Administration Of BQ-123 Research Articles

Endothelin-1 increases Na+-K+-2Cl- cotransporter-1 expression in cultured astrocytes and in traumatic brain injury model: An involvement of HIF1α activation.

Na+-K+-2Cl- cotransporter-1 (NKCC1) is present in brain cells, including astrocytes. The expression of astrocytic NKCC1 increases in the acute phase of traumatic brain injury (TBI), which induces brain edema. Endothelin-1 (ET-1) is a factor that induces brain edema and regulates the expression of several pathology-related genes in astrocytes. In the present study, we investigated the effect of ET-1 on NKCC1 expression in astrocytes. ET-1 (100 nM)-treated cultured astrocytes showed increased NKCC1 mRNA and protein levels. The effect of ET-1 on NKCC1 expression in cultured astrocytes was reduced by BQ788 (1 μM), an ETB antagonist, but not by FR139317 (1 μM), an ETA antagonist. The involvement of ET-1 in NKCC1 expression in TBI was examined using a fluid percussion injury (FPI) mouse model that replicates the pathology of TBI with high reproducibility. Administration of BQ788 (15 nmol/day) decreased FPI-induced expressions of NKCC1 mRNA and protein, accompanied with a reduction of astrocytic activation. FPI-induced brain edema was attenuated by BQ788 and NKCC1 inhibitors (azosemide and bumetanide). ET-1-treated cultured astrocytes showed increased mRNA and protein expression of hypoxia-inducible factor-1α (HIF1α). Immunohistochemical observations of mouse cerebrum after FPI showed co-localization of HIF1α with GFAP-positive astrocytes. Increased HIF1α expression in the TBI model was reversed by BQ788. FM19G11 (an HIF inhibitor, 1 μM) and HIF1α siRNA suppressed ET-induced increase in NKCC1 expression in cultured astrocytes. These results indicate that ET-1 increases NKCC1 expression in astrocytes through the activation of HIF1α.

Attenuation of opioid tolerance by ETA receptor antagonist, BQ123, administered intravenously in mice

Intracerebroventricular injection of endothelin-A receptor antagonist BQ123 potentiates opioid analgesia and reverses analgesic tolerance. This study explores whether these effects can be replicated by injecting BQ123 intravenously. Male Swiss-Webster mice were used. Morphine tolerance was induced using 3- or 7-day dosing. Intravenous BQ123 (8mg/kg) was injected only once on Day 1, 2, 3 or 4 (3-day studies), and on Day 4, 6 or 8 (7-day studies). On Day 4 or 8, respectively, tail-flick and hot-plate latencies were measured following a morphine challenge dose. Intravenous BQ123 increased the potency and duration of morphine antinociceptive responses. In the 3-day study, the antinociceptive response was unaffected by BQ123 given on Days 1 or 2. BQ123 treatment on Day 3 or 4 (Day 4, BQ123 given 15-min before morphine) significantly potentiated antinociceptive response versus vehicle-treated tolerant mice. In 7-day studies, the antinociceptive response was unaffected by BQ123 given on Day 4. BQ123 given on Day 6 or 8 (Day 8, BQ123 given 15-min before morphine) produced a >100% increase in antinociceptive response versus vehicle-treated tolerant mice for at least 48h. Intravenous administration of BQ123 is effective in potentiating morphine analgesia and restoring antinociceptive response in morphine-tolerant mice.

Down-regulation of astrocytic sonic hedgehog by activation of endothelin ETB receptors: Involvement in traumatic brain injury-induced disruption of blood brain barrier in a mouse model

In the adult brain, sonic hedgehog acts on cerebral microvascular endothelial cells to stabilize the blood-brain barrier. The expression of sonic hedgehog by astrocytes is altered during brain injury, and this change has been shown to affect permeability of blood-brain barrier. However, much remains unknown about the regulation of astrocytic sonic hedgehog production. Our results showed that endothelin-1 reduced sonic hedgehog mRNA expression and extracellular protein release in mouse cerebral cultured astrocytes, but had no effect in bEnd.3, a mouse brain microvascular endothelial-derived cell line. The effect of endothelin-1 on astrocyte sonic hedgehog expression was suppressed by an ETB antagonist BQ788, but was unchanged by the ETA antagonist FR139317. In cultured astrocytes and bEnd.3, endothelin-1 did not affect the expression of the sonic hedgehog receptor-related molecules, patched-1 and smoothened. In an animal model of traumatic brain injury, fluid percussion injury on the mouse cerebrum increased the expression of sonic hedgehog, patched-1, and smoothened. Repeated administration of BQ788 enhanced sonic hedgehog expression at 5 days after fluid percussion injury. Histochemical examination revealed sonic hedgehog expression in glial fibrillary acidic protein-positive astrocytes in the cerebrum after fluid percussion injury. Administration of exogenous sonic hedgehog and BQ788 suppressed Evans blue extravasation, an indicator of blood vessel permeability, induced by fluid percussion injury. The effects of BQ788 on fluid percussion injury-induced Evans blue extravasation were reduced by the administration of jervine, a sonic hedgehog inhibitor. Altogether, these results suggest that endothelin-1 down-regulates astrocytic sonic hedgehog to promote disruption of the blood-brain barrier during traumatic brain injury.

