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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.

PURPOSE: Oxygen deficiency in tumors reduces the efficacy of nonsurgical treatment modalities. We tested the hypothesis that electrical stimulation of the sciatic nerve could modify the oxygenation status and the blood flow of tumors implanted in the thigh of mice. MATERIALS AND METHODS: The sciatic nerve was electrically stimulated at 5 Hz. Local transplantable liver tumor (TLT) and fibrosarcoma (FSaII) tumor oxygen pressure (pO(2)) and perfusion measurements were carried out using electron paramagnetic resonance (EPR) oximetry and the OxyLite/OxyFlo technique. The radiosensitizing effect of the protocol was assessed by irradiating FSaII tumors with X-rays. RESULTS: Tumor pO(2) increased from approximately 3 mm Hg to approximately 8 mm Hg, and relative tumor blood flow was increased by 241% and 162% for TLT and FSaII tumor models, respectively. The effect on the tumor oxygenation was inhibited by a nitric oxide synthase (NOS) inhibitor, and an increase in the tumor nitric oxide (NO) content was observed using EPR spin-trapping. The tumor oxygen consumption rate was decreased after the stimulation protocol. In addition, the electrical stimulation of the host tissue increased regrowth delays by a factor of 1.65. CONCLUSIONS: This increase in tumor oxygenation is due to the temporary increase in tumor blood flow, but particularly to a decrease in the tumor oxygen consumption rate (inhibition of respiration) that is mediated by a local production of NO during the protocol. Those tumor hemodynamic changes resulted in a radiosensitizing effect.

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.

In most cell types a family of transcriptional regulatory proteins termed hypoxia-inducible factors, of which HIF-1 is the most prevalent, mediates the physiologic response to hypoxia. Although much has been learned over the past decade since the discovery of the first HIF family member, the proximal signaling events linking a decline in oxygen concentration to the activation of HIF-dependent signaling are only now being clarified. Activation of HIF-1 in eukaryotes induces expression of many genes that assist in adapting the organism to an environment in which oxygen is limiting, such as of genes involved in new blood vessel formation, including isoforms of vascular endothelial growth factor and angiopoietins, among others. Targeted expression of constitutively active HIF transgenes to ischemic tissues may be beneficial as a form of therapeutic angiogenesis.

Endothelial nitric oxide synthase (eNOS) phosphorylation increases nitric oxide formation, for example, after VEGF stimulation. We investigated whether nitric oxide formed after overexpression of VEGF or of phosphomimetic eNOS (S1177D) affects PMN-induced myocardial detriment after ischemia and reperfusion. Pigs (n=8 per group) were subjected to percutaneous liposome-based gene transfer by retroinfusion of the anterior interventricular vein 48 h before LAD occlusion (60 min) and reperfusion (24 h). Thereafter, regional myocardial function was assessed as subendocardial segment shortening (SES), and infarct size was determined. Tissue from the infarct region, the noninfarcted area at risk, and a control region was analyzed for NF-kappaB activation (EMSA), tumor necrosis factor (TNF)-alpha, and E-selectin mRNA and infiltration of polymorphonuclear neutrophils (PMN). L-NAME was applied in one group of VEGF-transfected animals. NF-kappaB activition, PMN infiltration in the infarct region, and AAR were reduced after transfection of VEGF or eNOS S1177D, but not after VEGF+L-NAME coapplication. Infarct size decreased, whereas SES improved after either VEGF or eNOS S1177D transfection, an effect inhibited by L-NAME coapplication. Retroinfusion of liposomal VEGF cDNA reduces NF-kappaB-dependent postischemic inflammation and subsequent myocardial reperfusion injury, an effect mediated at least in part by enhanced eNOS phosphorylation.

In a recent publication ([1][1]), we have documented the capacity of lactate to enter endothelial cells via the monocarboxylate transporter MCT1 and to signal by stimulating an autocrine proangiogenic NF-κB/interleukin-8 (IL-8) pathway. In this mechanistic study, the reason why we used human

Experimental diabetes induces functional and structural changes in the peritoneum.BackgroundPeritoneal dialysis (PD) is an established renal replacement therapy in diabetic patients, but the influence of diabetes on the peritoneal membrane (PM) remains debated. We have used functional, biochemical and molecular studies in vivo and in vitro to substantiate the changes induced by diabetes and hyperglycemia in the PM.MethodsPeritoneal equilibration tests were performed 2, 4, and 6 weeks after induction of diabetes with streptozotocin (STZ) in rats. Morphological analyses, determination of nitric oxide synthase (NOS) activities, and expression studies for NOS isoforms and advanced glycation end products (AGE) were performed in parallel. Additional studies were conducted in diabetic rats treated with insulin, non-diabetic rats fed with urea, and cultured bovine aortic endothelial cells (BAEC).ResultsIn comparison with controls, diabetic rats were characterized by: increased permeability for small solutes and decreased sodium sieving; capillary proliferation; increased endothelial NOS (eNOS) and AGE immunoreactivity; up-regulation of eNOS and down-regulation of neuronal NOS; and increased NOS activity in the PM. The changes, which culminated at week 6, were prevented by chronic insulin treatment in diabetic rats. In contrast to hyperglycemia, hyperosmolality alone did not induce functional or structural changes in the PM. Studies in BAEC showed that high glucose incubation led to increased activity and expression of eNOS, a prerequisite for vascular proliferation.ConclusionsThese data demonstrate that chronic hyperglycemia is associated with functional and structural changes in the peritoneum that parallel with selective regulation of NOS isoforms and AGE deposits. The alterations are prevented by insulin treatment, which suggests that adequate control of diabetes can preserve PM integrity in diabetic patients prior to PD.

