Ahj Danser

Effects of ionotropic glutamate receptor antagonists on rat dural artery diameter in an intravital microscopy model

Background and purpose:u2002 During migraine, trigeminal nerves may release calcitonin gene‐related peptide (CGRP), inducing cranial vasodilatation and central nociception; hence, trigeminal inhibition or blockade of craniovascular CGRP receptors may prevent this vasodilatation and abort migraine headache. Several preclinical studies have shown that glutamate receptor antagonists affect the pathophysiology of migraine. This study investigated whether antagonists of NMDA (ketamine and MK801), AMPA (GYKI52466) and kainate (LY466195) glutamate receptors affected dural vasodilatation induced by α‐CGRP, capsaicin and periarterial electrical stimulation in rats, using intravital microscopy.

THE HANDLE REGION PEPTIDE (HRP) COUNTERACTS THE BENEFICIAL EFFECTS OF THE RENIN INHIBITOR ALISKIREN IN SPONTANEOUSLY HYPERTENSIVE RATS (SHR): 5C.01

Objective: To investigate whether the putative (pro)renin receptor blocker, the handle region peptide (HRP), affects the blood pressure lowering and end-organ protective effects of the renin-inhibitor aliskiren in spontaneously hypertensive rats (SHR). Design and Method: Male SHR were implanted with telemetry transmitters to monitor heart rate (HR) and mean arterial pressure (MAP). After a 2-week recovery period, vehicle, the renin inhibitor (i) aliskiren 100 mg/kg.day, the handle region peptide (HRP) 1 mg/kg.day or a combination of HRP and aliskiren were infused for 3 weeks using an osmotic minipump. Vehicle-treated WKY rats served as controls. Subsequently, the heart was removed to study coronary function (using the Langendorff model). Results: Vehicle-treated SHR displayed a higher baseline MAP (146 ± 3 vs. 102 ± 2 mmHg) and lower baseline HR (326 ± 4 vs. 366 ± 2 beats/min) than WKY. HRP did not affect MAP whereas aliskiren and HRP+aliskiren lowered MAP (by maximally 29 ± 2 and 20 ± 1 mmHg respectively) without affecting HR. Aliskiren significantly reduced MAP throughout the 3-week infusion period whereas the blood pressure lowering effect of HRP+aliskiren returned to baseline within 2-weeks of treatment. The endothelium-dependent vasodilator bradykinin increased coronary flow (CF) in WKY and SHR by maximally 180 ± 25% and 82 ± 13% (P < 0.05). 3-week treatment with aliskiren improved the response to bradykinin in SHR to WKY level whereas the response was unaltered after treatment with HRP or HRP+aliskiren. Ang II decreased CF in WKY and SHR by maximally 42 ± 5% and 75 ± 3% (P < 0.05). 3-week treatment with aliskiren diminished the enhanced Ang II response in SHR whereas the response was unaltered after treatment with HRP or HRP+aliskiren. Conclusions: HRP counteracts the beneficial effects of aliskiren on blood pressure and coronary endothelial function.

A human capsaicin model to quantitatively assess salivary CGRP secretion

Background Capsaicin induces the release of calcitonin gene-related peptide (CGRP) via the transient receptor potential channel V1 (TRPV1). The CGRP response after capsaicin application on the tongue might reflect the “activation state” of the trigeminal nerve, since trigeminal CGRP-containing vesicles are depleted on capsaicin application. We tested (i) the quantitative CGRP response after oral capsaicin application; (ii) the optimal concentration of red chili homogenate; and (iii) the day-to-day variability in this response. Methods Saliva was collected for two consecutive days after oral application of eight capsaicin dilutions (red chili homogenates) of increasing concentrations in 13 healthy individuals. Effects of homogenate concentration were assessed. Consecutively, saliva was sampled after application of vehicle and undiluted homogenates. Results CGRP secretion (pg/ml) increased dose-dependently with homogenate concentration (pu2009<u20090.001). CGRP levels were highest after application of nondiluted homogenate (vs. baseline: 13.3 (5.0) vs. 9.7 (2.9); pu2009=u20090.003, as was total CGRP secretion in five minutes (pg) with undiluted (vs. baseline): 89.2 (44.1) vs. 14.1 (2.8); pu2009<u20090.001. The dose-dependent response in CGRP was not affected by day (pu2009=u20090.14) or day*concentration (pu2009=u20090.60). Increase in CGRP (undiluted – baseline; pg/ml) did not differ between measurements on dose-finding (pu2009=u20090.67) and follow-up days (pu2009=u20090.46). Conclusion Oral application of red chili homogenate is well tolerated and causes a dose-dependent CGRP release in saliva, without day-to-day effects in this response. This model could be used to noninvasively study the activation state of the trigeminal nerve innervating salivary glands.