ET-B receptors involvement in peripheral opioid analgesia induced by light-emitting diode photobiomodulation in male and female mice

Currently, photobiomodulation therapy (PBMT) is gaining space in the scientific and clinical environment. To help elucidate the importance of irradiance, this study evaluated the effect of two different PBMT irradiances (3.5 and 90 mW/cm2), given a fixed wavelength of 630 nm and a dose of 2 J/cm2, on mechanical hyperalgesia following Complete Freund's Adjuvant (CFA) intraplantar (i.pl.) injection in mice. Additionally, we investigated the role of peripheral opioid and endothelin-B receptors (ETB-R), as well as sex differences in treatment outcome. Different groups of male or female mice were evaluated 6 and 96 h after CFA. Mechanical hyperalgesia was evaluated 30 min after treatments. Naloxone or Bq-788 administration, fifteen minutes before PBMT or Sarafotoxin S6c, helped determine the involvement of peripheral opioid and ETB-Rs on PBMT. Lastly, ETB-Rs skin immunocontent in both sexes was quantified after PBMT consecutive daily treatments. PBMT at an irradiance of 90 mW/cm2, was more effective than 3.5 mW/cm2. Bq-788 and naloxone administration prevented the effects of PBMT and SRTX S6c; however, PBMT did not influence peripheral ETB-Rs immunocontent. The results suggest that irradiance influences PMBT effect; and that activation of ETB-R play a role in peripheral PBMT opioid induced analgesia. Lastly, PMBT effects do not appear to be sex-dependent.

The Role of Endothelin‐1 Receptor Subtypes in Limiting Insulin‐Stimulated Blood Flow in Middle‐Aged Adults

Beyond its metabolic actions, insulin stimulates peripheral vasodilation, which serves to increase glucose delivery to skeletal muscle. Insulin activates two distinct signaling pathways leading to the production of nitric oxide versus endothelin‐1 (ET‐1). Several lines of research suggest that insulin‐stimulated ET‐1 production may limit insulin‐stimulated nitric oxide‐mediated vasodilation. Recently, it was demonstrated that basal ET‐1‐mediated vasoconstricton is elevated in middle‐aged adults. However, no studies have examined the role of ET‐1 during hyperinsulinemia in middle‐aged individuals. Therefore, we tested the hypothesis that ET‐1 receptor antagonism would improve insulin‐stimulated blood flow (BF) in a cohort of middle‐aged adults. In 14 subjects (age: 54±2 years, BMI: 26±1 kg/m2) with a range of insulin resistance (glucose infusion rate range: 2.0 to 7.2 mg/kg/min), two femoral arterial catheters were placed. One catheter in the left leg for measurement of arterial blood pressure (BP) and one in the right leg for determination of arterial glucose concentrations and intra‐arterial administration (25nmol/min) of BQ‐123 (ETA antagonist) and BQ‐788 (ETB antagonist). In addition, a femoral venous catheter (right leg) was placed for determination of venous glucose concentrations, and one antecubital venous catheter was placed for infusion of insulin and glucose. Femoral artery BF (right leg: Doppler Ultrasound), BP, and glucose uptake (arterial‐venous glucose concentration x BF) were measured during a hyperinsulinemic‐euglycemic clamp alone (insulin: 40 mU/m2/min), followed by hyperinsulinemia with intra‐arterial BQ‐123 administered for one hour (Insulin+BQ‐123). BQ‐788 was then co‐infused with BQ‐123 for an additional hour (insulin+BQ‐123+BQ‐788; n=10). The infusion of BQ‐123 during hyperinsulinemia augmented leg BF (Insulin: 399±42 mL/min; Insulin+BQ‐123: 500±47 mL/min, P<0.001) and vascular conductance (Insulin: 4.3±0.5 mL/min/mmHg; Insulin+BQ‐123: 5.8±0.7 mL/min/mmHg, P<0.001), and increased leg glucose uptake (Insulin: 76±13 mg/min; Insulin+BQ‐123: 103±15 mg/min, P=0.02). Subsequently, co‐infusion of BQ‐788 provided no further increase in leg BF (Insulin+BQ‐123: 485±60 mL/min; Insulin+BQ‐123+BQ‐788: 511±70 mL/min, P=0.42), vascular conductance, or glucose uptake. Sub‐analysis of subjects with type 2 diabetes (T2D, n=7) versus non‐T2D (n=7) suggested no difference in responsiveness to either drug intervention between groups (both P>0.05). These data suggest ET‐1 signaling through ETA receptors plays an important role in limiting insulin‐stimulated BF in middle‐age, whereas ETB receptor signaling has little effect. These findings provide novel insight into the role of ET‐1 during hyperinsulinemia, favoring selective targeting of the ETA receptor for improving insulin‐stimulated BF and glucose uptake with aging.Support or Funding InformationSupported by UTA College of Nursing and Health Innovation