The effects of insulin on tumor oxygenation, perfusion, oxygen consumption,and radiation sensitivity were studied on two different mouse tumor models (TLT, a liver tumor, and FSAII, a fibrosarcoma). Anesthetized mice were infused with insulin i.v. at a rate of 16 milliUnits/kg/min for 25 min. Local tumor oxygenation measurements were carried out using two independent techniques: electron paramagnetic resonance oximetry and a fiber-optic device (OxyLite). Two complementary techniques were also used to assess the blood flow inside the tumor: a laser Doppler system (OxyFlo) and contrast-enhanced magnetic resonance imaging. The oxygen consumption rate of tumor cells after in vivo insulin infusion was measured using high frequency electron paramagnetic resonance oximetry. To know if insulin was able to enhance radiation-induced tumor regrowth delay, tumor-bearing mice were treated with 16 Gy of 250 kV radiation dose after insulin infusion. We provide evidence that insulin increases the local pressure of oxygen of tumors (from 0-3 mm Hg to 8-11 mm Hg) as well as the tumor response to irradiation (increasing regrowth delay by a factor of 2.11). We found that the insulin-induced increase of tumor pressure of oxygen: (a) is not caused by an increase in the tumor blood flow, which is even decreased after insulin infusion; (b) is because of a decrease in the tumor cell oxygen consumption (in vivo insulin consumed oxygen three times slower than control cells); and (c) is inhibited by a nitric oxide (NO) synthase inhibitor, Nomega-nitro-L-arginine methyl ester, when injected i.p. at 15 micromol/kg(-1), 1 h before insulin infusion. We demonstrate by immunoblotting that the NO pathway involves a phosphorylation of endothelial NO synthase and showed a concomitant increase in the cyclic GMP tumor level. These findings provide unique insights into biological processes in tumors, new possible management for treating cancer patients, and raise major questions about the role of insulin secretion (fasting status and diabetes) in the clinical response of tumors to radiation therapy.

Voltage-gated sodium channels play a key role in the excitability of myocardial cells and in impulse propagation.1,2⇓ An increase in Na+ current is, for example, responsible for the generation of the rapid upstroke of the action potential (phase 0). Mutations in the gene encoding this channel ( SCN5A ) have been linked to the pathogenesis of several cardiac channelopathies, including the long-QT and the Brugada syndromes,1,2⇓ the discoveries of which have helped in the functional characterization of these channels. In addition, the signaling pathways that regulate Na+ channel activity, such as the sympathetic nervous system, have been the subject of numerous studies.3–6⇓⇓⇓ Indeed, stimulation of β-adrenergic receptors can both enhance conduction in normal ventricular myocardium but also induce arrhythmic events in a number of cardiac disease states.1,2⇓ Several mechanisms that contribute to the regulation of Na+ channels by β-adrenergic stimulation have been documented (see Figure). All of them involve G proteins, although diffusible second messengers are not necessarily involved. For example, besides the coupling of Gαs to adenylate cyclase and downstream protein kinase A (PKA)-mediated phosphorylation events,3,4⇓ the so-called “membrane-delimited” pathway does not involve diffusible cytosolic factors. The Gαs subunit can, indeed, directly modify Na+ channel activity.5,6⇓ This type of regulation is not unique to Gαs, as the direct binding of the Gβγ protein subunit to several Ca2+, K+, and Cl− channels has been extensively documented (for review, see Dascal7). Schematic representation f the 2 major pathways regulating voltage-dependent Na+ channels (Na+Ch) in cardiac …

Background: In cardiovascular imaging, variations within and between observers, and between different acquisitions remain an important issue when interpreting in vivo data. Though there has recently been an increasing interest in ultra-high field MRI to characterize rodent models of cardiovascular diseases, reports concerning its reproducibility are scarce. The purpose of our work was to evaluate the intraobserver, interobserver and interexperiment variability of left ventricular (LV) mass and volumes measurements in mice using an 11.7T MRI scanner and to assess the validity of our results by comparing them with ex vivo measurements. Methods: 29 C57Bl6/J male mice aged 10-18 weeks were studied, including animals having previously undergone minimally invasive transverse aortic constriction. Imaging was performed on an 11.7 T MRI system (Biospec, Bruker, Germany). The LV systolic function was assessed from a stack of short-axis views obtained with a FLASH-cine sequence covering the entire ventricles. Using a dedicated software (SegmentTM, Medviso, Sweden), the following volumes (μl) were determined : end-diastolic, end-systolic and stroke volumes. LV ejection fraction (in %) and LV mass (mg) were subsequently deduced. Interobserver (n=24), intraobserver (n=24) and interexperiment (n=12) reproducibility were studied with the Bland and Altman method. LV masses obtained with the MRI were compared with ex vivo morphometric data. Results: Reproducibility was excellent for the LV mass and end-diastolic volume (EDV) (coefficient of variability - CoV - between 5,38 and 11,77%), good for end-systolic volume (ESV) (CoV between 15,24 and 16.81%) and moderate for data related to the smallest volumes, i.e. the stroke volume (SV) and ejection fraction (EF) (CoV between 14,73 and 20,93%). Importantly, we demonstrated an excellent correlation between LV mass measurements by weighing or imaging, with a Pearson correlation (r) of 0.92. Conclusions: In our study, we show that the mouse LV systolic function can be longitudinally assessed on an 11.7T MRI scanner, with excellent reproducibility of LV mass and EDV and a good correlation of LV mass results with ex vivo morphometric measurements. However, we highlight the need to take into account some shortcomings when interpreting MRI data, like moderate reproducibility of very small volumes, focusing on parameters that are the most reproducible or using alternative sequences with better resolution.