Influence of varying estrogen levels on trigeminal CGRP release in healthy women

Migraine is 2-3 times more prevalent in women than in men, with frequent perimenstrual attacks. TRPV1 receptors on sensory nerve endings of the trigeminal track are important in mediating migraine attacks by releasing the vasodilator calcitonin gene-related peptide (CGRP). Variation in estrogen levels during the menstrual cycle may have an influence on the sensitivity of the TRPV1 receptor or on the amount of CGRP in perivascular nerve terminals and hence on CGRP release. Capsaicin, the active ingredient of hot chili peppers, stimulates the TRPV1 receptor and causes CGRP-dependent vasodilatation [1]. We set up a model to study trigeminal CGRP release in humans. We compared the vasodilator effects of capsaicin application and electrical stimulation on the forehead skin. Healthy women, not using hormonal contraceptives (age: 18-45, n=14), were studied with a Laser Doppler Imager on day 19-21 of their menstrual cycle and on day 1-2 of their menstruation. A 0.2 mM and a 20 mM capsaicin solution were applied to the skin. In addition, iontophoresis of saline was performed as a TRPV1-independent stimulus. Increases in dermal blood-flow (DBF) were measured. Blood samples were collected to measure estrogen levels. We measured higher DBF responses to application of 0.2 mM capsaicin (Max:226±34 a.u.) and 20 mM capsaicin (Max:507±39 a.u.) during day 1-2 (low estrogen levels: 15±2 pg/ml) of the menstruation, than during day 19-21 (high estrogen levels: 67±9 pg/ml) of the menstrual cycle (Max:176±34 a.u. and 432±33 a.u. for 0.2 mM and 20 mM, respectively, P<0.05). There was no difference in DBF responses to electrical stimulation of the forehead skin, suggesting that the observed changes are related to the sensitivity of the TRPV1 receptor. Our results indicate an influence of variation in estrogen levels on trigeminal CGRP release, with the highest reactivity observed around the menstruation when estrogen levels are low. This mechanism may, at least partly, explain the high incidence of migraine attacks during the perimenstrual period.

Endothelin-converting-enzyme 1 inhibition and CGRP receptor recycling in human coronary and middle meningeal arteries

Although best known for its role in the conversion of big endothelin to endothelin-1, endothelin-converting enzyme 1 (ECE-1) also regulates the resensitization of certain neuropeptide receptors, including the receptor for calcitonin gene-related peptide (CGRP) ( Padilla et al., 2007 ). We investigated the role of ECE-1 in the resensitization of responses to CGRP in human coronary (HCA) and middle meningeal (HMA) arteries using the potent and selective ECE-1 inhibitor, SM-19712. Segments of HCA (O 0.5–1 mm) and HMA (O 0.5–1 mm) were mounted in organ baths and concentration response curves (CRCs) to CGRP were constructed in the absence or presence of the ECE-1 inhibitor SM-19712. After the first CRC to CGRP the segments were washed and after 30-45 minutes a second CRC was constructed in the absence or presence of SM-19712 to investigate ECE-1-dependent CGRP resensitization. Furthermore, CRCs to big endothelin were constructed in the presence or absence of SM-19712. In both HCA and HMA, no differences were seen between the initial responses to CGRP in the absence or presence of SM-19712 (HCA Emax+SM19712 94±8%, Emax–SM19712 93±5%; pEC50+SM19712 9.1±0.2, pEC50-SM19712 9.2±0.1; HMA Emax+SM19712 72±7%, Emax–SM19712 59±7%; pEC50+SM19712 8.5±0.4, pEC50-SM19712 8.1±0.8), as well as between the second CRCs to CGRP in the absence or presence of SM-19712 (HCA Emax+SM19712 110±13%, Emax–SM19712 78±22%; pEC50+SM19712 7.5±0.5, pEC50-SM19712 7.9±0.01; HMA Emax+SM19712 38±13%, Emax–SM19712 44±1%; pEC50+SM19712 8.6±0.5, pEC50-SM19712 7.8±0.9). Furthermore, contractions to big endothelin were not different in the absence or presence of SM-19712 in either HCA (Emax+SM19712 118±14%, Emax-SM19712 115±32%; pEC50+SM19712 6.0±0.5, pEC50-SM19712 6.9±0.2) or HMA (Emax+SM19712 121±1%, Emax–SM19712 147±19%; pEC50–SM19712 7.4±0.4, pEC50+SM19712 7.0±0.8). Our results indicate that ECE-1 does not regulate the resensitization of CGRP responses in HCA and HMA.

