Mice genetically inactivated in interleukin-17A receptor are defective in long-term control of Mycobacterium tuberculosis infection.

Interleukin-17A (IL-17A), a pro-inflammatory cytokine acting on neutrophil recruitment, is known to play an important role during Mycobacterium tuberculosis infection, but the role of IL-17A receptor signalling in immune defence against this intracellular pathogen remains poorly documented. Here we have analysed this signalling using C57BL/6 mice genetically inactivated in the IL-17 receptor A subunit (IL-17RA(-/-) ). Although early after infection bacterial growth was controlled to the same extent as in wild-type mice, IL-17RA(-/-) mice were defective in exerting long-term control of M. tuberculosis infection, as demonstrated by a progressively increasing pulmonary bacterial burden and shortened survival time. Compared with infected wild-type mice, IL-17RA(-/-) mice showed impaired recruitment of neutrophils to the lungs at the early but not the late stage of infection. Pulmonary tumour necrosis factor-α, IL-6 and particularly IL-10 levels were decreased in the absence of IL-17RA signalling, whereas IL-1ß was increased. CD4(+) -mediated and γδ-mediated IL-17A production was dramatically increased in IL-17RA(-/-) mice (confirming part of their phenotype), whereas production of interferon-γ and expression of the bactericidal enzyme inducible nitric oxide synthase were not affected. Collectively, our data suggest that early but not late neutrophil recruitment is essential for IL-17A-mediated long-term control of M. tuberculosis infection and that a functional interferon-γ response is not sufficient to control M. tuberculosis growth when the IL-17RA pathway is deficient. As treatment of auto-immune diseases with anti-IL-17A antibodies is actually being tested in clinical studies, our data suggest that caution should be taken with respect to possible reactivation of tuberculosis.

Induction of the specific allergic immune response is independent of proteases from the fungus Alternaria alternata.

We have analyzed the importance of proteases for the induction of allergic responses against the mold Alternaria alternata. Responses induced in vivo with untreated or heat treated (protease inactivated) extracts were compared in BALB/c, C57BL/6, TLR4 KO, and MyD88 KO mice. In BALB/c mice, both extracts induced similar lung inflammation, upregulation of inflammatory mediators, Th2 cytokines, and Alternaria-specific antibodies. However heat inactivation abrogated polyclonal IgE production. Similar results were obtained in C57BL/6 albeit lung expression of some Th2 mediators was decreased in mice stimulated with the heat-treated extract. Treatment of the extract with protease inhibitors did not affect the induction of the allergic response either, except again for the polyclonal IgE response. Th2 responses and lung inflammation were readily induced in TLR4 knockout mice. In contrast, lung inflammation, Th2 responses, cytokine productions, and antibody synthesis were strongly suppressed in MyD88-deficient mice. Early lung IL-33 and IL-1-α expression were also suppressed. In conclusion, albeit some heat labile proteases are required for the stimulation of the polyclonal IgE secretion, fungal proteases, and TLR4 signaling are not required while MyD88 is essential for triggering the systemic immune response and for the development of lung allergic inflammation in response to Alternaria extracts.

Heart 6-phosphofructo-2-kinase activation by insulin requires PKB (protein kinase B), but not SGK3 (serum- and glucocorticoid-induced protein kinase 3).

On the basis of transfection experiments using a dominant-negative approach, our previous studies suggested that PKB (protein kinase B) was not involved in heart PFK-2 (6-phosphofructo2-kinase) activation by insulin. Therefore we first tested whether SGK3 (serum- and glucocorticoid-induced protein kinase 3) might be involved in this effect. Treatment of recombinant heart PFK-2 with [γ-32P]ATP and SGK3 in vitro led to PFK-2 activation and phosphorylation at Ser466 and Ser483. However, in HEK-293T cells [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] co-transfected with SGK3 siRNA (small interfering RNA) and heart PFK-2, insulin-induced heart PFK-2 activation was unaffected. The involvement of PKB in heart PFK-2 activation by insulin was re-evaluated using different models: (i) hearts from transgenic mice with a muscle/heart-specific mutation in the PDK1 (phosphoinositide-dependent protein kinase 1)-substrate-docking site injected with insulin; (ii) hearts from PKBß-deficient mice injected with insulin; (iii) freshly isolated rat cardiomyocytes and perfused hearts treated with the selective Akti-1/2 PKB inhibitor prior to insulin treatment; and (iv) HEK-293T cells co-transfected with heart PFK-2, and PKBα/ß siRNA or PKBα siRNA, incubated with insulin. Together, the results indicated that SGK3 is not required for insulin-induced PFK-2 activation and that this effect is likely mediated by PKBα.

