bluuebunny/crossref_metadata_2025 huggingface dataset:
Administration of Control-Released Hepatocyte Growth Factor Enhances the Efficacy of Skeletal Myoblast Transplantation in Rat Infarcted Hearts by Greatly Increasing Both Quantity and Quality of the Graft
Authors: Tambara Keiichi , Premaratne Goditha U. , Sakaguchi Genichi , Kanemitsu Naoki , Lin Xue , Nakajima Hiroyuki , Sakakibara Yutaka , Kimura Yu , Yamamoto Masaya , Tabata Yasuhiko , Ikeda Tadashi , Komeda Masashi
Published: July 2021

Background—

We investigated whether simultaneous administration of control-released hepatocyte growth factor (HGF) enhances the efficacy of skeletal myoblast (SM) transplantation (Tx) through its antiapoptotic, angiogenic, and antifibrotic effects in myocardial infarction (MI).

Methods and Results—

Forty-eight Lewis rats with chronic MI were divided into 4 groups. In Group I (n=14), neonatal SMs (5×10
6
) were transplanted in the MI area with a gelatin sheet incorporating 40 μg (1 g/L) of HGF applied. Group II (n=14) had SM Tx and placement of a saline sheet. Groups III (n=10) and IV (n=10) had culture medium injection plus HGF and saline sheet application, respectively. Four rats each from Groups I and II were sacrificed at day 1 for TUNEL assay on donor SMs. The percentage of TUNEL-positive donor cells was much lower in Group I than in Group II (
P
<0.05). At 4 weeks, in Group I, left ventricular diastolic dimension was smallest in echocardiography, end-systolic elastance was highest, and τ was the lowest (both
P
<0.0005 in ANOVA) in cardiac catheterization. Vascular density inside the graft was higher in Group I than in Group II (
P
<0.0001). The percentage of fibrotic area inside the graft was smaller in Group I than in Group II (
P
<0.001). The graft volume as estimated by fast skeletal myosin heavy chain-positive areas was ≈7-fold larger in Group I than in Group II (
P
<0.0001).

Conclusions—

In SM Tx, HGF can greatly increase the graft volume and vascularity and reduce fibrosis inside the graft, which enhances the efficacy of SM Tx to infarcted hearts.
Regulation of Vascular Smooth Muscle Cell Proliferation by Cyclooxygenase-2-Derived Prostanoids.
Authors: Ferreri Nicholas R , Young Wilson , Haider Asifa , Li Irene
Published: July 2021

10

Tumor necrosis factor-alpha (TNF, 1 nM) and angiotensin II (Ang II, 100 nM) increased the DNA content of vascular smooth muscle cell (vsmc) derived from rat thoracic aorta (control: 3.7 ± 0.2, TNF: 5.7 ± 0.7, Ang II: 6.2 ± 0.5 arbitrary units; p<0.001). Cell number also increased in response to these molecules, indicative of a direct correlation between DNA content and cellular proliferation (control: 3.1 ± 0.1, Ang II: 4.4 ± 0.6 x 10
4
cells; p<0.001; control: 4.1 ± 0.3, TNF: 7.8 ± 0.4 x 10
4
cells; p<0.001). Increases in DNA content and cell number were preceded by increases in COX-2 mRNA accumulation and protein expression, and accumulation of COX-2 mRNA was prevented by pretreatment with losartan (1 μM) but not PD123319 (1 μM). COX-1 mRNA accumulation did not change after challenge with either TNF or Ang II for 0.5, 2, and 6 hr. Two different COX-2-selective inhibitors, NS-398 (0.1 μM) and nimesulide (1 μM) reduced TNF- and Ang II-mediated increases in DNA content and cell number by more than 95%. Prostacyclin (PGI
2
) synthesis was increased approximately 3- and 8-fold, in response to TNF or Ang II, respectively, (control: 200 ± 35, TNF: 705 ± 28 pg 6-keto PGF1α/ml; p<0.001; control: 190 ± 45, Ang II: 1645 ± 120 pg 6-keto PGF1α/ml, p<0.001. These increases were completely inhibited by either NS-398 or nimesulide. Basal PGI
2
levels also were significantly reduced by these inhibitors, suggesting a tight coupling of COX-2 to PGI
2
synthesis in these cells. TNF and Ang II induced modest increases in vsmc TXA
2
synthesis (control: 33 ± 3, TNF: 49 ± 3, Ang II: 46 ± 6; p<0.001). The increases were abolished by NS-398 and nimesulide, although basal TXA
2
synthesis was not affected. Despite the relatively small increases in TXA
2
synthesis, the TXA
2
receptor antagonist, BMS 180,291 (1 μM) inhibited the proliferative response to Ang II and TNF by >95%. These data suggest that a COX-2-dependent increase in TXA
2
synthesis may contribute to vsmc hyperplasia in pathophysiological conditions associated with elevated levels of either TNF or Ang II. In contrast, PGI
2
may contribute to a compensatory mechanism to limit the proliferative effects of cytokines such as TNF or vasoactive peptides such as Ang II.
Neuronal Control of Salt Sensitivity: Role of Sensory and Sympathetic Nerves
Authors: Wang Donna H , Wu Wei
Published: July 2021