Background Mesenchymal stem cells (MSCs) are widely used for cell therapy, particularly for the treatment of ischemic heart disease. Mechanisms underlying control of their proliferation capacity, which is known to be inversely correlated to their differentiation abilities, have not been fully characterized. The AMP-activated protein kinase (AMPK) controls several cell functions including cell proliferation. Objective We hypothesized that AMPK plays a crucial role in the control of MSC proliferation. Methods MSCs isolated from WT or AMPKα1 KO adult mouse bone marrow were stimulated with the specific AMPK activator, A769662. Protein phosphorylation state was determined by immunoblotting, cell viability was quantified by FACS after AnnexinV and Propidium Iodide staining, cell proliferation was quantified by EdU incorporation and gene expression was analyzed by real-time PCR. Results MSCs isolated from murine bone marrow exclusively expressed the AMPKa1 catalytic subunit. A769662 was able to induce a robust and sustained AMPK activation in WT MSCs but not in AMPKa1 KO MSCs. This A769662-dependent AMPK activation blocks WT MSC proliferation but not in KO MSCs without affecting their viability. Moreover, AMPKa1 deletion induces an increase of basal MSC proliferation. Among AMPK downstream targets proposed to regulate cell proliferation, we showed that neither the p70 ribosomal S6 protein kinase/eukaryotic elongation factor 2-dependent protein synthesis pathway nor p21 was involved, whereas p27 expression was increased by A-769662. Silencing p27 expression prevented the A-769662- dependent inhibition of MSC proliferation. Conclusions These results suggest that activating AMPK promotes a principal prerequisite for cell differentiation, the arrest of MSC proliferation via p27 regulation.

Background —Contrary to β 1 - and β 2 -adrenoceptors, β 3 -adrenoceptors mediate a negative inotropic effect in human ventricular muscle. To assess their functional role in heart failure, our purpose was to compare the expression and contractile effect of β 3 -adrenoceptors in nonfailing and failing human hearts. Methods and Results —We analyzed left ventricular samples from 29 failing (16 ischemic and 13 dilated cardiomyopathic) hearts (ejection fraction 18.6±2%) and 25 nonfailing (including 12 innervated) explanted hearts (ejection fraction 64.2±3%). β 3 -Adrenoceptor proteins were identified by immunohistochemistry in ventricular cardiomyocytes from nonfailing and failing hearts. Contrary to β 1 -adrenoceptor mRNA, Western blot analysis of β 3 -adrenoceptor proteins showed a 2- to 3-fold increase in failing compared with nonfailing hearts. A similar increase was observed for Gα i-2 proteins that couple β 3 -adrenoceptors to their negative inotropic effect. Contractile tension was measured in electrically stimulated myocardial samples ex vivo. In failing hearts, the positive inotropic effect of the nonspecific amine isoprenaline was reduced by 75% compared with that observed in nonfailing hearts. By contrast, the negative inotropic effect of β 3 -preferential agonists was only mildly reduced. Conclusions —Opposite changes occur in β 1 - and β 3 -adrenoceptor abundance in the failing left ventricle, with an imbalance between their inotropic influences that may underlie the functional degradation of the human failing heart.

Receptor-coupled contraction of smooth muscle involves recruitment to the plasma membrane of downstream effector molecules PKC alpha and rhoA but the mechanism of this signal integration is unclear. Caveolins, the principal structural proteins of caveolar plasma membrane invaginations, have been implicated in the organization and regulation of many signal transducing molecules. Thus, using laser scanning confocal immunofluorescent microscopy, we tested the hypothesis that caveolin is involved in smooth muscle signaling by investigating caveolin isoform expression and localization, together with the effect of a peptide inhibitor of caveolin function, in intact differentiated smooth muscle cells. All three main caveolin isoforms were identified in uterine, stomach, and ileal smooth muscles and assumed a predominantly plasma membranous localization in myometrial cells. Cytoplasmic introduction of a peptide corresponding to the caveolin-1 scaffolding domain-an essential region for caveolin interaction with signaling molecules-significantly inhibited agonist-induced translocation of both PKC alpha and rhoA. Translocation was unimpaired by a scrambled peptide and was unaltered in sham-treated cells. The membranous localization of caveolins, and direct inhibition of receptor-coupled PKC alpha and rhoA translocation by the caveolin-1 scaffolding domain, supports the concept that caveolins can regulate the integration of extracellular contractile stimuli and downstream intracellular effecters in smooth muscle. (C) 2000 Academic Press.

Publisher Summary Over the past two decades, the role of nitric oxide (NO) and related congeners in cardiovascular pharmacology have largely been elucidated. This chapter focuses on the extravascular production of NO in the control of cardiac contractile function. Cardiac muscle cells express the NO synthase isoform originally described in endothelial cells (eNOS), which is coupled to both β- adrenergic and cholinergic receptors in cardiac myocytes, including specialized pacemaker and conduction cells within the heart. The molecular pharmacology of NO generation in control of myocardial contractile function by the autonomic nervous system is described in detail, as is the targeting of eNOS to caveolar microdomains and its interactions with caveolin-3. Evidence pointing to the presence of another NOS isoform in the sarcoplasmic reticulum of cardiac myocytes is briefly reviewed. Response to systemic sepsis or localized infection is described.

