Endothelin-1 is a novel prognostic factor in non-small cell lung cancer

Endothelin-1 (ET-1) is a potent vasoactive peptide and a hypoxia-inducible angiogenic growth factor associated with the development and growth of solid tumours. This study evaluated the expression of big endothelin-1 (big ET-1), a stable precursor of ET-1, and ET-1 in non-small cell lung cancer (NSCLC). Big ET-1 expression was evaluated in paraffin-embedded tissue sections from 10 NSCLC tumours using immunohistochemistry and in situ hybridisation. The production of big ET-1 and ET-1 was studied in six established NSCLC cell lines. The plasma concentrations of big ET-1 were measured in 30 patients with proven NSCLC prior to chemotherapy by means of a sandwich enzyme-linked immunoassay and compared to levels in 20 normal controls. Big ET-1 immunostaining was detected in the cancer cells of all tumours studied. Using in situ hybridisation, tumour cell big ET-1 mRNA expression was demonstrated in all samples. All six NSCLC cell lines expressed ET-1, with big ET-1 being detected in three. The median big ET-1 plasma level in patients with NSCLC was 5.4 pg/mL (range 0-22.7 pg/mL) and was significantly elevated compared to median big ET-1 plasma levels in controls, 2.1 pg/mL (1.2-13.4 pg/mL) (p=0.0001). Furthermore, patients with plasma big ET-1 levels above the normal range (upper tertile) had a worse outcome (p=0.01). In conclusion, big ET-1/ET-1 is expressed by resected NSCLC specimens and tumour cell lines. Plasma big ET-1 levels are elevated in NSCLC patients compared to controls with levels >7.8 pg/mL being associated with a worse outcome. The development of selective ET-1 antagonists such as Atrasentan indicates that ET-1 may be a therapeutic target in NSCLC. © 2004 Wichtig Editore.

Vascular remodeling in the internal mammary artery graft and association with in situ endothelin-1 and receptor expression

BACKGROUND: The vasoconstricting peptide endothelin-1 (ET-1) has been associated with atherosclerotic cardiovascular disease, vascular smooth muscle cell (VSMC) growth stimulation, and intimal thickening. ET-1 binds 2 receptor subtypes, endothelin A and B, and the ETA receptor mediates vasoconstriction and VSMC growth. This study aims to quantitatively assess arterial remodeling variables and compare them with changes in ET-1, ETA, and ETB expression in the internal mammary artery (IMA). METHODS AND RESULTS: Specimens from 55 coronary artery disease (CAD) patients (45 men, 10 women; mean age 65 years) and 14 control IMA specimens (from 7 men and 7 women; mean age 45 years) were collected. IMA cross sections were assessed by histochemical and immunohistochemical staining methods to quantify the levels of medionecrosis, fibrosis, VSMC growth, ET-1, ETA, ETB, and macrophage infiltration. The percentage area of medionecrosis in the patients was almost double that in the controls (31.85+/-14.52% versus 17.10+/-9.96%, P=0.0006). Total and type 1 collagen was significantly increased compared with controls (65.8+/-18.3% versus 33.7+/-13.7%, P=0.07, and 14.2+/-10.0% versus 4.8+/-2.8%, P=0.01, respectively). Despite ACE and/or statin therapy, ET-1 expression and cell cycling were significantly elevated in the patient IMAs relative to the controls (46.27+/-18.46 versus 8.56+/-8.42, P=0.0001, and 37.29+/-12.88 versus 11.06+/-8.18, P=0.0001, respectively). ETA and ETB staining was elevated in the patient vessels (46.88+/-11.52% versus 18.58+/-7.65%, P=0.0001, and 42.98+/-7.08% versus 34.73+/-5.20%, P=0.0067, respectively). A mild presence of macrophages was noted in all sections. CONCLUSIONS: Elevated distribution of collagen indicative of fibrosis coupled with increased cell cycling and high levels of ET-1 and ETA expression in the absence of chronic inflammation suggests altered IMA VSMC regulation is fundamental to the remodeling process.