Myocardial injury induced by ultrasound-targeted microbubble destruction: evidence for the contribution of myocardial ischemia.

Ultrasound-targeted microbubble destruction (UTMD) can cause left ventricular (LV) dysfunction and tissue alterations in rats when high ultrasound (US) energy and long duration of imaging are used. However, the mechanism underlying these alterations remains unclear. The aim of the present work was to investigate the possible role of ischemia in the pathogenesis of the UTMD-induced LV damages in rats. To address this issue, rat hearts were exposed in situ to perfluorocarbon-enhanced sonicated dextrose albumin (PESDA) and US at peak negative pressures of 0.6, 1.2 or 1.8 MPa for 1, 3, 9, 15 or 30 min. Blood pressure and electrocardiogram were continuously recorded during insonation. LV function was assessed before and immediately after US exposure, as well as at 24 h and 7 d. At each time point, groups of rats were euthanized and their hearts were harvested for morphologic analysis. Rats exposed to either PESDA alone or US alone showed no functional or morphologic abnormalities. By contrast, rats exposed to both PESDA and US exhibited transient LV dysfunction, transient ST-segment elevation, premature ventricular contractions, microvascular ruptures, contraction band necrosis and morphologic tissue damage. These bio-effects were spontaneously and completely reversible by one week, except in the groups exposed to the highest peak negative pressure for the longest duration, in which mild dysfunction persisted and interstitial fibrosis developed. In conclusion, simultaneous exposure of rat hearts to PESDA and US in vivo results in significant bio-effects that are similar to myocardial ischemia, including transient regional LV dysfunction, transient ST-segment elevation and myocyte contraction band necrosis.

Comparison between adenoviral and retroviral vectors for the transduction of the thymidine kinase PET reporter gene in rat mesenchymal stem cells.

UNLABELLED: Mesenchymal stem cells (MSCs) are a promising cell line for the treatment of ischemic heart disease. To evaluate the success of their transplantation into living animals, noninvasive imaging techniques that are able to track the distribution and fate of those cells would be useful. The aim of this study was to investigate the feasibility of infecting rat MSCs with adenoviruses and retroviruses carrying the herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene; to compare the level of transgene expression induced by the 2 viral vectors; to evaluate the effects of viral transduction on cell phenotype, viability, proliferation rates, and differentiation capabilities; and to test the possibility of noninvasively imaging transduced MSCs using 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine (18F-FHBG) and small-animal PET after their transplantation into living rats. METHODS: We infected rat bone marrow MSCs with adenoviruses carrying the HSV1 mutant tk (Ad-HSV1-sr39tk) PET reporter gene (PRG) or with a retroviral construct expressing the wild-type HSV1-tk PRG. The efficacy and intensity of HSV1-sr39tk and HSV1-tk gene expression were determined by a direct comparison of [8-3H]-penciclovir ([8-3H]-PCV) cell uptake in both infected MSC populations and noninfected control MSCs. Small-animal PET studies were performed on living rats after an intramuscular injection of infected MSCs. The MSCs either have been incubated in advance with 18F-FHBG or they were administered and 18F-FHBG was thereafter intravenously administered [corrected] RESULTS: Both adenoviral and retroviral vectors can be used to introduce the tk PRG in MSCs. Neither adenovirus nor retrovirus infections significantly modify MSC phenotype, viability, proliferation, and differentiation capabilities. No significant 3H-PCV uptake was observed in noninfected MSCs. By contrast, after both adenoviral and retroviral infections, the infected MSC populations exhibited a similar, significantly higher, 3H-PCV accumulation. Small-animal PET images showed intense activity within the transplanted regions irrespective of the infected MSC population used. CONCLUSION: Our results demonstrate the feasibility of infecting MSCs with adenoviruses and retroviruses expressing the HSV1-tk PRG and suggest that infected MSCs can be noninvasively imaged with 18F-FHBG and small-animal PET after their transplantation into living animals.