Sumatriptan and dihydroergotamine in proximal and distal human isolated coronary arteries

Sumatriptan and dihydroergotamine (DHE) are both 5-HT receptor agonists and two of the most widely used drugs for the acute treatment of migraine. These drugs are contra-indicated in people with cardiovascular disease because of their vasoconstricting properties, as has previously been assessed in proximal coronary arteries. The effect of DHE in distal coronary arteries, however, has never been reported, although smaller coronary arteries might also account for angina-like symptoms, especially in women. The aim of this study was to compare the contractile effects of sumatriptan and DHE in proximal and distal human coronary arteries, and to relate our findings to the plasma concentrations obtained in clinical practice. Segments of proximal (O 3-5 mm) and distal (O 0.5–1 mm) human isolated coronary arteries were mounted in organ baths and concentration response curves for sumatriptan and DHE were constructed. In proximal coronary artery segments, maximal contractions to sumatriptan (16+/-18% of contraction to 100 mM KCl) and DHE (5+/-4%) were not significantly different. In contrast, in distal coronary arteries, the contractile responses to sumatriptan (18+/-11%) were significantly larger than those to DHE (4+/-2%). At clinically relevant concentrations (Cmax after different formulations), contractions to both sumatriptan and DHE in proximal as well as distal coronary arteries were below 6%. Thus, our results indicate that coronary artery contractions to DHE in distal coronary artery are smaller than those to sumatriptan, although in the clinical situation both drugs are likely to induce only a slight contraction.

17β-estradiol and methylation of migraine-related genes

Migraine is much more common in women than in men, especially during the reproductive part of their lives. Fluctuations in the female hormone 17β-estradiol throughout the menstrual cycle are thought to be an important factor in causing migraine attacks in women. Although it has been shown that 17β-estradiol can change vascular sensitivity to CGRP, it is still unknown how 17β-estradiol causes these effects. GWAS studies have identified genes that may be relevant for migraine, but an influence of environmental factors is likely. Interestingly, the prophylactic effectivity of valproate, a DNA methylation inhibitor, may point to the involvement of epigenetic mechanisms in migraine. 17β-estradiol is known to be involved in epigenetic mechanisms and recently it was shown that CGRP can be regulated via epigenetic mechanisms. Therefore, the aim of this study was to investigate the role of 17β-estradiol in the methylation of migraine-related genes. Female SD rats were ovariectomized (ovx) and treated with 17β-estradiol or placebo pellets. DNA was isolated from blood, aorta, dura mater, trigeminal caudal nucleus and trigeminal ganglion. PCR was performed for 10 migraine- and female hormone-related genes (MTHFR, eNOS, ESR1, GPER, CGRP, USF1, USF2, RAMP1, CRCP and CRLR) and DNA methylation was assessed through bisulfite treatment and sequenom mass spectrometry. No difference in DNA methylation was seen between control animals, ovx placebo-treated or ovx 17β-estradiol-treated animals for the MTHFR, GPER, eNOS, CGRP, USF1 and USF2 genes. Tissue-specific differences in methylation were seen for the ESR1, CRCP, eNOS and CRLR genes. Remarkably, our results point to an increase in methylation of the CRCP gene in the trigeminal caudal nucleus in the 17β-estradiol-treated animals. These esults indicate a possible epigenetic regulatory mechanism for one part of the CGRP receptor in the trigeminal caudal nucleus.