A review of the profile of endothelin axis in cancer and its management

The endothelins and their associated receptors are important controllers of vascular growth, inflammation and vascular tone. In cancer, they have roles in the control of numerous factors in cancer development and progression, including angiogenesis, stromal reaction, epithelial mesenchymal transitions, apoptosis, invasion, metastases and drug resistance. Also, we consider current information on the role of this signalling system in cancer and examine the state of the current cell, animal and clinical trials utilizing endothelin targeted drugs for cancer management. Although targeting the endothelin axis in cell lines and xenografts show some promise in retarding cellular growth, results from limited clinical trials in prostatic cancer are less encouraging and did not offer significant survival benefit. The ability to target both cancer cells and vasculature via endothelin is an important consideration that necessitates the further refining of therapeutic strategies as we continue to explore the possibilities of the endothelin axis in cancer treatment.

Expression profile of endothelin 1 and its receptor endothelin receptor A in papillary thyroid carcinoma and their correlations with clinicopathologic characteristics

The endothelin axis is a group of signaling molecules and their receptors that have been implicated in vascularization of cancers, with their expression being observed to change in different cancer types. In this research, we examined the expression of endothelin 1 and endothelin receptor A at the protein and messenger RNA (mRNA) levels in 123 papillary thyroid carcinomas and 40 matched lymph nodes with metastatic papillary thyroid carcinomas. We found altered endothelin axis mRNA expression in several clinicopathologic parameters with increased endothelin 1 expression in thyroid papillary carcinoma showing stromal calcification, cancers in men, and primary cancers with lymph node metastases. Increased endothelin receptor A mRNA expression was noted in the larger cancers. There is a significant correlation between expression of endothelin receptor A and endothelin 1 in papillary thyroid carcinoma. Both endothelin receptor A and endothelin 1 mRNA expressions were significantly higher in metastatic carcinoma in the lymph node than in primary thyroid cancer. The metastatic carcinoma in the lymph node had increased expression compared with matched primary thyroid carcinoma. Expressions of endothelin 1 and endothelin receptor A were also documented as being high at the protein level. Our results indicate that in thyroid cancer, endothelin 1 and endothelin receptor A are associated with growth in advanced stages and lymph node metastases, likely through known angiogenic linkages. Targeting the endothelin axis may be useful in planning angiogenesis therapy for thyroid cancer.

Changes of tissue endothelin-1 and nitric oxide synthase in a sheep model of large intestinal obstruction

Large intestinal obstruction (LIO) in farm animals can cause a ischaemic necrosis of intestinal tissue, eventually leading to death. The roles of endothelin-1 (ET-1) and nitric oxide (NO) are not well understood in the process of LIO, but evidence suggests that endothelial-derived mediators may participate. In the present study, ET-1 concentration and total nitric oxide synthase (NOS) activity were measured in heart, liver, pancreas, lung and kidney in a model of LIO in sheep. Our data demonstrated that ET-1 concentration and NOS activity were altered, with significant increases of ET-1 in heart, lung and kidney and of NOS activity in pancreas and kidney, but a marked decline of NOS activity in liver (p<0.05). It is postulated that these alterations in NOS activity and ET-1 concentration may contribute to the progressive loss of organ function, and finally lead to death in LIO in sheep.

Changes in biomechanical properties of the coronary artery wall contribute to maintained contractile responses to endothelin-1 in atherosclerosis.

AIMS: Our aim was to determine whether alterations in biomechanical properties of human diseased compared to normal coronary artery contribute to changes in artery responsiveness to endothelin-1 in atherosclerosis. MAIN METHODS: Concentration-response curves were constructed to endothelin-1 in normal and diseased coronary artery. The passive mechanical properties of arteries were determined using tensile ring tests from which finite element models of passive mechanical properties of both groups were created. Finite element modelling of artery endothelin-1 responses was then performed. KEY FINDINGS: Maximum responses to endothelin-1 were significantly attenuated in diseased (27±3 mN, n=55) compared to normal (38±2 mN, n=68) artery, although this remained over 70% of control. There was no difference in potency (pD2 control=8.03±0.06; pD2 diseased=7.98±0.06). Finite element modelling of tensile ring tests resulted in hyperelastic shear modulus μ=2004±410 Pa and hardening exponent α=22.8±2.2 for normal wall and μ=2464±1075 Pa and α=38.3±6.7 for plaque tissue and distensibility of diseased vessels was decreased. Finite element modelling of active properties of both groups resulted in higher muscle contractile strain (represented by thermal reactivity) of the atherosclerotic artery model than the normal artery model. The models suggest that a change in muscle response to endothelin-1 occurs in atherosclerotic artery to increase its distensibility towards that seen in normal artery. SIGNIFICANCE: Our data suggest that an adaptation occurs in medial smooth muscle of atherosclerotic coronary artery to maintain distensibility of the vessel wall in the presence of endothelin-1. This may contribute to the vasospastic effect of locally increased endothelin-1 production that is reported in this condition.