AMPKalpha2 counteracts the development of cardiac hypertrophy induced by isoproterenol.

As AMP-activated protein kinase (AMPK) controls protein translation, an anti-hypertrophic effect of AMPK has been suggested. However, there is no genetic evidence to confirm this hypothesis. We investigated the contribution of AMPKalpha2 in the control of cardiac hypertrophy by using AMPKalpha2-/- mice submitted to isoproterenol. The isoproterenol-induced cardiac hypertrophy, measured by left ventricular mass and histological examination, was significantly higher in AMPKalpha2-/- than in WT animals. Moreover, the intensification of cardiac hypertrophy found in AMPKalpha2-/- mice can be linked to the abnormal basal overstimulation of the p70 ribosomal S6 protein kinase, an enzyme known to regulate protein translation and cell growth. In conclusion, this work shows that AMPKalpha2 plays a role of brake for the development of cardiac hypertrophy.

Chronic intranasal administration of mould spores or extracts to unsensitized mice leads to lung allergic inflammation, hyper-reactivity and remodelling.

Allergic asthma is a serious multifaceted disease characterized by eosinophil-rich airway inflammation, airway hyperreactivity and airway wall modifications known as remodelling. We previously demonstrated that the spores of two allergenic moulds, Alternaria alternata and Cladosporium herbarum, were potent inducers of immunoglobulin E (IgE) production. Moreover, mice sensitized by two intraperitoneal injections before intranasal challenge with A. alternata or C. herbarum spores developed an allergic lung inflammation and hyperreactivity. Here we report on the effect of chronic intranasal administration of C. herbarum spores or A. alternata extracts to unsensitized BALB/c mice. Our results demonstrate that this chronic treatment led to an increase of total serum IgE and the appearance of specific IgE and IgG1. Total cell number in bronchoalveolar lavage fluid from treated mice was highly increased compared to phosphate-buffered-saline-treated mice because of the accumulation of macrophages, neutrophils, lymphocytes and eosinophils. Airway hyperreactivity appeared after 3 weeks (extract) and 7 weeks (spores) and was maintained during the whole treatment. Increased interleukin-13 mRNA expression in the lungs and T helper type 2 cytokines (interleukin-4, -5, -6 and -13) and transforming growth factor-beta secretion in bronchoalveolar lavage fluid were also observed. Lung hydroxyproline and fibronectin contents indicated increased fibrosis in mice treated with mould allergen. These observations were confirmed by histological analysis demonstrating airway wall remodelling and strong mucus production. These observations show that this model, using chronic intranasal administration of relevant particulate allergens, is an interesting tool for the study of mechanisms leading to allergic pulmonary diseases and lung remodelling.

Profibrotic effect of IL-9 overexpression in a model of airway remodeling.

IL-9 overexpression protects against alveolar fibrosis induced by crystalline silica particles. This cytokine is also involved in allergic asthma. In the present study, we examined the effect of IL-9 overexpression on the subepithelial fibrotic response, a feature of asthmatic remodeling, induced by chronic exposure to Alternaria alternata extract. IL-9-overexpressing mice (Tg5) and their wild-type counterparts (FVB) were intranasally exposed to A. alternata extract or PBS (controls) twice a week during 3 mo. At the end of the allergic challenge, enhanced pause (Penh) measured in response to methacholine and fibrotic parameters, such as collagen and fibronectin lung content, were significantly higher in Tg5 compared with FVB. Staining of lung sections with Masson's Trichrome also showed more collagen fibers in peribronchial areas of treated Tg5 mice. A similar recruitment of inflammatory cells was observed in challenged FVB and Tg5 mice, except for eosinophils, which were significantly more abundant in the lung of Tg5. High serum levels of IgE and IgG1 in both strains indicated that FVB and Tg5 developed a strong type 2 immune response. The concentration of the eosinophil chemoattractant RANTES and the profibrotic mediator connective tissue growth factor (CTGF) was higher in the BAL of challenged Tg5 than FVB. These results demonstrate a profibrotic role of IL-9 in an airway remodeling model, possibly involving eosinophils and CTGF. These data also highlight a dual role of IL-9 in lung fibrosis, being anti- or profibrotic depending on the alveolar or airway localization of the process, respectively.