Endothelial cells constitutively express the NOS isoform eNOS, which generates NO in response to specific extracellular signals to regulate vascular smooth muscle tone, vascular permeability, and platelet adhesion, among other actions. In addition to coronary vascular and endocardial endothelium, both atrial and ventricular myocytes express eNOS, the activation of which is also dependent on specific intracellular and extracellular signals. eNOS is targeted in cardiac myocytes to caveolae in plasma membranes and, in the case of cardiac myocytes, possibly T-tubular membranes as well. eNOS targeting to caveolae in cardiac myocytes requires co-translational myristoylation and subsequent palmitoylation for efficient targeting of the enzyme to the specialized lipid microdomains characteristic of caveolae. Although eNOS also contains a caveolin binding motif, this is insufficient for correct targeting of eNOS to caveolae. Recent evidence obtained from ventricular myocytes of mice with targeted disruption of the eNOS gene indicates that the lack of functional eNOS interrupts muscarinic cholinergic control of ICa-L in these cells. eNOS-/- mice are hypertensive and develop cardiac hypertrophy as they age, and these animals also exhibit an accelerated degree of vascular remodeling in response to injury. Reconstitution experiments confirm both the essential role of eNOS in coupling m2 AchR signaling to the control of ICa-L and myocyte automaticity and the importance of eNOS subcellular localization within caveolae in mediating this signal transduction pathway. It appears that translocation into caveolae is essential for signaling. However, this is not the case with all receptors associated with caveolae.

The isoform of nitric oxide synthase (eNOS or NOS3) originally described in endothelial cells is also expressed in a number of other cell types, including cardiac myocytes. eNOS is activated in both atrial and ventricular myocytes, including specialized pacemaker cells, by M2AChR agonists, among other stimuli. In cardiac myocytes, as in endothelial cells, eNOS is targeted to sarcolemmal caveolae, due to both co-translational myristoylation and later palmitoylation, and by the presence of a caveolin binding domain in eNOS which interacts with the caveolin scaffolding domain. In the absence of ligand, the M2AChR is not associated with caveolar microdomains, but translates into caveolae upon agonist (but not antagonist) binding. Finally, the role of M2AChR-induced eNOS activation in regulating I(Ca-L) via activation of guanylyl cyclase has been confirmed in ventricular myocytes of mice that lack functional eNOS (i.e., eNOS(null)).

Hypercholesterolemia is a central pathogenic factor of endothelial dysfunction caused in part by an impairment of endothelial nitric oxide (NO) production through mechanisms that remain poorly characterized. The activity of the endothelial isoform of NO synthase (eNOS) was recently shown to be modulated by its reciprocal interactions with the stimulatory Ca2+–calmodulin complex and the inhibitory protein caveolin. We examined whether hypercholesterolemia may reduce NO production through alteration of this regulatory equilibrium. Bovine aortic endothelial cells were cultured in the presence of serum obtained from normocholesterolemic (NC) or hypercholesterolemic (HC) human volunteers. Exposure of endothelial cells to the HC serum upregulated caveolin abundance without any measurable effect on eNOS protein levels. This effect of HC serum was associated with an impairment of basal NO release paralleled by an increase in inhibitory caveolin–eNOS complex formation. Similar treatment with HC serum significantly attenuated the NO production stimulated by the calcium ionophore A23187. Accordingly, higher calmodulin levels were required to disrupt the enhanced caveolin–eNOS heterocomplex from HC serum–treated cells. Finally, cell exposure to the low-density lipoprotein (LDL) fraction alone dose-dependently reproduced the inhibition of basal and stimulated NO release, as well as the upregulation of caveolin expression and its heterocomplex formation with eNOS, which were unaffected by cotreatment with antioxidants. Together, our data establish a new mechanism for the cholesterol-induced impairment of NO production through the modulation of caveolin abundance in endothelial cells, a mechanism that may participate in the pathogenesis of endothelial dysfunction and the proatherogenic effects of hypercholesterolemia.

Cardiac myocytes have been shown to express constitutively endothelial nitric oxide synthase (eNOS) (nitric oxide synthase 3), the activation of which has been implicated in the regulation of myocyte L-type voltage-sensitive calcium channel current (I Ca-L ) and myocyte contractile responsiveness to parasympathetic nervous system signaling, although this implication remains controversial. Therefore, we examined the effect of the muscarinic cholinergic agonist carbachol (CCh) on I Ca-L and contractile amplitude in isoproterenol (ISO)-prestimulated ventricular myocytes isolated from adult mice, designated eNOS null mice, with targeted disruption of the eNOS gene. Although both eNOS null and wild-type (WT) ventricular myocytes exhibited similar increases in I Ca-L in response to ISO, there was no measurable suppression of I Ca-L by CCh in cells from eNOS null mice, in contrast to cells from WT mice. These results were reflected in the absence of an effect of CCh on the positive inotropic effect of ISO in eNOS null myocytes. Also, unlike myocytes from WT animals, eNOS null myocytes failed to exhibit an increase in cGMP content in response to CCh. Nevertheless, the pharmacologic nitric oxide donors 3-morpholino-sydnonimine and S-nitroso-acetyl-cystein increased cGMP generation and suppressed ISO-augmented I Ca-L in eNOS null cells, suggesting that the signal transduction pathway(s) downstream of eNOS remained intact. Of importance, activation of the acetylcholine-activated K + channel by CCh was unaffected in atrial and ventricular eNOS null myocytes. These results confirm the obligatory role of eNOS in coupling muscarinic receptor activation to cGMP-dependent control of I Ca-L in cardiac myocytes.

