Eric Vicaut

Predominant role of A1 adenosine receptors in mediating adenosine induced vasodilatation of rat diaphragmatic arterioles: involvement of nitric oxide and the ATP‐dependent K+ channels

We investigated, by intravital microscopy in rats, the role of the subtypes of adenosine receptors A1 (A1/AR) and A2 (A2AR) in mediating adenosine‐induced vasodilatation of second and third order arterioles of the diaphragm. Adenosine, and the A1AR selective agonists R(−)‐N6‐(2‐phenylisopropyl)‐adenosine (R‐PIA) and N6‐cyclo‐pentyl‐adenosine (CPA) induced a similar concentration‐dependent dilatation of diaphragmatic arterioles. The non selective A2AR subtype agonist N6‐[2‐(3,5‐dimethoxyphenyl)‐2‐(2‐methylphenyl) ethyl]adenosine (DPMA) also dilated diaphragmatic arterioles but induced a significantly smaller dilatation than adenosine. By contrast the selective A2aAR subtype agonist 2‐[p‐(2‐carboxyethyl)phenyl amino]‐5′‐N‐ethyl carboxamido adenosine (CGS 21680) did not modify diaphragmatic arteriolar diameter. The non selective adenosine receptor antagonist 1,3‐dipropyl‐8‐p‐sulphophenylxanthine (SPX, 100 μM) and the selective A1AR antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (CPX, 50 nM) significantly attenuated adenosine‐induced dilatation of diaphragmatic arterioles. By contrast, adenosine significantly dilated diaphragmatic arterioles in the presence of A2AR antagonist 3,7‐dimethyl‐1‐propargylxanthine (DMPX, 10 μM). The dilatation induced by adenosine was unchanged by the mast cell stabilizing agent sodium cromoglycate (cromolyn, 10 μM). The nitric oxide (NO) synthase inhibitor Nω‐nitro‐L‐arginine (L‐NOARG, 300 μM) attenuated the dilatation induced by adenosine, and by the A1AR and A2AR agonists. The ATP‐dependent K+ channel blocker glibenclamide (3 μM) significantly attenuated diaphragmatic arteriolar dilatation induced by adenosine and by the A1AR agonists R‐PIA and CPA. By contrast, glibenclamide did not significantly modify arteriolar dilatation induced by the A2AR agonist DPMA. These findings suggest that adenosine‐induced dilatation of diaphragmatic arterioles in the rat is predominantly mediated by the A1AR, via the release of NO and activation of the ATP‐dependent K+ channels.

Prediction of Head-Up Tilt Test Result by Analysis of Early Heart Rate Variations

BACKGROUNDnHead-up tilt testing is a useful test for investigating vasovagal syncope. The determination of early, accurate, predictive criteria for a negative result would permit a reduction in the duration of the tilt test.nnnMETHODS AND RESULTSnPatients with no drug use and no illnesses other than recurrent unexplained syncope were recruited. In an initial study (110 consecutive patients), we aimed to determine a predictive criterion based on heart rate variations during the first minutes of upright tilting that could distinguish between patients with positive and negative tilt tests (patients with an early continual decrease in heart rate or blood pressure were excluded). Then we tested the predictive value of the established criterion in a second independent sample of patients with unexplained syncope (109 consecutive patients). An early sustained increase in heart rate < or = 18 bpm during the first 6 minutes of upright tilting at a 60 degree angle allowed us to predict negative tilt tests with 100% specificity, 100% positive predictive value, and 88.6% sensitivity. This criterion was validated in the second, prospective arm of the study (96.4% specificity, 98.4% positive predictive value, and 87.3% sensitivity), even with subsequent use of isoproterenol in low doses.nnnCONCLUSIONSnIn patients with no drug use and no illnesses other than recurrent unexplained syncope, a simple clinical criterion identifies patients who will not develop syncope during a prolonged upright tilt.

Arteriolar vasoconstriction and tachyphylaxis with intraarterial angiotensin II

Several aspects of the differences between the responses of the second- to fifth-order arterioles (A2 to A5) to intraarterial administration of angiotensin II (AII) were studied by intravital microscopy on an original preparation of rat cremaster muscle. Dose-response curves displayed a leftward shift when the arteriolar order increased. Doses inducing 50% vasoconstriction were 15.1, 0.51, and 0.08 micrograms for A3, A4, and A5, respectively. For A2, very small vasoconstriction was found even at the highest dose of angiotensin II. The dynamics of the response were also dependent on the arteriolar order. The duration of the peak of vasoconstriction increased from A3 to A5, and the interval between the contact of vascular wall with drug and the response was smaller in A4 and A5 than in A3. To understand the effect of diameter as a determinant of heterogeneity in the degree of arteriolar vasoconstriction, norepinephrine was administered under the same conditions as angiotensin II, and responses were measured on arterioles with the same morphological characteristics as those examined after angiotensin II. When comparing the regression curves for the percentage of vasoconstriction vs diameter, we found that this relationship was drug dependent. The significantly steeper slope for angiotensin II than for norepinephrine excluded the possibility that heterogeneity of the degree of vasoconstriction is solely due to differences between the morphological characteristics of the arterioles. Since tachyphylaxis to AII is considered to be a reflection of the drug-receptor interaction, we also studied the magnitude of this phenomenon from proximal to distal parts of the arteriolar network. We showed that the degree of partial tachyphylaxis after 1 microgram AII was dependent on the arteriolar order and a decreasing tachyphylaxis gradient was evidenced from A3 to A5 arterioles.