Anti-hypotensive treatment and endothelin blockade synergistically antagonize exercise fatigue in rats under simulated high altitude.

Rapid ascent to high altitude causes illness and fatigue, and there is a demand for effective acute treatments to alleviate such effects. We hypothesized that increased oxygen delivery to the tissue using a combination of a hypertensive agent and an endothelin receptor A antagonist drugs would limit exercise-induced fatigue at simulated high altitude. Our data showed that the combination of 0.1 mg/kg ambrisentan with either 20 mg/kg ephedrine or 10 mg/kg methylphenidate significantly improved exercise duration in rats at simulated altitude of 4,267 m, whereas the individual compounds did not. In normoxic, anesthetized rats, ephedrine alone and in combination with ambrisentan increased heart rate, peripheral blood flow, carotid and pulmonary arterial pressures, breathing rate, and vastus lateralis muscle oxygenation, but under inspired hypoxia, only the combination treatment significantly enhanced muscle oxygenation. Our results suggest that sympathomimetic agents combined with endothelin-A receptor blockers offset altitude-induced fatigue in rats by synergistically increasing the delivery rate of oxygen to hypoxic muscle by concomitantly augmenting perfusion pressure and improving capillary conductance in the skeletal muscle. Our findings might therefore serve as a basis to develop an effective treatment to prevent high-altitude illness and fatigue in humans.

Use of A192621 to provide evidence for involvement of endothelin ET(B)-receptors in endothelin-1-mediated cardiomyocyte hypertrophy.

Increased plasma levels of endothelin-1 correlate with the severity of left ventricular hypertrophy in vivo. The aim of the study was to determine the relative contribution of stimulation of endothelin ETA and endothelin ETB receptors, and the associated activation of protein kinase C, to the hypertrophic response initiated by endothelin-1 in adult rat ventricular cardiomyocytes maintained in culture (24 h). Endothelin-1 (10-7 M) increased the total mass of protein and the incorporation of [14C] phenylalanine into protein to 26% and 25% greater (P

Alterations in vascular matrix metalloproteinase due to ageing and chronic hypertension: effects of endothelin receptor blockade.

OBJECTIVE: To determine the effects of age and dual endothelin (ET)A/ETB receptor antagonism (bosentan) on aortic matrix metalloproteinase (MMP) abundance and tissue inhibitor of metalloproteinase (TIMP) expression in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). METHODS: Male SHR and control WKY rats were randomly assigned to receive placebo or bosentan (100 mg/kg per day) for 3 months. Animals were killed under terminal anaesthesia at either 20 weeks (adult) or 17-20 months (senescent). Aortic gelatinase activity was determined by zymography, whereas MT-1 MMP and TIMP-1 expression were assessed by immunoblotting. RESULTS: In WKY rats, aortic MMP-2 but not proMMP-2 activity was 3.6-fold higher (P <0.02) in the senescent compared with the adult group. TIMP-1 (twofold) and MT-1 MMP (3.8-fold) expression increased (P <0.05) with age in the WKY groups. Short-term hypertension (adult SHR versus adult WKY) increased MMP-2 to 74.7 +/- 14.1 from 18.9 +/- 3.5 arbitrary units (AU) (P = 0.0012), but did not alter proMMP-2 activity. This increased further on progression to chronic hypertension (117.4 +/- 12.2 versus 74.7 +/- 14.1 AU; P <0.02). Bosentan decreased MMP-2 (78.9 +/- 3.8 versus 117.4 +/- 12.2 AU; P = 0.014) and proMMP-2 activity (P <0.006) in the senescent SHR group. CONCLUSION: Ageing and the development/progression of hypertension are associated with increased MMP-2 activity in the aorta, which is consistent with ongoing remodelling of the vasculature. However, the underlying mechanisms regulating MMP-2 abundance in ageing and hypertension appear to be divergent, as MT-1 MMP expression is differentially altered. Dual ETA/ETB receptor antagonism did not alter the age-dependent increase in aortic MMP activity in normotensive rats. However, bosentan decreased pro and active MMP-2 activity in senescent SHR rats, indicating that ET modulates late events in vascular remodelling in hypertension.

