J.G.R. De Mey

Two-photon microscopy of vital murine elastic and muscular arteries : Combined structural and functional imaging with subcellular resolution

Understanding vascular pathologies requires insight in the structure and function, and, hence, an imaging technique combining subcellular resolution, large penetration depth, and optical sectioning. We evaluated the applicability of two-photon laser-scanning microscopy (TPLSM) in large elastic and small muscular arteries under physiological conditions. Elastic (carotid) and muscular (uterine, mesenteric) arteries of C57BL/6 mice were mounted in a perfusion chamber. TPLSM was used to assess the viability of arteries and to visualize the structural components elastin, collagen, nuclei, and endothelial glycocalyx (EG). Functionality was determined using diameter changes in response to noradrenaline and acetylcholine. Viability and functionality were maintained up to 4 h, enabling the assessment of structure-function relationships. Structural vessel wall components differed between elastic and muscular arteries: size (1.3 vs. 2.1 µm) and density (0.045 vs. 0.57 µm–2) of internal elastic lamina fenestrae, smooth muscle cell density (3.50 vs. 1.53 µm–3), number of elastic laminae (3 vs. 2), and adventitial collagen structure (tortuous vs. straight). EG in elastic arteries was 4.5 µm thick, covering 66% of the endothelial surface. TPLSM enables visualization and quantification of subcellular structures in vital and functional elastic and muscular murine arteries, allowing unraveling of structure-function relationships in healthy and diseased arteries.

Tissue-Specific Utilization of Menaquinone-4 Results in the Prevention of Arterial Calcification in Warfarin-Treated Rats

The effects of vitamin K (phylloquinone: K1 and menaquinone-4: MK-4) on vascular calcification and their utilization in the arterial vessel wall were compared in the warfarin-treated rat model for arterial calcification. Warfarin-treated rats were fed diets containing K1, MK-4, or both. Both K1 and MK-4 are cofactors for the endoplasmic reticulum enzyme γ-glutamyl carboxylase but have a structurally different aliphatic side chain. Despite their similar in vitro cofactor activity we show that MK-4 and not K1 inhibits warfarin-induced arterial calcification. The total hepatic K1 accumulation was threefold higher than that of MK-4, whereas aortic MK-4 was three times that of K1. The utilization of K1 and MK-4 in various tissues was estimated by calculating the ratios between accumulated quinone and epoxide species. K1 and MK-4 were both equally utilized in the liver, but the aorta showed a more efficient utilization of MK-4. Therefore, the observed differences between K1 and MK-4 with respect to inhibition of arterial calcification may be explained by both differences in their tissue bioavailability and cofactor utilization in the reductase/carboxylase reaction. An alternative explanation may come from an as yet hypothetical function of the geranylgeranyl side chain of MK-4, which is a structural analogue of geranylgeranyl pyrophosphate and could interfere with a critical step in the mevalonate pathway.

Hyperglycaemia-induced impairment of endothelium-dependent vasorelaxation in rat mesenteric arteries is mediated by intracellular methylglyoxal levels in a pathway dependent on oxidative stress

Aims/hypothesisImpaired nitric oxide (NO)-dependent vasorelaxation plays a key role in the development of diabetic vascular complications. We investigated the effect of hyperglycaemia on impaired vasoreactivity and a putative role therein of the AGE precursor methylglyoxal.MethodsThe effects of high glucose and methylglyoxal on NO-dependent vasorelaxation in isolated rat mesenteric arteries from wild-type and transgenic glyoxalase (GLO)-I (also known as GLO1) rats, i.e. the enzyme detoxifying methylglyoxal, were recorded in a wire myograph. AGE formation of the major methylglyoxal-adduct 5-hydro-5-methylimidazolone (MG-H1) was detected with an antibody against MG-H1 and quantified with ultra-performance liquid chromatography (tandem) mass spectrometry. Reactive oxygen species formation was measured with a 5-(and-6)-chloromethyl-2′7′-dichlorodihydrofluorescein diacetate acetyl ester probe and by immunohistochemistry with an antibody against nitrotyrosine.ResultsHigh glucose and methylglyoxal exposure of mesenteric arteries significantly reduced the efficacy of NO-dependent vasorelaxation (p < 0.05). This impairment was not observed in mesenteric arteries of GLO-I transgenic rats indicating a specific intracellular methylglyoxal effect. The diabetes-induced impaired potency (pD2) in mesenteric arteries of wild-type rats was significantly improved by GLO-I overexpression (p < 0.05). Methylglyoxal-modified albumin did not affect NO-dependent vasorelaxation, while under the same conditions the receptor for AGE ligand S100b did (p < 0.05). Methylglyoxal treatment of arteries increased intracellular staining of MG-H1 in endothelial cells and adventitia by fivefold accompanied by an eightfold increase in the oxidative stress marker nitrotyrosine. Antioxidant pre-incubation prevented methylglyoxal-induced impairment of vasoreactivity.Conclusions/interpretationThese data show that hyperglycaemia-induced impairment of endothelium-dependent vasorelaxation is mediated by increased intracellular methylglyoxal levels in a pathway dependent on oxidative stress.