Interleukin 10 antioxidant effect decreases leukocytes/endothelial interaction induced by tumor necrosis factor α.

ABSTRACT Little is known about the endothelial mechanisms involved in the anti-inflammatory effects of interleukin 10 (IL-10). The goal of this study was to evaluate the effects of IL-10 on endothelial oxidative stress and endothelial inflammation induced by tumor necrosis factor &agr; (TNF-&agr;). Production of reactive oxygen species (ROS) in perfused human umbilical vein endothelial cells (HUVECs) was studied by fluorescent microscopy using dichlorodihydrofluorescein diacetate. Tumor necrosis factor &agr; (1 ng/mL) was added to the perfusion medium in the absence and presence of IL-10 (1 ng/mL). The role of phosphatidylinositol 3-kinase (PI3-kinase) was assessed using wortmannin and LY 2940002 (inhibitors of PI3-kinase). Specific inhibition of p110 &agr; and p110 &ggr;/&dgr; PI3-kinase subunits was studied using A66 and TG100–115. As well, levels of ceramide and intercellular adhesion molecule 1 (ICAM-1) expression were measured. Finally, the effect of IL-10 on TNF-&agr;–induced leukocyte/endothelium interaction was examined using an ex vivo perfused vessel model. Interleukin 10 significantly reduced dichlorodihydrofluorescein diacetate fluorescence induced by TNF-&agr; in HUVECs (12.5% ± 3.2% vs. 111.7% ± 21.6% at 60 min). Pretreatment by LY2940002 or wortmannin restored ROS production induced by TNF-&agr; in the presence of IL-10. In HUVECs treated by TNF-&agr; + IL-10, inhibition of p110 &agr; PI3-kinase subunit significantly increased ROS production, whereas p110 &ggr;/&dgr; inhibition did not have a significant effect. Pretreatment with IL-10 significantly decreased TNF-&agr;–induced increased levels of ceramide (TNF-&agr; vs. TNF-&agr; + IL-10: 6,278 ± 1,013 vs. 1,440 ± 130 pmol/mg prot), as well as ICAM-1 expression and leukocyte adhesion (TNF-&agr; vs. TNF-&agr; + IL-10: 26.8 ± 2.6 vs. 6.7 ± 0.4 adherent leukocytes/field at 15 min). Interleukin 10 decreases the level of inflammation induced by TNF-&agr; in endothelial cells by reducing the TNF-&agr;–induced ROS production, ICAM-1 expression, and leukocyte adhesion to the endothelium. The antioxidant effect of IL-10 is mediated through PI3-kinase and is paralleled by a decrease in ceramide synthesis induced by TNF-&agr;.

Scanning laser ophthalmoscope imaging of fluorescein-labelled blood cells

Abstract• Purpose: To demonstrate the feasibility of a technique for the visualization by scanning laser ophthalmoscope (SLO) of fluores cein-labelled autologous leukocytes and platelets in retinal vessels. • Method: Individual blood samples from rats and rabbits were centrifuged to isolate platelets and leukocytes, then passively labelled with fluorescein and reinjected into the same animal. An SLO was used to visualize and record cell displacement in the retinal circulation. Labelled platelets were analysed by flow cytometry. • Results: By SLO, platelets appeared as a heterogeneous particle flow, and individual leukocytes appearing as brighter spots could easily be traced. Flow cytometry showed that after labelling platelets were well individualized and their size was slightly increased. • Conclusion: Circulating blood cells can be visualized in retinal vessels by a simple method consisting of passive labelling of autologous platelets and leukocytes by fluorescein. No platelet toxicity was detected. This method could be applied to the study of blood cell movement in human retinal vascular diseases.

An intact cremaster muscle preparation for studying the microcirculation by in vivo microscopy

We describe here in detail a new preparation which has the advantage of limiting muscle dissection drastically and of preserving all the vascular interconnections intact

Specific response of mouse tumor-feeding arterioles to stimulation by 5-HT1 agonists

