Inflammation and impaired endothelium-dependant vasodilatation in non obese women with gestational diabetes mellitus: preliminary results.

BACKGROUND: To evaluate whether abnormal endothelial function, a common finding in gestational diabetes mellitus (GDM) pregnancies, can be explained by inflammatory cytokines. METHODS: Forearm skin blood flow (FSBF), into response to acetylcholine (Ach) (endothelium-dependent vasodilatation), were measured in 24 pregnant control subjects and 28 gestational diabetes mellitus (GDM) women, in the third trimester of gestation. A fasting glycemic and lipidic panel was obtained, and inflammatory cytokines (TNF-α and IL-6) and adiponectin were also determined. RESULTS: FSBF is significantly reduced in GDM group compared with control subjects (344.59 ± 57.791 vs.176.38 ± 108.52, P < 0.05). Among all subjects, FSBF showed a strong negative correlation with TNF-α and IL-6 (r = -0.426, P < 0.0001 and r = -0.564, P < 0.0001, respectively) and positive correlation with adiponectin (r = 0.468, P < 0.0001). CONCLUSIONS: Endothelial function, an early marker of macrovascular disease, is present in non-obese pregnancies complicated by GDM. This alteration seems to be directly related to inflammatory status, which may represent a patho-physiological link between GDM and type 2 diabetes and, later on, metabolic syndrome.

Noninvasive assessment of endothelial function in the skin microcirculation.

BACKGROUND: The structure and function of blood vessels varies along the vascular tree. Endothelial dysfunction is a hallmark of increased cardiovascular (CV) risk that can be assessed by several methods, some of which are invasive and of restricted application. The aim of this study was to determine whether the laser Doppler response of skin microcirculation to acetylcholine, reflects that of conduit artery assessed by brachial artery flow-mediated dilation (FMD). METHODS: Noninvasive measurement of endothelium-dependent vasodilation in the skin microcirculation by laser Doppler flowmetry (LDF) in response to a local transdermal iontophoretic application of acetylcholine (Ach-SkBF) is an operator-independent method. Ach-SkBF and FMD were measured in the nondominant upper limb of 55 unselected consecutive patients admitted in our department for evaluation of CV risk factors. RESULTS: Ach-SkBF was (mean +/- s.d. (min-max)) 490 +/- 414%, (10-1667%) and FMD was 3.77 +/- 3.01% (0.91-10.91). A strong linear relationship was found between Ach-SkBF and FMD: Ach-SkBF = 122.7 FMD + 25.8 (r = 0.92, P < 0.0001). CONCLUSIONS: Endothelial dilatory response to increased blood flow and to acetylcholine are similar in large arteries and in the skin microvasculature. Thus, measurement of blood flow changes in the skin microcirculation using LDF coupled with acetylcholine iontophoresis represents a technically challenging and reliable noninvasive method for the assessment of endothelial function within a large range of normal and altered endothelium responses.

Serotonin and angiotensin receptors in cardiac fibroblasts coregulate adrenergic-dependent cardiac hypertrophy.

By mimicking sympathetic stimulation in vivo, we previously reported that mice globally lacking serotonin 5-HT(2B) receptors did not develop isoproterenol-induced left ventricular hypertrophy. However, the exact cardiac cell type(s) expressing 5-HT(2B) receptors (cardiomyocytes versus noncardiomyocytes) involved in pathological heart hypertrophy was never addressed in vivo. We report here that mice expressing the 5-HT(2B) receptor solely in cardiomyocytes, like global 5-HT(2B) receptor-null mice, are resistant to isoproterenol-induced cardiac hypertrophy and dysfunction, as well as to isoproterenol-induced increases in cytokine plasma-levels. These data reveal a key role of noncardiomyocytes in isoproterenol-induced hypertrophy in vivo. Interestingly, we show that primary cultures of angiotensinogen null adult cardiac fibroblasts are releasing cytokines on stimulation with either angiotensin II or serotonin, but not in response to isoproterenol stimulation, demonstrating a critical role of angiotensinogen in adrenergic-dependent cytokine production. We then show a functional interdependence between AT(1)Rs and 5-HT(2B) receptors in fibroblasts by revealing a transinhibition mechanism that may involve heterodimeric receptor complexes. Both serotonin- and angiotensin II-dependent cytokine production occur via a Src/heparin-binding epidermal growth factor-dependent transactivation of epidermal growth factor receptors in cardiac fibroblasts, supporting a common signaling pathway. Finally, we demonstrate that 5-HT(2B) receptors are overexpressed in hearts from patients with congestive heart failure, this overexpression being positively correlated with cytokine and norepinephrine plasma levels. Collectively, these results reveal for the first time that interactions between AT(1) and 5-HT(2B) receptors coexpressed by noncardiomyocytes are limiting key events in adrenergic agonist-induced, angiotensin-dependent cardiac hypertrophy. Accordingly, antagonists of 5-HT(2B) receptors might represent novel therapeutics for sympathetic overstimulation-dependent heart failure.