Interrelated overexpression of endothelial and inducible nitric oxide synthases, endothelin-1 and angiogenic factors in human papillary thyroid carcinoma.

OBJECTIVE: Nitric oxide (NO) and endothelin-1 (ET-1) are involved in carcinogenesis. Overexpression of the ET-1 axis has been demonstrated in papillary thyroid carcinoma (PTC). This study investigated the expression of NO synthases (NOS) and their relationship with expression of ET-1 and angiogenic markers in PTC. DESIGN AND PATIENTS: Expression of NOS, angiogenic markers [vascular endothelial growth factor (VEGF), angiopoietin-1 and angiopoietin-2] and their receptors was studied in surgical thyroid samples obtained from 22 patients aged 15-68 years. Three groups were constituted: normal thyroid (n = 5), Hashimoto's thyroiditis (n = 9) and PTC (n = 8). RESULTS: Immunohistochemistry disclosed NOS2 and NOS3 immunoreactivity in PTC cells, the percentage of positive cells being greater than normal (P < 0.02). Real-time quantitative polymerase chain reaction (RTQ-PCR) showed that NOS2 and NOS3 mRNA levels were, respectively, increased (P < 0.02) by 2.6 +/- 0.6 and 4.2 +/- 1.1 times in PTC. RTQ-PCR demonstrated that VEGF, its receptors VEGFR-1 and VEGFR-2, and angiopoietin-2 and its receptor (Tie2) were also overexpressed (P < 0.05) in PTC. Correlations were found between ET-1 expression and that of NOS2, angiopoietin-1 and -2 (P < 0.05). NOS2 mRNA levels also correlated with those of NOS3 and angiopoietin-2 (P < 0.05). In thyroiditis, NOS2 immunoreactivity was observed in inflammatory cells whereas NOS2 mRNA levels were 12.1 +/- 1.6 times higher than normal (P < 0.005). CONCLUSIONS: This study revealed an activation of the NO pathway in thyroid carcinoma, which is interrelated to the ET-1 axis, both systems being overexpressed in concert with angiogenic factors. This global system might play a role in carcinogenesis and constitutes a potential target for anticancer therapy.

Myocardial delivery of colloid nanoparticles using ultrasound-targeted microbubble destruction.

AIMS: Ultrasound (US)-targeted microbubble destruction (UTMD) is a promising method for delivering genetic material to the heart. The aim of this study was: (i) to test whether colloid nanoparticles can be delivered to the rat myocardium using UTMD; and (ii) to determine whether tissue damage and contractile dysfunction occurs in hearts exposed to UTMD in vivo. METHODS AND RESULTS: Hearts from anaesthetized rats were exposed to perfluorocarbon-enhanced sonicated dextrose albumin (PESDA) (at two different microbubble concentrations) and US at peak pressures of 0.6, 1.2, or 1.8 MPa for 1, 3, or 9 min. During US, pairs of 30 and 100 nm fluorescent nanospheres were infused intravenously. Left ventricular function was assessed before and immediately after US, as well as at 24 h and 7 days. At the end of the experiments, the number of ruptured microvessels and the amount of nanospheres deposited were quantified. Rats exposed to PESDA alone or US alone showed no functional abnormalities, no capillary ruptures, and no nanosphere delivery. In contrast, rats exposed to both PESDA and US exhibited microvascular ruptures and nanosphere deposits. They also showed transient contractile dysfunction and premature ventricular contractions. All these changes were time-, US peak pressure-, and PESDA concentration-dependent. CONCLUSION: UTMD allows colloid nanoparticles to be delivered to the rat myocardium through microvessel rupture sites. The efficacy of delivery depends on the peak pressure applied, the duration of US exposure, and contrast concentration. UTMD also causes time- and peak pressure-dependent contractile dysfunction, and tissue alterations that are spontaneously reversible over time.