18

Neonatal degeneration of capsaicin-sensitive sensory nerves renders a rat responsive to a salt load with an increase in blood pressure and a decrease in natriuretic response (Wang, et al, Hypertension 1998;32:649-653). To test the hypothesis that the sympathetic nervous system contributes to the development of salt sensitive hypertension induced by sensory denervation, newborn Wistar rats were given 50mg/kg capsaicin and/or 80mg/kg guanethidine s.c.. Control rats were treated with vehicle. After the weaning period, male rats were grouped as the following and given high sodium diet (4%) for 2 weeks: capsaicin and guanethidine co-administration (CAP-GUA), capsaicin only (CAP), guanethidine only (GUA), and vehicle control (CON). Norepinephrine concentrations in the atrium were significantly lower in CAP-GUA and GUA than in CON rats (p<0.05). Twenty-four hour urine and sodium excretion was significantly lower in CAP than in CAP-GUA, GUA and CON rats (p<0.05). Mean arterial pressure (mmHg) was significantly higher in CAP (180±10) than in CAP-GUA (106±1), GUA (133±5), and CON (122±3) rats (p<0.05). Thus, sympathectomy restores the natriuretic response to a high salt intake and prevents the development of salt sensitive hypertension induced by sensory denervation. These data indicate that sensory nerves counterbalances the pro-hypertensive effect of the sympathetic nerves to maintain blood pressure within normal range during salt loading.
Oxidative Stress in a Rat Model of Obesity-Induced Hypertension
Authors: Dobrian Anca D , Davies Michael J , Schriver Suzanne D , Lauterio Thomas J , Prewitt Russell L
Published: July 2021

45

The mechanisms underlying the development of hypertension in obesity are not yet fully understood. We recently reported the development of hypertension in a rat model of diet-induced obesity (Dobrian AD et al Hypertension 2000;35: in press). When Sprague-Dawley rats (n=60) are fed a moderately high fat diet(32kcal% fat) for 10-16 weeks, about half of them develop obesity (obesity-prone (OP) and mild hypertension (153±3.4 mmHg systolic pressure), whereas the other half (obesity-resistant, OR) maintains body weight equivalent to low fat control (C) and are normotensive. We examined the potential role of oxidative stress in the development of hypertension in this model. Lipid peroxides measured as thiobarbituric acid reactive substances (TBARS) are showing a significant increase in the LDL fraction of OP rats (2.8±0.32 nmol MDA/mg protein) compared to OR and C (0.9±0.3 nmol MDA/mg protein). Also, aortic and kidney TBARS showed a significant 3- and 5- fold increase in OP rats after 16 weeks of diet (3.3±0.44 aorta and 6.7±0.52 nmol MDA/mg protein kidney) vs OR. In addition, superoxide generation by aortic rings, measured by lucigenin (25μM) luminescence showed a 2-fold increase in the OP group (4325.6±174 RLU/15 min/mg DNA X10
5
) compared to both OR and C. Plasma renin activity was 2-fold increased in OP vs both OR and control groups.The urine nitrate/nitrite measured by LDH colorimetric method showed a 1.8-fold decrease in OP rats (2.4±0.17 μmoles) compared to OR. A similar 1.6-fold decrease was found for plasma nitrate/nitrite in OP rats vs OR and C. However, eNOS expression assessed by semiquantitative RT-PCR showed a strong increase in the OP rats vs OR and controls in both kidney cortex(eNOS/β-actin ratio of 0.78±0.21 in OP vs 0.32±0.16 in OR)and medulla(0.86±0.18 in OP vs 0.36±0.14 in OR), suggesting a possible shift toward superoxide vs NO production by the eNOS enzyme.Also, eNOS expression was increased ∼4.8-fold in the thoracic aorta of OP compared to OR rats. Collectively, data show a decreased NO production in OP animals, due in part to the increased oxidative stress, possibly generated by the activation of renin-angiotensin system and increased eNOS expression.
Changes in Vsmc Phenotypes in Ang Ii-Dependent Hypertension of TGR (mREN2) 27 (Tgr) Transgenic Rats
Authors: Rossi Gian Paolo P , Sartore Saverio , Colonna Stefania , Sacchetto Alfredo , Rizzoni Damiano , Mazzocchi Giuseppina , Belloni Anna S , Nussdorfer Gastone G , Pessina Achille C
Published: July 2021