Endothelin receptor antagonism prevents hypoxia-induced mortality and morbidity in a mouse model of sickle-cell disease.

Patients with sickle-cell disease (SCD) suffer from tissue damage and life-threatening complications caused by vasoocclusive crisis (VOC). Endothelin receptors (ETRs) are mediators of one of the most potent vasoconstrictor pathways in mammals, but the relationship between vasoconstriction and VOC is not well understood. We report here that pharmacological inhibition of ETRs prevented hypoxia-induced acute VOC and organ damage in a mouse model of SCD. An in vivo ultrasonographic study of renal hemodynamics showed a substantial increase in endothelin-mediated vascular resistance during hypoxia/reoxygenation-induced VOC. This increase was reversed by administration of the dual ETR antagonist (ETRA) bosentan, which had pleiotropic beneficial effects in vivo. It prevented renal and pulmonary microvascular congestion, systemic inflammation, dense rbc formation, and infiltration of activated neutrophils into tissues with subsequent nitrative stress. Bosentan also prevented death of sickle-cell mice exposed to a severe hypoxic challenge. These findings in mice suggest that ETRA could be a potential new therapy for SCD, as it may prevent acute VOC and limit organ damage in sickle-cell patients.

Ultrasound imaging of renal vaso-occlusive events in transgenic sickle mice exposed to hypoxic stress.

One of the major clinical manifestations of sickle cell disease (SCD) is vaso-occlusive crisis in response to hypoxic exposure, leading to acute and chronic organ damages, especially in kidneys. In a SCD transgenic murine model, ultrasound imaging allowed us to characterize the circulatory changes in renal arteries during vaso-occlusive crisis. Cardiac output, heart rate and renal blood flow velocities (BFV) were measured in 10 male transgenic and 10 male wild-type (WT) mice with a conventional echograph (Vivid 7, GE Medical), before and after hypoxic exposure (8%O(2), 18h). To assess entrapment of red cells, histologic study of the kidneys was performed in both groups. Hypoxic exposure decreased heart rates in both groups (-17%, p < 0.001). Cardiac output remained stable in WT, and decreased in transgenic (-26%, p < 0.01). Peak systolic BFV in the renal artery was not modified in both groups. End-diastolic and mean BFV remained stable in WT, but decreased in sickle transgenic (-56%, p < 0.01 and -47%, p < 0.001, respectively). Transgenic mice displayed marked congestion in peritubular capillaries and glomerular abnormalities with trapped sickle red cells, whereas WT did not present any histologic injury. Five hours after hypoxic exposure, blood flow velocities returned to basal values in both groups. Decrease in end-diastolic and mean BFV in absence of peak systolic BFV after hypoxic exposure strongly indicated that the increase in vascular resistance in kidneys related to sickling of red cells. Thus, ultrasound imaging of the renal artery in mouse is a powerful, noninvasive, easy-to-repeat method to evidence circulatory changes in murine models of vascular renal human diseases.

Ultrasonic assessment of cerebral blood flow changes during ischemia-reperfusion in 7-day-old rats.

A model of ischemic brain injury in 7-day-old rat pups has been developed to study perinatal ischemia. It combines permanent occlusion of the distal left middle cerebral artery (LMCA) and transient occlusion of homolateral common carotid artery (LCCA). At removal of the clip on LCCA, reflow allowed brain reperfusion through cortical anastomoses. In 10 rat pups, we measured blood flow velocities (BFV) in main cerebral arteries with 12-MHz ultrasound imaging. At basal states, peak systolic BFV in proximal LMCA was 16.0 +/- 3.0 cm.s(-1). Occlusion of LMCA did not yield significant modifications. Occlusion of LCCA involved only a decrease in BFV to 9.5 +/- 2.6 cm.s(-1) (p < 0.001). Indeed, LMCA was then supply by the right internal carotid and the vertebral arteries through the circle of Willis. In three rat pups, release of occlusion of LCCA was followed by restoration of BFV in the left internal carotid artery and in LMCA, in seven pups, by a reversed flow in the LICA and lower BFV in LMCA (11.9 +/- 2.3, p < 0.05). BFV returned to basal values from h5 to h48 in all animals. In addition, ultrasound imaging is a useful, reproducible, non invasive, easy-to-repeat, method to assess and monitor arterial cerebral blood flow supply in small animals. It helps to characterize changes occurring during cerebral ischemia and reperfusion, particularly the depth of the hypoperfusion, as well as the variability of reflow. In preclinical studies, this method could help to identify what can be assigned to a neuroprotective treatment and what depends on changes in cerebral blood flow supply.