Chronic Hypoxia Stimulates Periarterial Sympathetic Nerve Development in Chicken Embryo

BackgroundEpidemiological findings suggest an association between low-for-age birth weight and the risk to develop coronary heart diseases in adulthood. During pregnancy, an imbalance between fetal demands and supply may result in permanent alterations of neuroendocrine development in the fetus. We evaluated whether chronic prenatal hypoxia increases arterial sympathetic innervation. Methods and ResultsChicken embryos were maintained from 0.3 to 0.9 of the 21-day incubation period under normoxic (21% O2) or hypoxic conditions (15% O2). At 0.9 incubation, the degree of sympathetic innervation of the embryonic femoral artery was determined by biochemical, histological, and functional (in vitro contractile reactivity) techniques. Chronic hypoxia increased embryonic mortality (32% versus 13%), reduced body weight (21.9±0.4 versus 25.4±0.6 g), increased femoral artery norepinephrine (NE) content (78.4±9.4 versus 57.5±5.0 pg/mm vessel length), and increased the density of periarterial sympathetic nerve fibers (14.4±0.7 versus 12.5±0.6 counts/104 &mgr;m2). Arteries from hypoxic embryos were less sensitive to NE (pD2, 5.99±0.04 versus 6.21±0.10). In the presence of cocaine, however, differences in sensitivity were no longer present. In the embryonic heart, NE content (156.9±11.0 versus 108.1±14.7 pg/mg wet wt) was also increased after chronic hypoxia. ConclusionsIn the chicken embryo, chronic moderate hypoxia leads to sympathetic hyperinnervation of the arterial system. In humans, an analogous mechanism may increase the risk for cardiovascular disease in adult life.

Comparison of the Responsiveness of Cutaneous Veins of Dog and Rabbit to Adrenergic and Cholinergic Stimulation

The reactivity to sympathetic nerve activation and the metabolism of 3H-norepinephrine were compared in isolated saphenous veins of dogs and rabbits. The veins of both species were equally

Effects of chemical sympathectomy on angiotensin ii-induced neointimal growth in the balloon-injured rat carotid artery

To investigate the role of the sympathetic nervous system in angiotensin II (AngII)-stimulated medial and neointimal smooth muscle cell (SMC) replication, we sympathectomized rats with 6-hydroxydopamine (6-OHDA) in which the left carotid artery was injured by a balloon catheter. Balloon injury is associated with a loss of specific [3H]-prazosin binding. AngII (250 ng/kg/min), infused 2 weeks after balloon injury of the rat left carotid artery, increased systolic blood pressure by approximately 70 mm Hg. There was no effect of 6-OHDA on this pressor response. AngII increased the cumulative 5-bromo-2′-deoxyuridine (BrdU) labeling fraction (LF) in the uninjured right carotid media and the injured left carotid neointima as compared to controls (5.7 ± 1.6% vs. 0.4 ± 0.1%, p < 0.05; 10.6 ± 0.9% vs. 5.0 ± 0.8%, p < 0.05, respectively). 6-OHDA decreased the AngII-induced increase in LF in the media of the uninjured right carotid artery (AngII/6-OHDA 0.9 ± 0.2% vs. AngII 5.7 ± 1.6%, p < 0.05). 6-OHDA did not decrease the AngII-induced increase in LF in both the injured left carotid media and neointima at 4 weeks after balloon injury. The effects of chemical sympathectomy were comparable with those obtained 12 weeks after balloon injury. Thus, the data show that the sympathetic nervous system mediates the AngII-induced increase in SMC DNA synthesis, but only in the uninjured carotid media. This indicates a differential regulation of AngII-induced SMC replication in injured and uninjured vessels.