Cardiac myocytes express the nitric-oxide synthase isoform originally identified in endothelial cells, termed eNOS or NOS3, where it plays a role in regulating myocyte responsiveness to both adrenergic and muscarinic cholinergic autonomic nervous system agonists. eNOS in endothelial cells has been shown to undergo extensive post-translational processing, and in cardiac myocytes as well as endothelial cells, eNOS has been shown to be targeted to plasmalemmal caveolae, a process that is dependent on myristoylation and palmitoylation. Other post-translational modifications essential for the correct subcellular targeting of eNOS have not been described previously. We demonstrate, using [35S]methionine pulse-chase experiments, that native eNOS in adult rat ventricular myocytes is initially translated as a nonpalmitoylated 150-kDa isoform, which is associated with cytosolic and intracellular membrane-enriched fractions. This is subsequently processed to a palmitoylated 135-kDa isoform, which is found only in a sarcolemma-enriched membrane fraction. Forskolin, an agent that elevates intracellular cAMP, rapidly inhibited processing of the 150-kDa isoform to the 135-kDa isoform and transport of eNOS to the sarcolemma, effects paralleled by protein kinase A-dependent phosphorylation of the larger eNOS isoform. Forskolin also decreased palmitoylation of the 135-kDa isoform, although it did not accelerate depalmitoylation of sarcolemmal eNOS, as determined by pulse-chase experiments with [3H]palmitate. Thus, post-translational processing of a 150-kDa isoform of myocyte eNOS appears to be necessary for intracellular trafficking of the enzyme to sarcolemmal caveolae. Both the post-translational processing and subcellular targeting of eNOS appear to be modified by changes in intracellular cAMP, an effect that may have important implications for cardiac myocyte responsiveness to autonomic agonists in vivo.

La philosophie d'Ernst Cassirer constitue 1'une des plus importantes pensées du XXe siècle — et aussi 1'une des plus méconnues. Partant de la critique de la vieille ontologie substantialiste,Cassirer s'appuie sur les nouvelles théories de la connaissance pour proposer un pluralisme épistémologique qui consacre la fonction médiatrice de la sensibilité. L'abandon du concept d'être au profit d'une théorie de la culture se fonde désormais sur 1'incarnation symbolique de la raison.

The expression by tumor cells of proteins with aberrant structure, expression or distribution accounts for the development of a humoral immune response. Autoantibodies (aAb) directed against tumor-associated antigens (TAA) may thus be particularly relevant for early detection of cancer. Serological proteome analysis (SERPA) aims to identify such circulating aAb through the immunoblotting of 2D-separated tumor cell proteins with cancer patient serum and the consecutive MS identification of proteins in reactive spots. This method has the advantage to use post-translationally modified proteins as a source of potential TAA. Here, we applied this strategy by using colorectal tumor cells pre-exposed to hypoxia in order to promote the expression of a pattern of TAA more likely to represent in vivo conditions. We used two human HCT116 and HT29 colorectal cancer cell lines exposed for 48 hours to 1% O2. Spots positive after immunoblotting of 2D-separated lysates of hypoxic cells with the sera of tumor-bearing mice, were collected and analysed by MS for protein identification. Among the hypoxia-specific immunogenic proteins, we identified a phosphorylated form of eukaryotic translation elongation factor 2 (phospho-Thr56 eEF2). We confirmed the increased phosphorylation of this protein in hypoxic colorectal tumor cells as well as in mouse tumors. Using a specific immunoassay, we could detect the presence of corresponding anti-phospho-Thr56 eEF2 aAb in the serum of tumor-bearing mice (vs healthy mice). We further documented that the detection of these aAb preceded the detection of a palpable tumor mass in mice and validated the presence of anti-phospho-Thr56 eEF2 aAb in the serum of patients with adenomatous polyps and colorectal carcinoma. In conclusion, this study validates a phosphorylated form of eEF2 as a new TAA and more generally, provides evidence that integrating hypoxia upstream of SERPA offers a more relevant repertoire of TAA able to unmask the presence of circulating aAb.

Cell detachment is a procedure routinely performed in cell culture and a necessary step in many biochemical assays including the determination of oxygen consumption rates (OCR) in vitro. In vivo, cell detachment has been shown to exert profound metabolic influences notably in cancer but also in other pathologies, such as retinal detachment for example. In the present study, we developed and validated a new technique combining electron paramagnetic resonance (EPR) oximetry and the use of cytodex 1 and collagen-coated cytodex 3 dextran microbeads, which allowed the unprecedented comparison of the OCR of adherent and detached cells with high sensitivity. Hence, we demonstrated that both B16F10 melanoma cells and human umbilical vein endothelial cells (HUVEC) experience strong OCR decrease upon trypsin or collagenase treatments. The reduction of cell oxygen consumption was more pronounced with a trypsin compared to a collagenase treatment. Cells remaining in suspension also encounter a marked intracellular ATP depletion and an increase in the lactate production/glucose uptake ratio. These findings highlight the important influence exerted by cell adhesion/detachment on cell respiration, which can be probed with the unprecedented experimental assay that was developed and validated in this study.

In physiological or pathophysiological conditions, vascular tone is regulated by various neural, endocrine, paracrine, and autocrine factors that may exert immediate or delayed effects. In this chapter, we report experiments undertaken to characterize immediate effects by examining the role of endothelium-derived relaxing factors (EDRFs) and of endothelin. We also summarize studies on long-term effects by examining calcium channels and regulation of vascular tone in hypertension, considering comparison of contractile properties of arteries from normotensive and hypertensive rats and their relation with calcium channels. We discuss the following points: differences in postcontraction tone, in response to Cat+-channel activator, in the effect of amlodipine added in vitro on the Bay K 8644 concentration—effect curve, and in dihydropyridine-binding sites. We show the influence of salt loading on the cardiac and renal preproendothelin-1mRNA expression and on its consequences in stroke-prone spontaneously hypertensive rats, and report its blood pressure-independent inhibition by lacidipine, a long-lasting calcium antagonist.