Myocardial expression of the endothelin system in endotoxin-treated rats.

Although circulating plasma levels of endothelin (ET)-1 are elevated in endotoxemia, little is known about the myocardial expression of the ET system in endotoxic shock. We assessed the temporal mRNA expression pattern of key components of the ET system (pre-pro ET (ppET) -1, -2, ET-converting enzyme-1, ETA and ETB receptors) by reverse transcription polymerase chain reaction in a rat model of early endotoxic shock. Lipopolysaccharide (5 mg/kg, i.p.) caused a transient increase (p 12-fold increase; p

Evidence for two endothelin Et(A) receptor subtypes in rabbit arteriolar smooth muscle.

1. Effects of endothelin-1 (Et-1) were studied on membrane currents in choroidal arteriolar smooth muscle by using perforated patch-clamp recordings. 2. Et-1 (10 nM) activated oscillatory Ca(2+)-activated Cl(-)-currents (I(Cl(Ca))) which could not be reversed by washing out. 3. Currents through L-type Ca(2+) channels were resolved in a divalent free medium (I(Ca(L)Na)). Et-1 reduced I(Ca(L)Na) by 75 +/- 7% within 30 s and this effect faded over 5 min, when the depression remained constant. On washing out Et-1, I(Ca(L)Na) almost completely recovered within 10 s. 4. BQ123 (1 microM), a peptide Et(A) receptor blocker, prevented the activation of I(Cl(Ca)), but failed to inhibit I(Cl(Ca)) transients once they had been initiated. In contrast, BQ123 not only prevented but also reversed the inhibition of I(Ca(L)Na) by Et-1. BQ788 (1 microM), an Et(B) receptor antagonist, did not prevent the activation of I(Cl(Ca)) or the inhibition of I(Ca(L)Na) by Et-1. 5. ABT-627 (10 nM), a non-peptide Et(A) receptor antagonist also blocked the activation of I(Cl(Ca)). However, on I(Ca(L)Na), ABT-627 (10 nM) mimicked the action of Et-1 an effect blocked by BQ123 suggesting that ABT-627 acted as an agonist. 6. The data are consistent with choroidal arteriolar smooth muscle cells having two types of Et(A) receptor, one where BQ123 is an antagonist and ABT-627 an agonist, where ligands dissociate freely and this receptor is coupled to inhibition of L-type Ca(2+) channels. In the other, BQ123 and ABT-627 are both antagonists and with Et-1 the receptor converts to a high affinity state producing the classical irreversible activation I(Cl(Ca)).

Endothelin 1 Stimulates Ca2+-Sparks and Oscillations in Retinal Arteriolar Myocytes via IP3R and RyR-Dependent Ca2+ Release

PURPOSE:
To investigate endothelin 1 (Et1)-dependent Ca(2+)-signaling at the cellular and subcellular levels in retinal arteriolar myocytes.
METHODS:
Et1 responses were imaged from Fluo-4-loaded smooth muscle in isolated segments of rat retinal arteriole using confocal laser microscopy.
RESULTS:
Basal [Ca(2+)](i), subcellular Ca(2+)-sparks, and cellular Ca(2+)-oscillations were all increased during exposure to Et1 (10 nM). Ca(2+)-spark frequency was also increased by 90% by 10 nM Et1. The increase in oscillation frequency was concentration dependent and was inhibited by the EtA receptor (Et(A)R) blocker BQ123 but not by the EtB receptor antagonist BQ788. Stimulation of Ca(2+)-oscillations by Et1 was inhibited by a phospholipase C blocker (U73122; 10 µM), two inhibitors of inositol 1,4,5-trisphosphate receptors (IP(3)Rs), xestospongin C (10 µM), 2-aminoethoxydiphenyl borate (100 µM), and tetracaine (100 µM), a blocker of ryanodine receptors (RyRs).
CONCLUSIONS:
Et1 stimulates Ca(2+)-sparks and oscillations through Et(A)Rs. The underlying mechanism involves the activation of phospholipase C and both IP(3)Rs and RyRs, suggesting crosstalk between these Ca(2+)-release channels. These findings suggest that phasic Ca(2+)-oscillations play an important role in the smooth muscle response to Et1 within the retinal microvasculature and support an excitatory, proconstrictor role for Ca(2+)-sparks in these vessels.