57

Arterial hypertension is associated with vascular smooth muscle cells (VSMC) phenotypic differentiation, but the role of Ang II and ET-1 is still unclear. Thus, we investigated the changes of VSMC phenotypes in Ang II-dependent hypertension and the role of ET-1 and its A receptor (ET
A
). Four-week old heterozygote male TGR rats (n=24) were body weight (BW)- and blood pressure (BP)-matched and randomly allocated to receive orally a placebo (Group P), the mixed ET
A
/ET
B
antagonist Bosentan (100 mg/Kg, Group B) the Ang II AT-1 specific receptor antagonist Irbesartan (50 mg/Kg BW, Group I) or the ET
A
selective antagonist BMS-182874 (52 mg/Kg BW, Group BMS). After 4-wk of treatments, during which BW and BP were measured weekly, animals were euthanized; the iliac artery and mesenteric arterioles were collected. In the latter the structural changes were assessed with a myograph. Immunohistochemistry with a panel of different antibodies specific for ET-1, ET
A
, smooth muscle (SM) myosin, SM actin, SM 22, myosin heavy chains Apla 22 and fibronectin EIIIA was carried out. The fetal, neonatal and adult aorta from normotensive Sprague-Dawley rats were studied as control. Compared to all other groups, Group I rats showed significantly (p<0.001) lower systolic BP (161±8 mmHg, vs 269±23 Group P; vs 254±21 Group BMS), LV weight (2.28±0.15 mg/g BW vs 3.71±0.26, 3.38±0.27 and 3.96±0.51), and normalized media thickness of the mesenteric arterioles (22.3±0.6 μm vs 25.3±0.5, 25.5±0.7 and 24.1±1.5). Hypertension, LVH and medial arterioles hypertrophy in group P TGR were paralleled by a shift of VSMC toward a fetal phenotype in the iliac artery, despite no change in the expression of both irET-1 and ET
A
. The VSMC phenotypic shift was prevented by both irbesartan and Bosentan, but not by the ET
A
-selective antagonist BM 182874. Thus, Ang II-dependent hypertension of TGR is associated with both vascular hypertrophy and a shift of VSMC toward a fetal phenotype, which occurs through AT-1- and ET
B
- but not ET
A
-mediated mechanisms.
Aldosterone Receptor Antagonism Normalizes Vascular Function in 11β-Hydroxysteroid Dehydrogenase Deficient Hypertension
Authors: Quaschning Thomas , Ruschitzka Frank T , Lunt Carolyn Mb , Niggli Bernhard , Shaw Sidney , Luscher Thomas Felix
Published: July 2021