The regulation of L-type voltage-dependent Ca2+ channels by chronic depolarization was studied in the aortic smooth muscle A7r5 and neuron-type PC12 cell lines, by probing the expression and the functional state of their constitutive alpha-1 subunits. PC12 cells showed, after prolonged exposure to a high-K+ depolarizing solution, a 25% reduction of the functional Ca2+ channel density which was accompanied by a decrease of the alpha-1 subunit mRNA expression. In A7r5 cells submitted to a similar protocol of depolarization, 45Ca2+ uptake measurements revealed a fall in the functional activity of L-type Ca2+ channels which was not related to a modulation of their mRNA expression, but arose from a long-term voltage-dependent channel inactivation. Accordingly, the lag time and the mechanisms of recovery were different in the two cell types. In PC12 cells, when restoring physiological culture conditions, de novo synthesis of alpha-1 subunits allowed the recovery of the original density of L-type Ca2+ channels at the membrane surface. As for the A7r5 cells, we showed that after chronic depolarization, the complete restoration of the resting membrane potential and the related Ca2+ channel activity required a 2-day incubation in physiological medium and could probably be related to a normalization of the increased intracellular Ca2+ concentration. In contrast, it is noteworthy that, in PC12 cells, the only transient increase of intracellular Ca2+ content in the first hours of depolarization could account for the long-term down-regulation of L-type Ca2+ channels.

Endothelin-1 is a potent vasoactive peptide which may play a role in the regulation of vascular resistance through its autocrine/paracrine effects. We have investigated the influence of salt loading on the renal and cardiac production of endothelin-1 in stroke prone spontaneously hypertensive rats, a classical model of hypertension. The results show that the dietary salt intake did not change systolic blood pressure nor the renal expression of the preproendothelin-1 mRNA but increased cardiac expression of the endothelin-1 gene transcript and a concomitant ventricular hypertrophy.

In rat brain, [3H]glibenclamide binds with high affinity to sulfonylurea receptors associated with ATP-sensitive potassium (KATP) channels. KATP channels may play a modulatory role in neurotransmitter release and are involved in acute pathological events occurring in the brain. Fluorescein derivatives, which are suitable tools for the labelling of nucleotide binding sites, influence KATP channels and sulfonylurea receptors properties in insulinoma and cardiac cells. In this study, a negative allosteric action of fluorescein derivatives on glibenclamide binding sites has been shown in rat cortical neurons. This supports the hypothesis of interactions between nucleotide- and sulfonylurea-binding sites within the sulfonylurea receptor.

Stroke-prone spontaneously hypertensive rats receiving a high salt diet were orally treated by the calcium antagonist lacidipine, at a dose which did not reduce systolic blood pressure. We observed that lacidipine inhibited the salt-induced cardiac hypertrophy and the concomitant increase of mRNA transcripts for preproendothelin-1 in ventricles. These data show that elevated blood pressure cannot necessarily account for cardiac hypertrophy and indicate that the therapeutic action of lacidipine is not only related to its haemodynamic properties, but also to the inhibition of the gene expression of growth factors such as endothelin.

We investigated the influence of salt loading on the renal and cardiac production of endothelin-1 (ET-1) in stroke-prone, spontaneously hypertensive rats (SHR-SP). The results show that the dietary salt intake did not change systolic blood pressure or the renal expression of the prepro-ET-1 mRNA but increased cardiac expression of the ET-1 gene transcript with concomitant ventricular hypertrophy. These changes were prevented by oral treatment with lacidipine, a long-lasting calcium antagonist, at a dose that did not reduce systolic blood pressure. This indicates that the cardioprotective properties of lacidipine may be dissociated from its blood pressure-lowering effect and could be related to inhibition of endothelin gene expression.

Background The incidence of melanoma is considerably increasing worldwide. Frequent failing of classical treatments led to development of novel therapeutic strategies aiming at managing advanced forms of this skin cancer. Additionally, the implication of the endocannabinoid system in malignancy is actively investigated. Methods We investigated the cytotoxicity of endocannabinoids and their hydrolysis inhibitors on the murine B16 melanoma cell line using a MTT test. Enzyme and receptor expression was measured by RT-PCR and enzymatic degradation of endocannabinoids using radiolabeled substrates. Cell death was assessed by Annexin-V/Propidium iodine staining. Tumors were induced in C57BL/6 mice by s.c. flank injection of B16 melanoma cells. Mice were injected i.p. for six days with vehicle or treatment, and tumor size was measured each day and weighted at the end of the treatment. Haematoxylin-Eosin staining and TUNEL assay were performed to quantify necrosis and apoptosis in the tumor and endocannabinoid levels were quantified by HPLC-MS. Tube formation assay and CD31 immunostaining were used to evaluate the antiangiogenic effects of the treatments. Results The N-arachidonoylethanolamine (anandamide, AEA), 2-arachidonoylglycerol and N- palmitoylethanolamine (PEA) reduced viability of B16 cells. The association of PEA with the fatty acid amide hydrolase (FAAH) inhibitor URB597 considerably reduced cell viability consequently to an inhibition of PEA hydrolysis and an increase of PEA levels. The increase of cell death observed with this combination of molecules was confirmed in vivo where only co-treatment with both PEA and URB597 led to decreased melanoma progression. The antiproliferative action of the treatment was associated with an elevation of PEA levels and larger necrotic regions in the tumor. Conclusions This study suggests the interest of targeting the endocannabinoid system in the management of skin cancer and underlines the advantage of associating endocannabinoids with enzymatic hydrolysis inhibitors. This may contribute to the improvement of long-term palliation or cure of melanoma.