Angiopoietin-1/Tie-2 signal after focal traumatic brain injury is potentiated by BQ788, an ETB receptor antagonist, in the mouse cerebrum: Involvement in recovery of blood-brain barrier function.

Angiopoietin-1, an angiogenic factor, stabilizes brain microvessels through Tie-2 receptor tyrosine kinase. In traumatic brain injury, blood-brain barrier (BBB) disruption is an aggravating factor that induces brain edema and neuroinflammation. We previously showed that BQ788, an endothelin ETB receptor antagonist, promoted recovery of BBB function after lateral fluid percussion injury (FPI) in mice. To clarify the mechanisms underlying BBB recovery mediated by BQ788, we examined the involvements of the angiopoietin-1/Tie-2 signal. When angiopoietin-1 production and Tie-2 phosphorylation were assayed by quantitative reverse transcription polymerase chain reaction and western blotting, increased angiopoietin-1 production and Tie-2 phosphorylation were observed in 7-10days after FPI in the mouse cerebrum, whereas no significant effects were obtained at 5days. When BQ788 (15nmol/day, i.c.v.) were administered in 2-5days after FPI, increased angiopoietin-1 production and Tie-2 phosphorylation were observed. Immunohistochemical observations showed that brain microvessels and astrocytes contained angiopoietin-1 after FPI, and brain microvessels also contained phosphorylated Tie-2. Treatment with endothelin-1 (100nM) decreased angiopoietin-1 production in cultured astrocytes and the effect was inhibited by BQ788 (1μM). Five days after FPI, increased extravasation of Evans blue dye accompanied by reduction in claudin-5, occludin, and zonula occludens-1 proteins were observed in mouse cerebrum while these effects of FPI were reduced by BQ788 and exogenous angiopoietin-1 (1μg/day, i.c.v.). The effects of BQ788 were inhibited by co-administration of a Tie-2 kinase inhibitor (40nmol/day, i.c.v.). These results suggest that BQ788 administration after traumatic brain injury promotes recovery of BBB function through activation of the angiopoietin-1/Tie-2 signal.

Blockage of spinal endothelin A receptors attenuates bone cancer pain via regulation of the Akt/ERK signaling pathway in mice

Bone cancer pain (BCP) is a common source of pain in patients with advanced stage and metastatic cancer; however, existing treatment for this kind of pain remains deficient. Being closely related to sensory change and inflammatory pain in both the central and peripheral nervous systems, endothelin A receptor (ETAR) plays an essential role in pain processing. As a result, ETAR antagonist has been reported to alleviate both neuropathic and inflammatory pain. Thus far, the role of ETAR in the process of BCP is still ambiguous. In this study, by using a BCP mouse model, the analgesic effect and molecular mechanism of the ETAR antagonist BQ-123 was investigated. Pain sensation in the BCP mouse model was investigated by the number of spontaneous flinches (NSF) and pain withdrawal threshold (PWT), and the mechanism of BCP was assessed by measuring p-Akt, p-Akt/t-Akt, p-ERK-1/2 and p-ERK-1/2/t-ERK-1/2 levels in L4–6 segments of the spinal cord. Our results demonstrated that BCP mice showed a higher NSF and a lower PWT score than Sham mice. In addition to the development of nociceptive sensitization, p-Akt, p-Akt/t-Akt, p-ERK-1/2 and p-ERK-1/2/t-ERK-1/2 were up-regulated correspondingly in L4–6 segments of the spinal cord in BCP mice. BQ-123 treatment showed a promising analgesic effect, and the effect was correlated to the down-regulation of p-Akt, p-Akt/t-Akt, p-ERK-1/2 and p-ERK-1/2/t-ERK-1/2 in spinal cord cells. The results suggested that intrathecal administration of BQ-123 was able to relieve BCP in mice as a consequence of suppressing the Akt and ERK signalling pathways.