Using intravital microscopy, we compared the responses to 5-HT1 receptor stimulation by the host-modified arterioles feeding a Meth-A tumor implanted in the flank of female Balb/c mice with the responses of tumor-independent arterioles (TIA) and those of control arterioles from mice without tumor. Topical administration of 5 x 10(-5) M serotonin in the presence of 10(-4) M ketanserin (5-HT2 receptors inhibitor) induced arteriolar vasodilation in TIA (+13%) and in the control arterioles (+19%), but induced constriction (-14%) in the tumor-feeding arterioles (TFA). Topical administration of the general 5-HT1 agonist 5-carboxamidotryptamine maleate (10(-6) to 10(-4) M) or the 5-HT1A agonist buspirone (2 x 10(-6) to 2 x 10(-4) M) induced vasoconstriction that was dramatically higher in TFA than in TIA or control arterioles (p < 0.0001 in both cases). In addition, topical administration of the 5-HT1B agonist M-trifluoromethylphenylpiperazine (2 x 10(-6) to 2 x 10(-4) M) produced opposite responses, i.e., dose-dependent vasodilation in TIA and control arterioles, and dose-dependent constriction in TFA. Since we observed the same degree of vasodilation in response to 10(-4) M acetylcholine in all three groups of arterioles, the differences between the responses to 5-HT1 receptor stimulation were not due to the absence of endothelial-dependent dilatory mechanisms in the tumor-feeding arterioles. We conclude that 5-HT1 agonists are interesting pharmacologic tools for the modulation of tumoral blood flow, since they more dramatically constrict the microvasculature feeding the tumors than that feeding normal tissue.

A preparation for in vivo study of the diaphragmatic microcirculation in the rat.

A new preparation is described for the study of the microcirculation of the rat diaphragm by in vivo microscopy. After midline laparotomy, the abdominal site of the diaphragm muscle was exposed. The rat was mechanically ventilated and placed in the Trendelenburg position, thus allowing a microscope placed on a three-dimensional articulated system to be set up perpendicular to the diaphragm. The diaphragm was then transilluminated by inserting fiberoptic microprobes into the thorax cavity by thoracotomy in the fifth intercostal space. This preparation allowed us to describe the morphological characteristics of the arteriolar network in vivo. As regards the venular network, two parts were distinguished: one part collects the blood of the external half of the diaphragm, runs roughly parallel to the arterioles, and converges toward the internal mammary and intercostal veins; the other part collects the blood of the central half of the diaphragm and converges on the central venous arcade along its central tendon. Anastomotic channels between these two parts were observed, as well as spontaneous inversion of the direction of the blood flow, indicating the presence of unsteady pressure gradients in some branches of the venular network. Capillary density was also studied by measuring intercapillary distance, whose mean value was 21.43 +/- 0.67 microns. No differences in intercapillary distance were found between the external and central parts of the diaphragm. In conclusion, we describe a preparation which allowed us to study the diaphragmatic microcirculation for at least 2 hr under good hemodynamic conditions. The study of this specific microcirculation is important because the diaphragms metabolism and functions are specific and because it is essential to life that its perfusion should be adapted to its specific metabolic requirements.

Theophylline dilates rat diaphragm arterioles via the prostaglandins pathway.

1 We investigated by intravital microscopy in rats, the in vivo direct effects of theophylline on the diameters of second and third order diaphragm arterioles. 2 Theophylline (1–100 μm) dilated second and third order diaphragm arterioles significantly, and with an amplitude which was not statistically different from the one obtained with adenosine (1–100 μm). Enprofylline (1–100 μm), a theophylline analogue with poor adenosine‐receptor antagonism but with similar or higher phosphodiesterases inhibition properties than theophylline, also dilated diaphragm arterioles, causing however, a significantly smaller dilatation than theophylline. 3 Neither the A1 adenosine receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (CPX, 50 nm), nor the A2 adenosine receptor antagonist 3,7‐dimethyl‐1‐proparglyxanthine (DMPX, 10 μm) reduced significantly theophylline‐induced arteriolar dilatation. 4 Theophylline (100 nm) abolished adenosine‐induced arteriolar dilatation. 5 The dilatation induced by theophylline was unchanged by the nitric oxide (NO) synthase inhibitor Nω‐nitro‐l‐arginine (NNA, 300 μm). 6 Theophylline‐induced arteriolar dilatation was abolished by the prostaglandin synthesis inhibitors mefenamic acid or indomethacin (20 μm). 7 These findings show that theophylline induced a significant dilatation of diaphragm arterioles via the release of prostaglandins.

Characterization of the specific response to serotonin of mouse tumour-feeding arterioles

PURPOSEnTo investigate the role of tumour versus non-tumour factors in the specific response to serotonin (5-HT) of tumour-feeding arterioles (TFA).nnnMATERIALS AND METHODSnUsing mouse models of intra-vital microscopy, the response to topical administration of 5-HT was studied in arterioles feeding tumours: fibrosarcoma (Meth A), murine mammary adenocarcinoma (EMT6) and human colo-rectal carcinoma (HRT18) intra-cutaneously implanted.nnnRESULTSnFor all types of tumour, 5-HT induced a far more pronounced constriction of TFA than of control arterioles. The presence of a tumour implanted in the connective tissue between the skin and the cremaster muscle also affected the reactivity of muscle arterioles. Conversely, the response to serotonin by neovessels grown after implantation of an exogenous element under the skin did not differ from that of control arterioles.nnnCONCLUSIONSnChanges in reactivity to serotonin were not dependent on the type of tumour implanted in the skin and were not present for a non-tumour implant. The presence of the tumour can alter the reactivity of vessels from tissue in contact with the tumour even if these vessels did not feed the tumour. This phenomenon is local and was not found in the vessels at a distance from the tumour.