Vasodilator Reactivity to Calcitonin Gene-Related Peptide Is Increased in Mesenteric Arteries of Rats during Early Pregnancy

The objective of the present study was to determine the effect of early pregnancy on the sensitivity to, and endogenous production of calcitonin gene-related peptide (CGRP). Contractile responses of arteries of 10-day pregnant and nonpregnant rats were studied in myographs. During contractions induced by 40 mmol/l K+, exogenous CGRP elicited an approximately 30% stronger relaxation in mesenteric arteries in pregnancy, an effect not seen in renal and uterine arteries. Capsaicin treatment during K+-induced contractions caused a persistent potentiation of the contractile response in mesenteric arteries, indicating that K+ stimulates the endogenous release of CGRP. This potentiation was similar in the pregnant and nonpregnant state (+81 ± 23% and +82 ± 23%, respectively), suggesting no effect of pregnancy on the endogenous CGRP release. The latter was paralleled by comparable CGRP content in the arteries of both groups, indicating similar tissue CGRP availability. The results of this study support the concept that early pregnancy is associated with a rise in the vascular sensitivity to CGRP in selected areas of the vascular bed without concomitant increase in the vascular CGRP production and release.

Role of newly formed platelets in thrombus formation in rat after clopidogrel treatment: comparison to the reversible binding P2Y12 antagonist ticagrelor

Platelet P2Y₁₂ receptors play an important role in arterial thrombosis by stimulating thrombus growth. Both irreversibly (clopidogrel) and reversibly binding (ticagrelor, AZD6140) P2Y₁₂ antagonists are clinically used for restricted periods, but possible differences in platelet function recovery after drug cessation have not been investigated. We treated WKY rats with a single, high dose of 200 mg/kg clopidogrel or 40 mg/kg ticagrelor. Blood was collected at different time points after treatment. Flow cytometry confirmed full platelet protection against ADP-induced αIIbβ₃ activation shortly after clopidogrel or ticagrelor treatment. At later time points after clopidogrel treatment, a subpopulation of juvenile platelets appeared that was fully responsive to ADP. Addition of ticagrelor to clopidogrel-treated blood reduced αIIbβ₃ activation of the unprotected platelets. In contrast, at later time points after ticagrelor treatment, all platelets gradually lost their protection against ADP activation. Perfusion experiments showed abolishment of thrombus formation shortly after clopidogrel or ticagrelor treatment. Thrombus formation on collagen was determined under high shear flow conditions. At later time points, large thrombi formed in the clopidogrel but not in the ticagrelor group, and unprotected, juvenile platelets preferentially incorporated into the formed thrombi. However, platelets from both groups were still similarly reduced in assays of whole blood aggregation. Conclusively, recovery of rat platelet function after ticagrelor differs mechanistically from that after clopidogrel. This difference is masked by conventional platelet aggregation methods, but is revealed by thrombus formation measurement under flow. Juvenile platelets formed at later time points after clopidogrel treatment promoted thrombus formation.

Mechanisms Leading to Increased Vasodilator Responses to Calcitonin-Gene-Related Peptide in Mesenteric Resistance Arteries of Early Pregnant Rats

The objective of this study was to explore the mechanism responsible for the higher relaxing responses of mesenteric arteries to calcitonin-gene-related peptide (CGRP) in pregnancy. We performed myograph and ligand binding studies to determine the role of matrix metalloproteinase-2 (MMP-2) and CGRP receptor density. MMP activity was manipulated in isolated arteries by exposing them to the blocking effects of doxycycline. Vascular activity of MMP-2 was studied by gelatin zymography, and CGRP receptor density was determined by ligand binding analysis. Compared to nonpregnant rats, CGRP elicited stronger arterial relaxation in pregnant rats. The latter effect was neither accompanied by a change in relaxing responses to direct activation of adenylyl cyclase by forskolin nor by a change in the response to stimulation of G-protein-coupled adrenergic receptors by isoproterenol. Doxycycline did not affect the stronger arterial relaxation in pregnancy in spite of the observed more than threefold higher arterial MMP-2 activity. Density of binding sites for [125I]CGRP in arteries from pregnant rats (64 ± 14 fmol/mg protein) and from virgin rats (54 ± 5 fmol/mg protein) were comparable. The results of this study provide evidence for increased coupling of CGRP receptors to adenylyl cyclase in early pregnancy.

Endothelin-1 and endothelin-2 initiate and maintain contractile responses by different mechanisms in rat mesenteric and cerebral arteries

Endothelin (ET)‐1 and ET‐2 cause potent long‐lasting vasoconstrictions by tight binding to smooth muscle ETA receptors. We tested the hypotheses that different mechanisms mediate initiation and maintenance of arterial contractile responses to ET‐1 and ET‐2 and that this differs among vascular beds.