Delayed Administration of BQ788, an ETB Antagonist, after Experimental Traumatic Brain Injury Promotes Recovery of Blood-Brain Barrier Function and a Reduction of Cerebral Edema in Mice.

Traumatic brain injury (TBI) is induced by immediate physical disruption of brain tissue, and causes death and disability. Studies on experimental TBI animal models show that disruption of the blood-brain barrier (BBB) underlies brain edema and neuroinflammation during the delayed phase of TBI. In neurological disorders, endothelin-1 (ET-1) is involved in BBB dysfunction and brain edema. In this study, the effect of ET antagonists on BBB dysfunction and brain edema were examined in a mouse focal TBI model using lateral fluid percussion injury (FPI). ET-1 and ETB receptors were increased at 2-7 days after FPI, which was accompanied by extravasation of Evans blue (EB) and brain edema. Repeated intracerebroventricular administration of BQ788 (15 nmol/day), an ETB antagonist, from 2 days after FPI promoted recovery of EB extravasation and brain edema, while FR 139317, an ETA antagonist, had no effect. Delayed intravenous administration of BQ788 also promoted recovery from FPI-induced EB extravasation and brain edema. While FPI caused decreases in claudin-5, occludin, and zonula occludens-1 proteins, BQ788 reversed FPI-induced reductions of them. Immunohistochemical observation of the cerebrum after FPI showed that ETB receptors are predominantly expressed in glial fibrillary acidic protein (GFAP)-positive astrocytes. BQ788 reduced FPI-induced increases in GFAP-positive astrocytes. GFAP-positive astrocytes produced vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase-9 (MMP9). FPI-induced increases in VEGF-A and MMP-9 production were reversed by BQ788. These results suggest that ETB receptor antagonism during the delayed phase of focal TBI promotes recovery of BBB function and reduction of brain edema.

Endothelin‑1 induces oncostatin M expression in osteoarthritis osteoblasts by trans‑activating the oncostatin M gene promoter via Ets‑1.

Oncostatin M (OSM) contributes to cartilage degeneration in osteoarthritis (OA) and was demonstrated to be expressed in OA osteoblasts. Endothelin‑1 (ET‑1) is implicated in the degradation of OA articular cartilage, and osteoblast proliferation and bone development. In the present study, the effects of ET‑1 on OSM expression in human OA osteoblasts were investigated, to the best of our knowledge, for the first time. Primary human OA osteoblasts were treated with ET‑1 (1, 5, 10, 20 and 30 nM) for 0.5, 1, 2, 3 and 4 h with or without the selective ETA receptor (ETAR) antagonist, BQ123, ETB receptor antagonist, BQ788 or the phosphatidylinositol 3‑kinase (PI3K) inhibitor, BKM120. ET‑1 treatment induced OSM mRNA expression, and the intracellular and secreted protein levels of OA osteoblasts in a dose‑dependent manner. This effect was suppressed by BQ123 and BKM120, but not BQ788 administration. In combination with electrophoretic mobility shift assays, deletional and mutational analyses on the activity of a human OSM promoter/luciferase reporter demonstrated that ET‑1 induced OSM expression in OA osteoblasts by trans‑activating the OSM gene promoter through specific binding of Ets‑1 to an Ets‑1 binding site in the OSM promoter in an ETAR‑ and PI3K‑dependent manner. Furthermore, ET‑1 treatment increased the expression of Ets‑1 in a dose‑dependent manner, however the knockdown of Ets‑1 suppressed the ET1‑induced expression of OSM in OA osteoblasts. In conclusion, the present study demonstrated that ET‑1 induces the expression of OSM in OA osteoblasts by trans‑activating the OSM gene promoter primarily through increasing the expression level of Ets‑1 in an ETAR‑ and PI3K‑dependent manner. The current study suggested novel insights into the mechanistic role of ET‑1 in the pathophysiology of OA.