THE VEGF-RECEPTOR INHIBITOR SUNITINIB CAUSES A PREECLAMPSIA-LIKE SYNDROME IN RATS: 2D.01

Objectives: Inhibition of angiogenesis by means of blocking vascular endothelial growth factor (VEGF)-mediated signaling is an established treatment for various forms of cancer, but, unexpectedly, this therapy is associated with hypertension and renal toxicity in a substantial proportion of patients. To obtain more insight in the underlying mechanism, the effect of the VEGF-receptor antagonist sunitinib on blood pressure (BP) and renal function and histology were investigated in male Wistar-Kyoto rats. Methods: In 16 rats BP and heart rate (HR) were monitored telemetrically during administration of sunitinib by oral gavage until a plateau in BP rise occurred, at which time animals were euthanized and blood and kidneys were collected. Control rats (n = 6) were administered vehicle. In a second experiment (n = 7), sunitinib, after reaching the BP-plateau, was withdrawn for 11 days to assess reversibility of adverse effects. In both experiments 24-hour urine samples were collected for measurement of proteinuria. Results: A plateau in BP rise was found after 6 days of treatment. At that time mean arterial BP had increased from 98.3 ± 0.3 mmHg to 126 ± 1.5 mmHg and HR had decreased from 319.3 ± 1.4 bpm to 271.6 ± 3.0 bpm (both p < 0.001), while BP and HR did not change in the control group. Serum creatinine, proteinuria and plasma endothelin-1 (ET-1) concentration increased 3-fold in the sunitinib-group. Histological evaluation of the kidneys revealed marked endothelial and epithelial cell swelling, intra-epithelial protein droplets and effacement and fusioning of podocyte foot processes. After sunitinib-withdrawal for 11 days BP, HR, ET-1, proteinuria and renal histological abnormalities all normalized, but serum creatinine remained elevated. Conclusions: VEGF receptor inhibition with sunitinib induces a reversible rise in BP, ET-1 levels, proteinuria and marked renal histological abnormalities. These findings are comparable to features of preeclampsia, a pregnancy-related disorder characterized by hypertension and proteinuria as well as increased plasma ET-1 concentrations. Evidence suggests that preeclampsia is caused by increased placental production of sFlt-1, the extracellular domain of the VEGF-receptor-1, which binds circulating VEGF thereby inhibiting VEGF-signaling.

ADMINISTRATION OF SUNITINIB, AN ORAL ANGIOGENESIS INHIBITOR, IS ASSOCIATED WITH HYPERTENSION, BUT NOT WITH STRUCTURAL CARDIAC CHANGES OR CARDIOMYOCYTE MITOCHONDRIAL DYSFUNCTION: PP.22.363