Antitumor effects of in vivo caveolin gene delivery are associated with the inhibition of the proangiogenic and vasodilatory effects of nitric oxide.

In tumors, caveolin-1, the structural protein of caveolae, constitutes a key switch through its function as a tumor suppressor and a promoter of metastases. In endothelial cells (EC), caveolin is also known to directly interact with the endothelial nitric oxide synthase (eNOS) and thereby to modulate nitric oxide (NO)-mediated processes including vasodilation and angiogenesis. In this study, we examined whether the modulation of the stoichiometry of the caveolin/eNOS complex in EC lining tumor blood vessels could affect the tumor vasculature and consecutively tumor growth. For this purpose, we used cationic lipids, which are delivery systems effective at targeting tumor vs. normal vascular networks. We first documented that in vitro caveolin transfection led to the inhibition of both VEGF-induced EC migration and tube formation on Matrigel. The DNA-lipocomplex was then administered through the tail vein of tumor-bearing mice. The direct interaction between recombinant caveolin and native eNOS was validated in coimmunoprecipitation experiments from tumor extracts. A dramatic tumor growth delay was observed in mice transfected with caveolin- vs. sham-transfected animals. Using laser Doppler imaging and microprobes, we found that in the early time after lipofection (e.g., when macroscopic effects on the integrity of the tumor vasculature were not detectable), caveolin expression impaired NO-dependent tumor blood flow. At later stages post-transfection, a decrease in tumor microvessel density in the central core of caveolin-transfected tumors was also documented. In conclusion, our study reveals that by exploiting the exquisite regulatory interaction between eNOS and caveolin and the propensity of cationic lipids to target EC lining tumor blood vessels, caveolin plasmid delivery appears to be a safe and efficient way to block neoangiogenesis and vascular function in solid tumors, independently of any direct effects on tumor cells.

Cardiomyocyte-restricted overexpression of endothelial nitric oxide synthase (NOS3) attenuates beta-adrenergic stimulation and reinforces vagal inhibition of cardiac contraction.

BACKGROUND: In the heart, nitric oxide synthases (NOS) modulate cardiac contraction in an isoform-specific manner, which is critically dependent on their cellular and subcellular localization. Defective NO production by NOS3 (endothelial NOS [eNOS]) in the failing heart may precipitate cardiac failure, which could be reversed by overexpression of NOS3 in the myocardium. METHODS AND RESULTS: We studied the influence of NOS3 in relation to its subcellular localization on the function of cardiomyocytes isolated from transgenic mice overexpressing NOS3 under the alpha-myosin heavy chain promoter (NOS3-TG). Immunoblot analysis demonstrated moderate (5-fold) NOS3 overexpression in cardiomyocytes from NOS3-TG heterozygotes. Caveolar localization of transgenic eNOS was demonstrated by immunofluorescence, coimmunoprecipitation with caveolin-3, sucrose gradient fractionation, and immunogold staining revealed by electron microscopy. Compared with wild-type littermate, contractility of NOS3-TG cardiomyocytes analyzed by videomicroscopy revealed a lower incidence of spontaneous arrhythmic contractions (n=32, P<0.001); an attenuation of the beta-adrenergic positive inotropic response (isoproterenol, 10(-7) mol/L: 62.1+/-7.8% versus 90.8+/-8.0% of maximum Ca2+ response; n=10 to 17; P<0.05); a potentiation of the muscarinic negative chronotropic response (carbamylcholine, 3.10(-8) mol/L: -63.9+/-14% versus -27.7+/-5.6% of basal rate; n=8 to 10; P<0.05), confirmed by telemetry in vivo; and an attenuation of the accentuated antagonism of beta-adrenergically stimulated contraction (-14.6+/-1.5% versus -3.5+/-1.5; n=7 to 11; P<0.05). Cardiomyocyte NOS inhibition reversed all 4 effects (P<0.05). CONCLUSIONS: Moderate overexpression of NOS3, targeted to caveolae in murine cardiomyocytes, potentiates the postsynaptic muscarinic response and attenuates the effect of high concentrations of catecholamines. Cardiomyocyte NOS3 may represent a promising therapeutic target to restore the sympathovagal balance and protect the heart against arrhythmia.