Abstract 10198: Endothelin B Receptor Agonist, IRL-1620 is an Effective Neurovascular Remodeling Agent in Diabetic Rats With Cerebral Ischemia

Endothelin B (ETB) receptor agonist, IRL-1620 administered at 2, 4, 6 hr post-cerebral ischemia, has been shown to significantly aid in neuroprotection and neurovascular remodeling in normal rats. The incidence of type 2 diabetes mellitus (diabetes) is on the rise and it can be expected that there will be an upsurge of associated diseases, such as ischemic stroke in patients with diabetes. The present study was conducted to determine the efficacy and neurovascular remodeling effect of IRL-1620 in normal (non-diabetic) and diabetic rats following cerebral ischemia induced by right middle cerebral artery (MCAO). The induction of diabetes was achieved through a high-fat diet and single low dose (45 mg/kg) of streptozotocin. IRL-1620 (5 μg/kg) was administered intravenously at 4, 6, and 8 hr after MCAO on day 0, 3 and 6 (total of 15 μg/kg in three divided doses on day 0, 3 and 6). A mortality of 46% was observed by day 7 in normal rats with MCAO when treated with vehicle, while it was 17% when treated with IRL-1620. In diabetic rats 90% mortality was observed by day 7 post-MCAO when treated with vehicle, while it was 45% when treated with IRL-1620. Functional outcome for the IRL-1620 group was significantly better in both non-diabetic and diabetic rats compared to the vehicle group. In both non-diabetic (80.81%; p=0.02) and diabetic (69.58%; p=0.04) rats a significant reduction in infarct volume was seen in the IRL-1620 group compared to the vehicle group. Significantly more blood vessels/30μm brain slice were observed in the IRL-1620 versus vehicle group. Immunofluorescence staining revealed a significant increase in ETB receptor expression for rats treated with IRL-1620, compared to vehicle. Co-localization of VEGF+ve and endothelial cells in diabetic rats was higher (p=0.0009) in the IRL-1620 group (15.5±1.1%) compared to the vehicle group (10.2±0.81%). In diabetic rats, the IRL-1620 group had significantly (p=0.0012) more neurons in the cortex compared to the vehicle group as well as the highest levels of neuronal replication. Administration of BQ-788, a specific ETB receptor antagonist, attenuated the effects of IRL-1620. It is concluded that IRL-1620 is an effective neurovascular remodeling agent and may be useful in the treatment of cerebral ischemia.

Improvement of cold injury-induced mouse brain edema by endothelin ETB antagonists is accompanied by decreases in matrixmetalloproteinase 9 and vascular endothelial growth factor-A.

Brain edema is a potentially fatal pathological state that often occurs after brain injuries such as ischemia and trauma. However, therapeutic agents that fundamentally treat brain edema have not yet been established. We previously found that endothelin ETB receptor antagonists attenuate the formation and maintenance of vasogenic brain edema after cold injury in mice. In this study, the effects of ETB antagonists on matrixmetalloproteinase (MMP)9 and vascular endothelial growth factor (VEGF)-A expression were examined in the cold injury model. Cold injury was performed in the left brain of male ddY mice (5-6 weeks old) for the induction of vasogenic edema. Expression of MMP9 and VEGF-A mRNA in the mouse cerebrum was increased by cold injury. Immunohistochemical observations showed that the MMP9 and VEGF-A were mainly produced in reactive astrocytes in the damaged cerebrum. Intracerebroventricular administration of BQ788 (10 μg) or IRL-2500 (10 μg) (selective ETB antagonists) attenuated brain edema and disruption of the blood-brain barrier after cold injury. BQ788 and IRL-2500 reversed the cold injury-induced increases in MMP9 and VEGF-A expression. The induction of reactive astrocytes producing MMP9 and VEGF-A in the damaged cerebrum was attenuated by BQ788 and IRL-2500. These results suggest that attenuations of astrocytic MMP9 and VEGF-A expression by ETB antagonists may be involved in the amelioration of vasogenic brain edema.

Cytokines, but not corticotropin-releasing factor and endothelin-1, participate centrally in the febrile response in zymosan-induced arthritis in rats

Recent literature has revealed that centrally generated prostaglandins participate in the febrile response in zymosan-induced arthritis in rats. However, it is not clear whether other centrally acting pyrogenic mediators such as cytokines, endothelins (ETs), and the corticotropin-releasing factor (CRF) contribute to the febrile response in this model. In the present study, rats were pretreated with intracerebroventricular (i.c.v.) injections of soluble TNF receptor I (sTNFRI), recombinant IL-1 receptor antagonist (IL-1ra), anti-rat IL-6 monoclonal antibody (AbIL-6), α-helical CRF9–41 (a nonselective CRF1/CRF2 receptor antagonist), BQ-123 (an ETA receptor antagonist), BQ-788 (an ETB receptor antagonist), and artificial cerebrospinal fluid (aCSF, control) prior to an intra-articular zymosan (4mg) injection. Rectal temperatures were measured with a telethermometer. The administration of IL-1ra (200µg), sTNFRI (500ng), and AbIL-6 (5µg) attenuated body temperature elevations after a zymosan injection. The administration of BQ-788 (3pmol), BQ-123 (3pmol), and α-helical CRF9-41 (25µg) did not affect the zymosan-induced febrile response. All the compounds used to pretreat the animals did not significantly alter their basal body temperatures. Together, the results here demonstrate that the febrile response in zymosan-induced arthritis in rats depends on the centrally acting pyrogenic cytokines TNF-α, IL-1β, and IL-6, but does not depend on either CRF or ET-1.