Ultrasonic assessment of hepatic blood flow as a marker of mouse hepatocarcinoma.

Two-dimensional color-coded pulsed Doppler ultrasonography (US) with a 12-MHz linear transducer was used to follow tumor growth and neoangiogenesis development in 12 transgenic mice developing a whole liver hepatocellular carcinoma (HCC) induced by the expression of SV40-T antigen. In this model, male mice developed HCC at various temporal and histologic stages (hyperplastic, four-eight wk; nodular, 12 wk; diffuse carcinoma, 16-20 wk), whereas female mice remained tumor free. Seven age-matched tumor-free mice were used as controls. Liver volume was calculated from B-mode images of the abdomen. Blood flow waveforms were recorded from the hepatic tumor-feeding artery upstream from the tumor vessels, allowing quantitative blood flow velocity measurements. Measurements were performed every four weeks from four to 20 weeks. As early as the hyperplastic stage (eight weeks), liver volume was increased by 2.7-fold, hepatic artery peak-systolic blood flow velocities (BFV) by 1.5-fold, end-diastolic BFV by 1.6-fold and mean BFV by 2.0-fold compared with control values (p < 0.001). Differences increased until 20 weeks and peak-systolic reached 90 +/- 6, end-diastolic 54 +/- 5 and mean BFV 48 +/- 5 cm s(-1). Successive measurements of BFV were reproducible and intraobserver repeatability coefficient values were <3 cm s(-1). In contrast, mesenteric artery BFV, which did not supply tumor region, did not show any significant difference with respect to control values. Thus, an increase in BFV constitutes a functional evaluation of tumor vascularity. In preclinical studies in small animals, measurements of liver volume and blood flow velocities in hepatic tumor-feeding artery provide a useful, reproducible, noninvasive, easy-to-repeat tool to monitor tumor growth and neoangiogenesis in hepatocellular carcinoma in mice.

Increased skin capillary density in treated essential hypertensive patients.

BACKGROUND: Microvascular rarefaction is a hallmark of essential hypertension. We measured the skin capillary density in nondiabetic hypertensive subjects with effective antihypertensive treatment and evaluated possible correlations with arterial blood pressure (BP). METHODS: This cross-sectional observational study included 76 (55 +/- 1 years) consecutive outpatients with essential hypertension under chronic antihypertensive drug treatment (BP < 140/90 mm Hg), 24 age- and sex-matched patients with recently discovered and never-treated hypertension and 70 normotensive (BP < 140/90 mm Hg) age- and sex-matched healthy controls. We used intravital video-microscopy to measure basal and maximal (during venous congestion) skin capillary densities in the dorsum of the fingers. Aortic stiffness was evaluated using pulse wave velocity and central aortic pressure calculated from radial artery applanation tonometry. RESULTS: Baseline and maximal capillary densities (number/mm2) were significantly lower (59.6 +/- 2.0 and 62.0 +/- 1.9) in untreated than in treated hypertensive patients (74.0 +/- 1.4 and 79.4 +/- 1.5; P < .001) and than in normotensives (68.2 +/- 1.5 and 72.4 +/- 1.5; P < .001). Based on multiple regression analysis, after adjustment to tobacco consumption, aortic (and not brachial) systolic BP was inversely correlated with basal and postocclusive capillary densities in normotensive subjects. In hypertensives, this correlation disappears and capillary density was influenced by two independent variables, antihypertensive drug treatment and overweight. CONCLUSIONS: In nondiabetic hypertensive patients, capillary density is reduced in association with a cluster of cardiovascular risk factors involving tobacco consumption and obesity. The finding of an increased capillary density in effectively treated antihypertensives suggests that a cause-to-effect relationship between BP and capillary density should be evaluated in a long-term prospective follow-up.