76

Background: The enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD2) provides mineralocorticoid receptor specificity for aldosterone by metabolising glucocorticoids to their receptor inactive 11-dehydro derivatives. The present study investigated the effect of the aldosterone receptor antagonist spironolactone on endothelial function in liquorrhice induced hypertension. Methods: Glycyrrhizic acid (GA), a recognised inhibitor of 11β-HSD2 was supplemented to the drinking water (3g/L) of Wistar Kyoto rats over a period of 21 days. From day 8 to 21 spirolonoactone (5.8±0.6 mg/kg/d) or placebo was added to chow (n=7/group). Endothelium-dependent and -independent vascular function was assessed as relaxation of preconstricted aortic rings to acetylcholine (10
-10
-10
-5
mol/L) or sodium nitroprusside (10
-10
-10
-5
mol/L). Furthermore aortic eNOS protein content, nitrate tissue levels, endothelin-1 (ET-1) protein levels were determined. Results: GA application increased SBP to 185±9 mmHg vs 142±8 mmHg in control animals, p<0.01). In the GA group endothelium-dependent relaxation was impaired as compared to controls (73±6% vs 99±5% of norepinephrine 3x10
-7
mol/L), whereas endothelium independent relaxation remained unchanged. In the aorta of 11β-HSD2 deficient rats, eNOS protein content and nitrate tissue levels (1114±128 vs 518±77μg/g protein, p<0.05) decreased. In contrast, aortic endothelin-1 (ET-1) protein levels were enhanced by GA. (308±38 vs 497±47 pg/mg tissue, p<0.05). Treatment with spironolactone normalized blood pressure in animals on GA (142±9 mmHg vs 189±8 mmHg in the placebo group; p<0.01) and restored endothelium-dependent relaxation (96±3%, p<0.01 vs placebo). Spironolactone furthermore blunted the decrease in vascular eNOS protein content and nitrate tissue levels as well as the elevation of ET-1 protein levels. Conclusion: In 11β-HSD2-deficient hypertension, aldosterone receptor antagonism normalizes blood pressure, prevents up-regulation of vascular ET-1 and restores NO-mediated endothelial dysfunction and therefore may advance as a novel therapeutic approach.
Angiotensin II Increases Vesicular Trafficking in Live Noradrenergic Brain Neurons
Authors: Raizada Mohan K , Wang Xianyu Wang , Yang Hong
Published: July 2021

P1

Angiotensin II (Ang II) interacts with the AT
1
receptor subtype and stimulates turnover of norepinephrine (NE) by regulating its synthesis, release, and uptake. The cellular mechanism by which Ang II regulates this neurotransmission is not well understood. The aim of this study was to test the hypothesis that Ang II increases vesicular trafficking to increase synaptic release of NE. cDNA encoding full length rat dopamine beta-hydroxylase (DβH) fused to Green Fluorescence Protein (GFP) was cloned in pCl-Neo vector under the control of CMV promoter and used to tag synaptic vesicles. Transfection of cells with this plasmid resulted in the expression of green fluorescence and GFP-DβH fusion protein which was functional as judged by an increase in DβH activity. Transfected neurons expressed bright green fluorescence representing GFP-DβH that was predominantly localized in the cell soma. Few neurites also displayed the fluorescence. Real time confocal microscopic examination of live neurons indicated that Ang II caused a time-dependent increase in both the intensity and the number of neurites depicting green fluorescence. An increase in GFP-DβH in the neurites became apparent within one minute and persisted for 15-20 minutes. A 2.5 fold increase in the GFP-DβH positive neurites was observed by 100 nM Aug II in 15 min [control, 2±0.5 (n=20) versus Ang II treatment, 5±0.4 (n=25)]. Ang II-induced GFP-DβH redistribution was punctate, co-localized with synaptophysin and was blocked by losartan, and not by PD123319. Release of Ang II induced NE paralleled its effect on GFP-DβH distribution. PMA treatment caused translocation of GFP-DβH in a fashion comparable with Ang II. In addition, inactivation of protein kinase C by a prolonged incubation of neurons with PMA, completely blocked the translocation of GFP-DβH from the cell body to neurites. These observations demonstrate that Ang II stimulates trafficking of NE vesicles by a PKC-dependent mechanism. Thus, an increased trafficking of NE containing vesicles from the neuronal cell body to the neurites may be key to Ang II stimulated release of NE from a noradrenergic neuron.
The Involvement of Oxidation-Sensitive Mechanisms in the Pathophysiology of Renovascular Hypertension
Authors: Lerman Lilach O , Nath Karl A , Rodriguez-Porcel Martin G , Schwartz Robert S , Romero Carlos J
Published: July 2021