Amelioration of cold injury-induced cortical brain edema formation by selective endothelin ETB receptor antagonists in mice.

Brain edema is a potentially fatal pathological condition that often occurs in stroke and head trauma. Following brain insults, endothelins (ETs) are increased and promote several pathophysiological responses. This study examined the effects of ETB antagonists on brain edema formation and disruption of the blood-brain barrier in a mouse cold injury model (Five- to six-week-old male ddY mice). Cold injury increased the water content of the injured cerebrum, and promoted extravasation of both Evans blue and endogenous albumin. In the injury area, expression of prepro-ET-1 mRNA and ET-1 peptide increased. Intracerebroventricular (ICV) administration of BQ788 (ETB antagonist), IRL-2500 (ETB antagonist), or FR139317 (ETA antagonist) prior to cold injury significantly attenuated the increase in brain water content. Bolus administration of BQ788, IRL-2500, or FR139317 also inhibited the cold injury-induced extravasation of Evans blue and albumin. Repeated administration of BQ788 and IRL-2500 beginning at 24 h after cold injury attenuated both the increase in brain water content and extravasation of markers. In contrast, FR139317 had no effect on edema formation when administrated after cold injury. Cold injury stimulated induction of glial fibrillary acidic protein-positive reactive astrocytes in the injured cerebrum. Induction of reactive astrocytes after cold injury was attenuated by ICV administration of BQ788 or IRL-2500. These results suggest that ETB receptor antagonists may be an effective approach to ameliorate brain edema formation following brain insults.

Endothelin-1 Activation of the Endothelin B Receptor Modulates Pulmonary Endothelial CX3CL1 and Contributes to Pulmonary Angiogenesis in Experimental Hepatopulmonary Syndrome

Hepatic production and release of endothelin-1 (ET-1) binding to endothelin B (ETB) receptors, overexpressed in the lung microvasculature, is associated with accumulation of pro-angiogenic monocytes and vascular remodeling in experimental hepatopulmonary syndrome (HPS) after common bile duct ligation (CBDL). We have recently found that lung vascular monocyte adhesion and angiogenesis in HPS involve interaction of endothelial C-X3-C motif ligand 1 (CX3CL1) with monocyte CX3C chemokine receptor 1 (CX3CR1), although whether ET-1/ETB receptor activation influences these events is unknown. Our aim was to define if ET-1/ETB receptor activation modulates CX3CL1/CX3CR1 signaling and lung angiogenesis in experimental HPS. A selective ETB receptor antagonist, BQ788, was given for 2 weeks to 1-week CBDL rats. ET-1 (±BQ788) was given to cultured rat pulmonary microvascular endothelial cells overexpressing ETB receptors. BQ788 treatment significantly decreased lung angiogenesis, monocyte accumulation, and CX3CL1 levels after CBDL. ET-1 treatment significantly induced CX3CL1 production in lung microvascular endothelial cells, which was blocked by inhibitors of Ca(2+) and mitogen-activated protein kinase (MEK)/ERK pathways. ET-1-induced ERK activation was Ca(2+) independent. ET-1 administration also increased endothelial tube formation in vitro, which was inhibited by BQ788 or by blocking Ca(2+) and MEK/ERK activation. CX3CR1 neutralizing antibody partially inhibited ET-1 effects on tube formation. These findings identify a novel mechanistic interaction between the ET-1/ETB receptor axis and CX3CL1/CX3CR1 in mediating pulmonary angiogenesis and vascular monocyte accumulation in experimental HPS.

Effects of endothelin A receptor blockade in patients with ST-elevation acute coronary syndrome — A rhythmologic substudy