Objectives: Inhibition of angiogenesis by blocking vascular endothelial growth factor (VEGF)-mediated signaling is an established treatment for various forms of cancer. This therapy is frequently associated with hypertension, but may also cause cardiac toxicity, mainly a decreased left ventricular ejection fraction (LVEF). Cardiomyocyte hypertrophy and changes in cardiac mitochondrial structure (swelling) have been reported in mice, suggesting that impaired adenosine triphosphate (ATP) generation secondary to mitochondrial dysfunction underlies the decrease in LVEF. We aimed to assess the effects of sunitinib on blood pressure (BP), cardiac structure and mitochondrial function in an ex-vivo rodent model. Methods: Normotensive male Wistar-Kyoto rats were administered sunitinib (n = 10) or vehicle (n = 6) by oral gavage and BP and heart rate (HR) were monitored telemetrically until a plateau in BP occurred at which time animals were euthanized. Blood and hearts were collected for determination of brain natriuretic peptide (BNP)-45 levels, heart weight, cardiomyocyte size and isolation of cardiomyocyte mitochondria for measurement of complex I- and II-dependent ATP production. Results: A plateau in BP rise was found after 6 days of treatment. At that time, mean arterial BP had increased from 98.3 ± 0.3mmHg to 126 ± 1.5mmHg and HR had decreased from 319.3 ± 1.4bpm to 271.6 ± 3.0bpm (both p < 0.001), while BP and HR did not change in the control group. Circulating BNP-45 levels were almost 2-fold (163.9 ± 33.9ng/ml vs 92.7 ± 15.0ng/ml; p = 0.03) and serum creatinine levels (28.8 ± 6.4 vs 8.0 ± 2.7 μmol/l; p = 0.03) almost threefold higher in the sunitinib compared to the control group. Heart weight-to-body weight ratio and cardiomyocyte area were not different between groups. Complex I-dependent ATP production between the sunitinib and control group (659.7 ± 88.1; vs 1028 ± 278 nmol/mg mitochondrial protein/30 min; p = 0.26) and complex II-dependent ATP-production (955.6 ± 93.3 vs 1409 ± 255 nmol/mg mitochondrial protein/30 min; p = 0.15) did not differ. Conclusions: Although circulating BNP-45 levels were increased in sunitinib-administered rats, no evidence for structural cardiac abnormalities or impaired cardiomyocyte mitochondrial ATP production was found. The rise in BNP-45 might be a consequence of the BP-rise and the decrease in renal function.

ENDOTHELIAL DYSFUNCTION AND DEFECTIVE SMOOTH MUSCLE CONTRACTILITY IN ANEURYSMAL FIBULIN-4 DEFICIENT MICE: 8A.05

Objective: Using Fibulin-4 knockdown mice (Fibulin-4R/R), we previously showed that the dosage of Fibulin-4 can determine the severity of aneurysm formation. Strikingly, even a modest reduction in expression of Fibulin-4 in the heterozygous Fibulin-4+/R mice occasionally resulted in small aneurysm formation. Aortic pulse pressure was 2 to 3-fold higher in Fibulin-4R/R mice, resulting from increased aortic stiffness. Here, we analyzed the altered biological pathways in aneurismal disease at the molecular, cellular and functional level. Methods: Aorta transcriptome changes of Fibulin-4+/R and Fibulin-4R/R aortas were performed using ANOVA and Ingenuity Pathway Analysis. Histology was applied to verify cellular abnormalities. Functional analysis occurred by measuring isometric forces of thoracic aortas in vitro and aortic pressures with a pressure transducer catheter in vivo. Results: Ingenuity Pathway Analysis identified three major dysregulated pathways, including TGFβ signaling, immune response and specifically Ca2+ signaling genes involved in the maintenance of contractile function. Histological analysis and á-smooth muscle actin immunostaining showed loss of smooth muscle cells (SMCs), which coincided with cartilage bone formation, already in Fibulin-4+/R animals. Severe loss of SMCs and increase of extracellular matrix depositions resulted in overall thickening of the aortic wall in Fibulin-4R/R mice. In addition, Fibulin-4R/R mice showed increased endothelial damage, endothelial cellular proliferation, as evidenced by BrdU labeling and thrombus formation, indicative for endothelial dysfunction. In vitro, maximum contractility was determined for phenylephrine and compared to KCl (100 mM). KCl responses showed a reduced contractile capacity of ascending aortas in Fibulin-4R/R mice correlating with a reduction of SMCs in this area. In descending aortas, phenylephrine-induced contractility decreased from 54 ± 7% in wildtypes to 13 ± 2% in Fibulin-4R/R mice. Endothelium-dependent vasorelaxation by acetylcholine, following preconstruction with U46619, was strongly reduced in Fibulin-4R/R mice. Finally, with reducing expression of fibulin-4, diastolic blood pressure decreased from 63 ± 3 in Fibulin-4+/+ to 60 ± 5 in Fibulin-4+/R and 41 ± 7 in Fibulin-4R/R mice. Conclusion: Our results uncover a role for Fibulin-4 in the maintenance of SMC contractile function and endothelial responsiveness.