Physiological noise in murine solid tumours using T2*-weighted gradient-echo imaging: a marker of tumour acute hypoxia?

T2*-weighted gradient-echo magnetic resonance imaging (T2*-weighted GRE MRI) was used to investigate spontaneous fluctuations in tumour vasculature non-invasively. FSa fibrosarcomas, implanted intramuscularly (i.m.) in the legs of mice, were imaged at 4.7 T, over a 30 min or 1 h sampling period. On a voxel-by-voxel basis, time courses of signal intensity were analysed using a power spectrum density (PSD) analysis to isolate voxels for which signal changes did not originate from Gaussian white noise or linear drift. Under baseline conditions, the tumours exhibited spontaneous signal fluctuations showing spatial and temporal heterogeneity over the tumour. Statistically significant fluctuations occurred at frequencies ranging from 1 cycle/3 min to 1 cycle/h. The fluctuations were independent of the scanner instabilities. Two categories of signal fluctuations were reported: (i) true fluctuations (TFV), i.e., sequential signal increase and decrease, and (ii) profound drop in signal intensity with no apparent signal recovery (SDV). No temporal correlation between tumour and contralateral muscle fluctuations was observed. Furthermore, treatments aimed at decreasing perfusion-limited hypoxia, such as carbogen combined with nicotinamide and flunarizine, decreased the incidence of tumour T2*-weighted GRE fluctuations. We also tracked dynamic changes in T2* using multiple GRE imaging. Fluctuations of T2* were observed; however, fluctuation maps using PSD analysis could not be generated reliably. An echo-time dependency of the signal fluctuations was observed, which is typical to physiological noise. Finally, at the end of T2*-weighted GRE MRI acquisition, a dynamic contrast-enhanced MRI was performed to characterize the microenvironment in which tumour signal fluctuations occurred in terms of vessel functionality, vascularity and microvascular permeability. Our data showed that TFV were predominantly located in regions with functional vessels, whereas SDV occurred in regions with no contrast enhancement as the result of vessel functional impairment. Furthermore, transient fluctuations appeared to occur preferentially in neoangiogenic hyperpermeable vessels. The present study suggests that spontaneous T2*-weighted GRE fluctuations are very likely to be related to the spontaneous fluctuations in blood flow and oxygenation associated with the pathophysiology of acute hypoxia in tumours. The disadvantage of the T2*-weighted GRE MRI technique is the complexity of signal interpretation with regard to pO2 changes. Compared to established techniques such as intravital microscopy or histological assessments, the major advantage of the MRI technique lies in its capacity to provide simultaneously both temporal and detailed spatial information on spontaneous fluctuations throughout the tumour.

Endothelin-1 is a critical mediator of myogenic tone in tumor arterioles: implications for cancer treatment.

Although derived from the host tissue, the tumor vasculature is under the influence of the tumor microenvironment and needs to adapt to the resistance to blood flow inherent to the dynamics of tumor growth. Such vascular remodeling can offer selective targets to pharmacologically modulate tumor perfusion and thereby improve the efficacy of conventional anticancer treatments. Radiotherapy and chemotherapy can, indeed, take advantage of a better tumor oxygenation and drug delivery, respectively, both partly dependent on the tumor blood supply. Here, we showed that isolated tumor arterioles mounted in a pressure myograph have the ability, contrary to size-matched healthy arterioles, to contract in response to a transluminal pressure increase. This myogenic tone was exquisitely dependent on the endothelin-1 pathway because it was completely abolished by the selective endothelin receptor A (ETA) antagonist BQ123. This selectivity was additionally supported by the large increase in endothelin-1 abundance in tumors and the higher density of the ETA receptors in tumor vessels. We also documented by using laser Doppler microprobes and imaging that administration of the ETA antagonist led to a significant increase in tumor blood flow, whereas the perfusion in control healthy tissue was not altered. Finally, we provided evidence that acute administration of the ETA antagonist could significantly stimulate tumor oxygenation, as determined by electron paramagnetic resonance oximetry, and increase the efficacy of low-dose, clinically relevant fractionated radiotherapy. Thus, blocking the tumor-selective increase in the vascular endothelin-1/ETA pathway led us to unravel an important reserve of vasorelaxation that can be exploited to selectively increase tumor response to radiotherapy.