AimsVentricular arrhythmias are common after acute myocardial infarction (AMI). Endothelin (ET) is a mediator of microvascular dysfunction and cardiac remodeling with arrhythmogenic potential. The aim of this study was to assess safety and feasibility of selective ET-A receptor blockade in ST-elevation acute coronary syndrome (STE-ACS) within a larger randomized trial. Main methodsPatients with posterior-wall STE-ACS were randomly assigned to receive intravenous BQ-123 at 400nmol/min or placebo over 60min, starting immediately prior to primary percutaneous coronary intervention. Twenty-four hour Holter recordings were performed during hospitalization for STE-ACS and after 6–8weeks. The predefined primary endpoint was the documentation of ventricular tachycardia and/or late potentials at follow-up. Key findingsThere was no significant difference in the predefined primary endpoint at 45 (33–62) days (0/16 (0%) in BQ-123 treated patients vs. 1/14 (7%) in the placebo group, p=0.465). At 2 (1–3) days, an increase in the total number of supraventricular extrasystoles (SVES)/24h in patients randomized to BQ-123 (45 (17–165) beats vs. 11 (5–72) beats in placebo treated patients, p=0.025) occurred. This increase was also observed at 45days (105 (37–216) beats vs. 11 (3–98) beats in placebo treated patients, p=0.037). There was no significant difference regarding other rhythmologic secondary endpoints between the two groups. SignificanceBased on the analysis of long-term ECG data, short-term administration of BQ-123 after AMI was safe. Because of the small sample size, no firm conclusion regarding antiarrhythmic efficacy can be drawn.

Interplay between Endothelin and Erythropoietin in Astroglia: The Role in Protection against Hypoxia

We show that, under in vitro conditions, the vulnerability of astroglia to hypoxia is reflected by alterations in endothelin (ET)-1 release and capacity of erythropoietin (EPO) to regulate ET-1 levels. Exposure of cells to 24 h hypoxia did not induce changes in ET-1 release, while 48–72 h hypoxia resulted in increase of ET-1 release from astrocytes that could be abolished by EPO. The endothelin receptor type A (ETA) antagonist BQ123 increased extracellular levels of ET-1 in human fetal astroglial cell line (SV-FHAS). The survival and proliferation of rat primary astrocytes, neural precursors, and neurons upon hypoxic conditions were increased upon administration of BQ123. Hypoxic injury and aging affected the interaction between the EPO and ET systems. Under hypoxia EPO decreased ET-1 release from astrocytes, while ETA receptor blockade enhanced the expression of EPO mRNA and EPO receptor in culture-aged rat astroglia. The blockade of ETA receptor can increase the availability of ET-1 to the ETB receptor and can potentiate the neuroprotective effects of EPO. Thus, the new therapeutic use of combined administration of EPO and ETA receptor antagonists during hypoxia-associated neurodegenerative disorders of the central nervous system (CNS) can be suggested.

Peri-interventional endothelin-A receptor blockade improves long-term outcome in patients with ST-elevation acute myocardial infarction

Endothelin (ET)-1 is a pro-fibrotic vasoconstrictive peptide causing microvascular dysfunction and cardiac remodelling after acute ST-elevation myocardial infarction (STEMI). It acts via two distinct receptors, ET-A and ET-B, and is involved in inflammation and atherogenesis. Patients with posterior-wall STEMI were randomly assigned to intravenous BQ-123 at 400 nmol/minute (min) or placebo over 60 min, starting immediately prior to primary percutaneous coronary intervention (n=54). Peripheral blood samples were drawn at baseline as well as after 24 hours and 30 days. Myeloperoxidase (MPO), as a marker of neutrophil activation and matrix metalloproteinase 9 (MMP-9), a marker of extracellular matrix degradation were measured in plasma. Clinical follow-up was conducted by an investigator blinded to treatment allocation over three years. During the median follow-up period of 3.6 years (interquartile range [IQR] 3.3-4.1) we observed a longer event-free survival in patients randomised to receive BQ-123 compared with patients randomised to placebo (mean 4.5 years (95% confidence interval: 3.9-5) versus mean 3 years (2.2-3.7), p=0.031). Patients randomised to ET-A receptor blockade demonstrated a greater reduction of MPO levels from baseline to 24 hours compared to placebo-treated patients (-177 ng/ml (IQR 103-274) vs -108 ng/ml (74-147), p=0.006). In addition, a pronounced drop in MMP-9 levels (-568 ng/ml (44-1157) vs -117 ng/ml (57-561), p=0.018) was observed. There was no significant difference in amino-terminal propetide of pro-collagen type III levels. In conclusion, short-term administration of BQ-123 leads to a reduction in MPO, as well as MMP-9 plasma levels and to a longer event-free survival in patients with STEMI.