P16

The mechanisms responsible for the maintenance of chronic renovascular hypertension (RVH) remain undefined. Excess systemic or local generation of angiotensin II (AII) may lead to enhanced release of reactive oxygen radical species, vasoconstrictor activity, and parenchymal injury. To examine the potential involvement of oxidation-sensitive mechanisms, blood samples were collected from 7 RVH pigs with unilateral renal artery stenosis (achieved by a local-irritant stent) 5 and 10 weeks after induction of RVH, and from 7 normal pigs. Shock-frozen renal cortical tissue was analyzed post mortem. At 5 weeks after induction of RVH, plasma renin activity (PRA) was elevated in RVH pigs and correlated with an increase in MAP of 27±11% (r=0.85). PGF2 alpha isoprostanes, markers of oxidative status and potent vasoconstrictors, were also increased compared to normal (157±21 vs. 99±16 pg/mL, p<0.05), and correlated with the concurrent increase in PRA (r=0.99) and MAP (r=0.84). By 10 weeks, MAP was still elevated (150±13 mmHg), but PRA returned to baseline, and they no longer correlated. Isoprostanes tended to further increase (to 187±29 pg/mL, p=0.07 compared to 5 weeks) and still correlated directly with the change in MAP from baseline (r=0.75). Plasma thiobarbituric acid-reactive substances, markers of increased oxidation, were also elevated (3.9±0.5 vs. 2.1±0.2 nmol/mL, p=0.005). In tissue of both kidneys, endogenous intracellular radical scavengers (glutathione peroxidase, catalase, CuZn- and Mn- superoxide dismutase) were significantly decreased (all p<0.001 compared to normal pigs), as well as natural antioxidants (alpha-tocopherol and ascorbate, p<0.03 compared to normal). This study demonstrates, for the first time, that in the early phase of RVH an increase in PRA and MAP is associated with increased plasma oxidation. When systemic PRA later declines, isoprostanes remain elevated, possibly due to local activation and/or slow responses to AII, and may potentially mediate sustenance of MAP. Moreover, oxidation-sensitive mechanisms may conceivably play a role in progression of ischemic and hypertensive renal tissue injury observed in RVH.
Plasma Angiotensin-(1-7) Is Decreased in Preeclampsia
Authors: Brosnihan K. Bridget , Karoly Michael , Merrill David
Published: July 2021

P18

Angiotensin-(1-7)[Ang-(1-7)] is a bioactive component of the renin-angiotensin system (RAS), which has depressor, vasodilatory, and antihypertensive actions. In normal pregnancy we questioned whether the known rise in plasma Ang II is counterbalanced by an increase in plasma Ang-(1-7) and whether plasma Ang-(1-7)levels are decreased in preeclampsia and may thus be a factor involved in the development of hypertension. Nulliparous preeclamptic patients (PREE) and third trimester normotensive pregnant contols (NPC)(matched for parity, race, and gestational age) were enrolled (n=15/group). A nonpregnant group (CON)(n=15) was also included for comparison. PREE had no previous history of hypertension. Mean gestational age of preeclamptic subjects was 33.9±1.2 vs 33.7±1.2 weeks for normotensive pregnant subjects (n.s.,p=0.9). PREE subjects had significant hypertension (159±3/98±2 mmHg) and all had ≥3† proteinuria. Plasma Ang I, Ang II, and renin activity (PRA) were significantly elevated in normal pregnancy as compared to nonpregnant CON subjects; plasma Ang-(1-7) was increased by 51%(p<0.05). In PREE subjects all components of the RAS were reduced as compared to NPC; however, plasma Ang II remained elevated as compared to nonpregnant CON subjects. These studies confirm that the RAS is activated in the third trimester of normal pregnancy,including an increase in plasma Ang-(1-7)levels. In preeclampsia, the decreased levels of plasma Ang-(1-7)in the presence of persistent elevated plasma Ang II are consistent with the development of hypertension.
Angiotensin II Attenuates Vasodilatation Induced by No-Donor, Glyceryl Trinitrate: Role of Super Oxide and Angiotensin Type 1 Receptor
Authors: Wada Atsushi , Ueda Shinichiro , Masumori-Maemoto Satoko , Kuji Naomitsu , Umemura Satoshi
Published: July 2021