Switching to a glycolytic metabolism is a rapid adaptation of tumor cells to hypoxia. Although this metabolic conversion may primarily represent a rescue pathway to meet the bioenergetic and biosynthetic demands of proliferating tumor cells, it also creates a gradient of lactate that mirrors the gradient of oxygen in tumors. More than a metabolic waste, the lactate anion is known to participate to cancer aggressiveness, in part through activation of the hypoxia-inducible factor-1 (HIF-1) pathway in tumor cells. Whether lactate may also directly favor HIF-1 activation in endothelial cells (ECs) thereby offering a new druggable option to block angiogenesis is however an unanswered question. In this study, we therefore focused on the role in ECs of monocarboxylate transporter 1 (MCT1) that we previously identified to be the main facilitator of lactate uptake in cancer cells. We found that blockade of lactate influx into ECs led to inhibition of HIF-1-dependent angiogenesis. Our demonstration is based on the unprecedented characterization of lactate-induced HIF-1 activation in normoxic ECs and the consecutive increase in vascular endothelial growth factor receptor 2 (VEGFR2) and basic fibroblast growth factor (bFGF) expression. Furthermore, using a variety of functional assays including endothelial cell migration and tubulogenesis together with in vivo imaging of tumor angiogenesis through intravital microscopy and immunohistochemistry, we documented that MCT1 blockers could act as bona fide HIF-1 inhibitors leading to anti-angiogenic effects. Together with the previous demonstration of MCT1 being a key regulator of lactate exchange between tumor cells, the current study identifies MCT1 inhibition as a therapeutic modality combining antimetabolic and anti-angiogenic activities.

observed decreased p16. Detailed analysis showed that HLX1 affects INK4a expression at the mRNA level and HLX1 binds to the INK4a promoter as observed by ChIP. We also observed increased levels of the repressive H3K27me3 mark and recruitment of PRC2 components at the Ink4a/Arf locus correlating with HLX1 expression. Co-immunoprecipitation studies showed that HLX1 associates with the PRC2, in particular with Suz12. RNAi studies showed that the repression of p16 by HLX1 is dependent of PRCs. In an attempt to understand if the repression of HLX1 was a property shared by other homeobox genes, we tested 20 homeoboxcontaining genes and identified that multiple homeobox can also repress p16. Conclusions: We identified the homeobox protein HLX1 as a novel p16 repressor. HLX1 binds to the INK4a promoter region and recruits Polycomb repressive complexes. Multiple homeobox proteins can also regulate p16 expression, which implies a conserved role for this family oftranscription factors in regulating the Ink4a/Arf locus, highlighting its potential physiological relevance for both senescence and carcinogenesis.

4622 Background: Dysregulation of mammalian Target of Rapamycin (mTOR) pathway plays a role in TCC development. We assessed the efficacy and toxicity of Everolimus (E), an oral mTOR inhibitor, in p...

Abstract 3336 Introduction: The search for new anticoagulants is a major challenge in medicine. Contact factors have never been considered as interesting targets for the development of new anticoagulant agents since they are not required for in vivo coagulation (i.e. deficiencies affecting these factors do not cause excessive bleeding). The discovery that FXI and FXII deficiency protects against thrombosis without causing spontaneous bleeding in mice makes FXII a unique and ideal target for drug design. We demonstrated in vitro that Ir-CPI, a 67 amino acids recombinant Kunitz-type protein from Ixodes ricinus, specifically interacted with activated human contact phase factors (FXIIa, FXIa, and kallikrein). Aims: The goal of this study was to investigate the potential anticoagulant and antithrombotic efficacy of Ir-CPI. Methods: The effects of Ir-CPI were investigated on the thrombin activity during the coagulation process in human plasma using the CAT method. Three different inducers were employed to specifically trigger the coagulation pathways and to generate thrombin. A standard concentration of 5 pM TF with 4 μM PL and 16.7 mM CaCl2 was used to activate the TF pathway whereas a lower TF concentration of 1 pM with 4 μM PL and 16.7 mM CaCl2 was selected for stimulating the TF pathway in combination with the contact phase pathway through a thrombin-mediated positive feedback. The contact phase pathway was also triggered alone by ellagic acid, PL and 16.7 mM CaCl2 (25-fold diluted APTT reagent Actin FS). The effect of Ir-CPI on venous thrombus formation was assessed using a rat thrombosis model induced by complete venous stasis in the posterior vena cava and FeCl3 topical application on the outer vessel wall. Results: When stimulating plasma coagulation trough the contact pathway, Ir-CPI caused a concentration-dependent prolongation of the lag time and the Tmax and a concentration-dependent decrease in the Cmax compared to the control curve (i.e. without inhibitor). When the coagulation cascade was triggered by the TF pathway (5 or 1 pM TF), only a slight concentration-dependent decrease of the Cmax and a concentration-dependent prolongation of the lag time and the Tmax were observed. For comparison purpose, the effects of the specific competitive FXIIa inhibitor corn trypsin inhibitor (CTI) were also investigated using the three same inducers than for Ir-CPI. For the contact pathway, a concentration-dependent decrease of the Cmax and a concentration-dependent prolongation of the lag time and the Tmax were found. When stimulating the TF pathway (5 or 1 pM TF), no modification of the thrombin generation curves was observed with the tested concentrations of CTI. When comparing the results of the two inhibitors acting trough the delay of the contact pathway, we found that Ir-CPI was about 30-fold more potent than CTI. We further evaluated the antithrombotic effect of Ir-CPI on a rat venous thrombosis model induced by endothelial damage and vessel ligation, close to the physiological venous thrombus formation in humans. We showed that Ir-CPI reduced thrombosis in a dose-dependent manner with an ED50 close to 65 μg/kg. A maximum effect starting from 0.5 mg/kg was observed with a mean reduction in the clot weight/body weight of 75 ± 7%. This antithrombotic effect of Ir-CPI was exclusively mediated through the inhibition of thrombin generation since it did not interference with collagen-induced platelet aggregation. This is the first time that an inhibitor of the coagulation contact phase was shown to protect against the formation of venous thrombi. The antithrombotic effect of Ir-CPI was also confirmed using other venous and arterial thrombosis models. We also showed that the effective antithrombotic dose of Ir-CPI in these tests did not promote bleeding or impair blood coagulation parameters. Conclusions: The present study demonstrated that Ir-CPI, a recombinant protein from the tick Ixodes ricinus targeting the contact factors (FXII, FXI and kallikrein), displayed an anticoagulant activity mainly through the delay of the contact pathway induced thrombin generation. This drug also exhibited an antithrombotic activity in our venous and arterial thrombosis model. This drug may thus provide an interesting and innovative therapeutic tool for the prevention and the treatment of thromboembolic diseases with a minimal risk of therapy-associated bleeding. Disclosures: No relevant conflicts of interest to declare.