Early activation of cardiac and renal endothelin systems in experimental heart failure.

We investigated the time course of the expression of cardiac and renal endothelin systems in tachycardia-induced heart failure in dogs. Eleven beagles underwent rapid pacing at a progressively increased rate over a period of 5 wk, with a weekly clinical examination, echocardiography, measurement of circulating and urinary endothelin-1 (ET-1), and myocardial and renal tissue biopsies. Real-time quantitative PCR was used for determinations of tissue prepro-ET-1 (ppET-1), ET-1-converting enzyme (ECE-1), and ETA and ETB receptor mRNA. Cardiac and renal tissue ET-1 contents were evaluated by immunostaining and measured by radioimmunoassay at autopsy. Rapid pacing caused a progressive increase in end-systolic and end-diastolic ventricular volumes (P < 0.05) from week 2 together with a decrease in ejection fraction and in mean velocity of circumferential shortening (P < 0.05) from week 1. These changes were tightly correlated to myocardial ppET-1 and renal ETA receptor mRNA and less so to myocardial ECE-1 mRNA, and they occurred before any increase in plasma and urinary ET-1 (P < 0.05 from week 4) and clinical signs of heart failure. Renal ppET-1 did not change. Both cardiac and renal ET-1 peptide contents were increased at autopsy. We conclude that tachycardia-induced heart failure in dogs is characterized by an early activation of the cardiac and renal tissue endothelin systems, which occurs before any changes in circulating and urinary ET-1 and is closely related to altered ventricular function.

Increased expression of endothelin-1 and its mitogenic receptor ETA in human papillary thyroid carcinoma.

OBJECTIVE: Since the isolation of endothelin-1 (ET-1) in 1988, there has been tremendous interest in the pathophysiological roles of ET-1 as a vasoconstrictive and mitogenic peptide. Whereas ET-1 is mainly released by vascular endothelial cells, it also proved to be produced by various tissues including the thyroid. Because of its mitogenic properties in malignancy and its role as an inflammatory modulator, ET-1 could be involved in thyroid carcinogenesis and thyroiditis. DESIGN AND PATIENTS: Studies were performed in human thyroid samples obtained at the time of surgery from 39 men and women aged 15-72 years. Thyroid samples were classified in four groups according to conventional histology: normal thyroid (n = 7) papillary thyroid carcinoma (n = 12), Hashimoto's thyroiditis (n = 9) and benign nontoxic nodular goitres (n = 11). Immunohistochemistry and real-time quantitative polymerase chain reaction were used to determine the expression of ET-1 and its receptors (ETAR and ETBR). RESULTS: ET-1 and ETAR mRNA levels were, respectively, 3.8 +/- 1.3 and 4.1 +/- 1.5 times greater (P < 0.001) in papillary thyroid carcinoma than in normal thyroid. Expression of ETBR was unaltered. In Hashimoto's thyroiditis, ET-1 and ETAR were also overexpressed (P < 0.005). Furthermore, immunohistochemistry demonstrated a greater percentage of ET-1-positive follicular cells in these conditions (P < 0.001). In nodular goitres, the expression was increased by 1.7 +/- 0.7 times (P < 0.05) but expression of receptors remained unchanged. CONCLUSIONS: ET-1 and ETAR overexpression observed in thyroid carcinoma suggest a mitogenic role of ET-1 that theoretically could be countered by ETAR antagonists. ET-1 and ETAR overexpression in thyroiditis supports a role of ET-1 in the inflammatory process.