Endothelin-A receptor blockade and long-term outcome in patients with ST-elevation acute coronary syndrome

Background: ST-Elevation Acute Coronary Syndrome (STE-ACS) is characterized by thrombotic coronary occlusion compromising blood flow at the epicardial and microvascular levels. Coronary thrombi are a source of large amounts of Endothelin-1 (ET-1), a pro-fibrotic vasoconstrictor and a mediator of microvascular dysfunction and cardiac remodeling. As part of a randomized clinical trial where we assessed the effect of the ET-A receptor Blocker BQ-123 on myocardial perfusion in patients with STE-ACS, we conducted a long-term substudy to assess the effect of BQ-123 treatment on the predefined combined endpoint survival or cardiovascular rehospitalization. Methods: Patients with posterior-wall STE-ACS were randomly assigned to intravenous BQ-123 at 400nmol/minute or placebo over 60 minutes, starting immediately prior to primary percutaneous coronary intervention (PCI, n=54). During a three-year follow-up period, patients were followed and kept on optimal medical treatment by an investigator who was blinded to the acute treatment allocation. Results: During the median follow-up period of 3.3 years (IQR 2.9-3.7), no deaths occurred. The reasons for rehospitalisation (n=19) were unplanned coronary revascularization (n=10, 52%), worsening angina (n=3, 17%), hypertensive urgency (n=2, 11%), as well as stroke (n=1), dyspnoea (n=1), ventricular tachycardia (n=1) and cerebrovascular disease (n=1). We observed a longer event-free survival in patients randomized to receive BQ-123 compared with patients randomized to placebo (3.8 years (95% CI: 3.3-4.2) for BQ-123 versus 2.8 years (2.1-3.4) for placebo, p=0.032, Figure 1). ![Figure][1] Conclusion: Short-term administration of BQ-123 in patients undergoing primary PCI for STE-ACS leads to a longer cardiovascular event-free survival. [1]: pending:yes

Antenatal betamethasone has a sex-dependent effect on the in vivo response to endothelin in adult sheep

Antenatal steroid administration is associated with multiple cardiometabolic alterations, including hypertension; however, the mechanisms underlying this phenomenon are unclear. The aim of the present study was to ascertain, in vivo, the contribution of the endothelin system to the development of hypertension in the adult offspring and the signaling pathway involved. Pregnant sheep were treated with two doses of betamethasone (n = 23) or vehicle (n = 22) at 80 days (~0.55) gestation and allowed to deliver at term. Adult sheep were chronically instrumented under general anesthesia to place vascular catheters and a femoral artery flow probe. Blood pressure and flow were recorded continuously, and femoral artery vascular resistance was calculated before and during administration of endothelin 1 (ET-1). Selective blockers (dantrolene, BQ123, niacinamide) or saline were administered simultaneously. Betamethasone-exposed animals exhibited a significant elevation in mean blood pressure (female: 98 ± 1.8 vs. 92 ± 2.1; males: 97 ± 3.4 vs. 90 ± 2.3; mmHg; P < 0.05). ET-1 elicited a significant increase in blood pressure (F = 56.4; P < 0.001) and in vascular resistance (F = 44.3; P < 0.001) in all groups. A betamethasone effect in the vascular resistance response to ET-1 (F = 25.7; P < 0.001) was present in females only, and the effect was partially blunted by niacinamide (F = 6.6; P < 0.01). Combined administration of niacinamide and BQ123, as well as of dantrolene abolished the betamethasone effect on vascular resistance. No significant differences in mRNA expression of ET(A) or ET(B) in endothelial or smooth muscle cells of resistance-size arteries were observed. We conclude that the betamethasone effect on vascular resistance is mediated by an enhanced response to ET-1 through ET(A) receptor via the cyclic ADPR/ryanodine pathway.

Effects of Repeated Central Administration of Endothelin Type A Receptor Antagonist on the Development of Neuropathic Pain in Rats

Endothelin-1 (ET-1) predominates in the endothelin family effectively in vascular tone control, mitogenesis, and neuromodulation. Its receptors are widespread in the central nervous system (CNS) associated with endogenous pain control, suggesting an important role of ET-1 in central pain processing. This study aimed to evaluate the effect of central ET-1 on the development of neuropathic pain behaviour by repeated intrathecal administration of endothelin type A receptor (ETAR) antagonist (BQ-123) in a sciatic nerve ligation (SNL) animal model. BQ-123 was administered intrathecally to rats at dosages 15 μg, 20 μg, 25 μg, and 30 μg, daily for 3 days. Mechanical allodynia was assessed daily 30 minutes before/after injection, 1 hour after injection of BQ-123 from post-SNL day 4 to day 6, and once on day 7 (without BQ-123 administration) before rats were sacrificed. Increasing trends of mechanical threshold were observed, and they reached significance at all dosages on post-SNL day 7 (P < 0.05 at dosage 15 μg and P < 0.001 at dosages 20 μg, 25 μg, and 30 μg) in comparison to control group. BQ-123 at dosage 30 μg showed the most stable and significant mechanical threshold rise. Repeated central administration of BQ-123 alleviated mechanical allodynia after SNL. Our results provide insight into the therapeutic strategies, including timing, against neuropathic pain development with ETAR antagonist.