Early detection of tumor-specific autoantibodies (auto-Abs) has the potential to be used for cancer screening and diagnosis. Whether auto-Ab may be useful to track metastatic progression or response to treatment is, however, largely unknown. To address these issues, the serological proteome was analyzed in an invasive but treatment-responsive mouse tumor model. Among 40 serum-reactive proteins identified by multiplex analysis, we chose to focus on glucose-regulated protein 78 (GRP78), a chaperone protein involved in the endoplasmic reticulum stress response. We first validated GRP78 as a protein overexpressed and mislocalized in tumor cells. We then documented that an increase in GRP78 auto-Ab titer preceded the detection of a palpable tumor mass, correlated with metastatic progression, and was influenced by the onset of tumor neovascularization. We also found that chemotherapy and radiotherapy, both leading to inhibition of tumor growth, oppositely influenced the anti-GRP78 immune response. Whereas radiation increased the concentration of GRP78 auto-Ab by three-fold, the auto-Ab titer was reduced in response to bolus or metronomic administration of cyclophosphamide. Finally, we established a decrease in auto-Ab-producing B lymphocytes in response to chemotherapy and the overexpression of GRP78 together with a strong immunoglobulin response in irradiated tumors. In conclusion, we identified GRP78 auto-Ab as an early marker of tumor and metastatic progressions. However, the multiple influences of anticancer treatments on the humoral immune system calls for caution when exploiting such auto-Ab as markers of the tumor response. © 2010 Neoplasia Press, Inc.

Purpose: Cyclic hypoxia in tumors originates from heterogeneities in red blood cell flux and influences tumor cells but also endothelial cells lining tumor blood vessels. Whether pO2 fluctuations, in particular transient reoxygenation periods, alter the well-known hypoxia-inducible factor (HIF)-dependent gene program is largely unknown. Experimental design: We compared the transcriptomic profiles of endothelial and tumor cells exposed to cyclic hypoxia vs continuous hypoxia to uncover a possible differential impact on angiogenesis and metastases. Results: Microarray analyses identified early genes that were selectively induced by cyclic hypoxia in endothelial cells. Among them, we focused on PTGS2 since the observed increase in mRNA expression led to a significant increase in the expression and the activity of COX-2 (the protein product of PTGS2). HIF-1α was shown to be stabilized by cyclic hypoxia (despite reoxygenation periods) and to favor COX-2 induction, as validated by the use of echinomycin and HIF-1α targeting siRNA. Using a specific COX-2 inhibitor and a dedicated COX-2-targeting siRNA, we documented that COX-2 accounted for the higher endothelial cell survival and angiogenic potential conferred by cyclic hypoxia. Cyclic hypoxia also led to a preferential COX-2 induction in tumor cells and, contrary to continuous hypoxia, fostered a higher metastatic take of pre-challenged tumor cells. Conclusions: Our study documents that PTGS2/COX-2 is part of a cyclic hypoxia gene signature and largely accounts for the unique phenotype of endothelial and tumor cells exposed to fluctuations in pO2, thereby offering new perspectives for the clustering of tumors expressing COX-2 together with other cyclic hypoxia-responsive genes. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 461.

1. The interaction of pinaverium bromide, a quaternary ammonium compound, with binding sites for (L-type) calcium channel blockers was investigated in rat ileum smooth muscle. 2. Pinaverium inhibited [3H]-(+)-PN200-110 ([3H]-(+)-isradipine) specific binding to tissue homogenates incompletely (Ki 0.38 microM; maximal inhibition 80%). In contrast, binding to single cell preparations (obtained by collagenase treatment) and to saponin-treated homogenates was completely inhibited. These data are compatible with the view that, in untreated homogenates, 20% of [3H]-(+)-isradipine binding sites are not accessible to pinaverium because it is associated with sealed inside-out vesicles. 3. Pinaverium bromide increased the apparent KD of [3H]-(+)-isradipine binding to saponin-treated homogenates but did not significantly affect the Bmax value. Moreover, the dissociation rate constant of [3H]-(+)-isradipine binding was not changed by pinaverium. These data suggest that pinaverium interacts with the dihydropyridine binding site in a competitive manner. However, in contrast to uncharged dihydropyridine calcium antagonists, pinaverium inhibited, rather than stimulated, [3H]-diltiazem binding to rat brain membranes (at 30-37 degrees C). 4. Although Bmax values of [3H]-(+)-isradipine were similar in homogenates prepared from tissue and cells (collagenase-treated), the KD value was significantly higher in cell homogenates (166 vs 95 pM). Similarly, the Ki value of pinaverium was higher in cell preparations than in tissue homogenates (0.77 vs 0.38 microM). Thus, collagenase can significantly modify the dihydropyridine recognition site.5. The competitive interaction of pinaverium, a permanently charged drug, with [3H]-(+)-isradipine bound to intact cells and its absence of interaction with [3H]-(+)-isradipine bound to sealed inside-out vesicles imply that the dihydropyridine receptor lies near the external surface of the plasma membrane.