Irradiation-induced angiogenesis through the up-regulation of the nitric oxide pathway: implications for tumor radiotherapy.

The combination of radiotherapy and antiangiogenic strategies has been shown to increase the tumor response in various experimental models. The rationale for this cotherapy was initially related to the expected gain in efficacy by acting on two different targets, e.g., tumor cells and endothelial cells (ECs). However, recent studies have documented more than additive effects due to apparent mutual potentiation of these approaches. In this study, we tested the hypothesis that these synergistic effects could stem from the stimulatory effects of ionizing radiations on angiogenesis, which would then need to be restrained to avoid tumor regrowth after irradiation. We found that irradiation dose-dependently induced the activation of the proangiogenic NO pathway in ECs through increases in endothelial nitric oxide synthase abundance and phosphorylation. Using 2- and 3-dimensional cultures of ECs and isolated mouse tumor arterioles, we documented that the irradiation-induced enhanced production of NO accounted for EC migration and sprouting. Irradiation was also shown to stimulate the colonization of Matrigel plugs implanted in mouse by ECs, where they formed capillary-like structures in a NO-dependent manner. These findings were confirmed by documenting the NO-mediated infiltration of CD31-positive ECs after local irradiation of Lewis lung carcinoma tumor-bearing mice. Finally, we measured a consistent increase in endothelial nitric oxide synthase mRNA by real-time PCR experiments in human biopsies of head and neck squamous cell carcinoma after low-dose irradiation. In conclusion, we have demonstrated that the potentiation of the NO signaling pathway after irradiation induces profound alterations in the EC phenotype leading to tumor angiogenesis. Moreover, our demonstration that the inhibition of NO production suppresses these provascular effects of irradiation highlights new potentials for the coordinated use of antiangiogenic strategies and radiotherapy in clinical practice.

Differential regulation of nitric oxide synthases and their allosteric regulators in heart and vessels of hypertensive rats.

OBJECTIVE: Nitric oxide synthase (NOS)-derived nitric oxide (NO) production is regulated posttranslationally through enzyme's inhibitory interaction with the caveolar coat protein, caveolin and stimulatory interaction with the chaperone heat shock protein, Hsp90. However, changes in the expression of these regulators with the development of hypertrophic cardiomyopathy are unknown. METHODS: Histochemical and immunoblotted signals for the NOS isoforms, caveolin and Hsp90 were compared in left ventricle (LV) and aortic or mesenteric vessels between spontaneously hypertensive rats (SHR; 18 and 63 weeks old) and age-matched normotensive Wistar-Kyoto (WKY) rats. To assess functional impacts on downstream NO signaling, superoxide anions (O(2)(-)) and cGMP contents were measured in the same tissues by oxidative fluorescent hydroethidine staining and enzyme immunoassay, respectively. RESULTS: Compared with levels in age-matched WKY rats, endothelial NOS (eNOS) proteins were increased in aorta of SHR at 18 weeks. Conversely, aortic caveolin-1 and -3 were decreased in SHR, whereas Hsp90 remained unchanged. In LV tissue of SHR at 18 weeks, caveolin-1 and -3 were similarly decreased, but Hsp90 upregulated, together with a downregulation of eNOS. However, at 63 weeks, both eNOS and neuronal NOS (nNOS) were markedly upregulated in the LV of SHR, together with an upregulation of Hsp90. No difference in cardiac and aortic cGMP contents was found between the two strains. In LV sections, O(2)(-) generation was higher in older compared with younger rats from both strains and highest in 63 weeks SHR. CONCLUSIONS: Changes in NOS protein abundance in SHR rats compared with WKY controls are differentially regulated according to the age of hypertension and the tissue examined and are not necessarily correlated with cGMP contents. The coordinate expressional changes in NOS isoforms and their allosteric regulators, such as caveolin and Hsp90, may act as a compensatory mechanism to maintain the production of bioactive NO in the face of increased oxidant stress.