% NaCl in drinking water) increased eNOS expression in THALs by 125 +/- 31%. High salt increased outer medullary osmolality from 362 +/- 13 to 423 +/- 6 mosmol/kg H(2)O (P < 0.05). Bosentan, a dual-ET receptor antagonist, blocked the increase in THAL eNOS expression caused by high salt (2.66 +/- 0.44 absorbance units with bosentan vs. 5.15 +/- 0.67 for vehicle; P < 0.05). Conscious systolic blood pressure did not differ between the two groups. In primary cultures of medullary THALs, raising osmolality from 300 to 350 and 400 mosmol/kg H(2)O using NaCl increased eNOS expression by 39 +/- 11% (P < 0.05) and 71 +/- 16%, respectively (P < 0.05). In primary cultures of THALs, raising osmolality from 300 to 400 mosmol/kg H(2)O for 1 h increased ET-1 release from 62 +/- 7 to 113 +/- 2 pg/mg protein (P < 0.05). BQ-788, an ET(B) receptor antagonist (1 muM), blocked the stimulatory effect of 400 mosmol/kg H(2)O on eNOS expression (70 +/- 13% vs. -5 +/- 10%; paired difference, 74 +/- 15%; P < 0.05). BQ-788 alone had no significant effect. We concluded that high salt stimulates THAL eNOS expression by increasing outer medullary osmolality, ET-1 release by the THAL and ET(B) receptor activation. This may be an important regulatory mechanism of THAL NaCl absorption when dietary salt intake is increased.