P25

We investigated effects of subpressor dose of angiotensin II (ANG II) on the vasodilating effect of Glyceryl trinitrate (GTN)in 9 normotensive healthy males. GTN at 100, 250, 1000 ng/min was intra-arterially infused with ANG II at 1, 5 pmol/min or placebo (protocol 1) with noradrenaline (NA) at 50 pmol/min or placebo (protocol 2). Effect of intra-arterial infusion of vitamin C at 25 mg/min on the interaction between GTN and ANG II was also tested (protocol 3).Protocol 1 was repeated after single oral dose of angiotensin type 1 receptor blocker, Candesartan (8mg) or matched placebo (protocol 4). Forearm blood flow (FBF) was measured by strain gauge plethymograph. ANG II significantly and dose-dependently attenuated the vasodilating effect of GTN (means±S.D. of % changes of FBF: 28±8, 79±22, 208±28 % at 100, 250, 1000 pmol/min of GTN with placebo, 8±6, 47±13, 173±31 with ANG II 1 pmol/min, 2±10, 39±13, 132±23 with ANG II 5 pmol/min. P=0.0057 vs placebo by ANOVA).NA did not affect the vasodilating effect of GTN. Co-infusion of vitamin C (25 mg/min)completely abolished the the interaction between ANG II and GTN. Attenuation of the effect of GTN by ANG II disappeared after the single dose of Candesartan. In summary, subpressor dose of ANG II attenuated the effect of GTN. There was a lack of effect of ANG II in the presence of either vitamin C or Candesartan. Our results suggest that ANG II may stimulate superoxide production through AT1 receptor which inactivates NO by forming peroxynitrite.
Cytochrome P450 Isoform Expression in Human Vascular Endothelial Cells
Authors: Barbosa-Sicard Eduardo , Kaergel Eva , Muller Dominik N , Honeck Horst , Luft Friedrich C , Schunck Wolf-Hagen
Published: July 2021

P29

Epoxy derivatives of arachidonic acid may act as important autocrine and paracrine mediators of endothelial function including regulation of vascular tone and control of inflammation. To identify potential candidates for catalyzing the synthesis of these and further arachidonic acid metabolites, we studied human vascular endothelial cells for the expression of individual cytochrome P450 isoforms belonging to the CYP families 1, 2, 3 and 4. An RT-PCR screening performed with subfamily- and isoform-specific primer pairs revealed mRNAs for the P450 forms 1A1, 1B1, 2C8, 2E1, 2J2, 3A7, 4A11 and 4F2. The identity of the RT-PCR products was confirmed by DNA sequencing. In addition, P450 1A2 mRNA was detected after induction with β-naphthoflavone which also enhanced the expresion of P450s 1A1 and 1B1. P450s 2B6 and 3A4 were not detectable. Similar P450 isoform patterns were obtained analyzing primary human endothelial cells originating from aorta, coronary arteries, dermal microvessels and umbilical veins, as well as an immortalized human endothelial cell line (HMEC-1). Further studies with HMEC-1 cells showed the expression of all human members of the P450 2C subfamily (2C8, 2C9, 2C18 and 2C19). We next used gaschromatography-mass spectrometry to identify the regioisomeric epoxeicosatrienoic acids produced by HMEC-1 cells. Among the P450 forms detected by the RT-PCR screening, P450 2C8 and 2J2 are the leading candidates for producing vasoactive epoxyeicosatrienoic acids. Using recombinant human P450 1A1, we then found that this P450 form catalyzes the formation of various regioisomeric hydroxy derivatives of arachidonic acid. We conclude that P450 1A1 known primarily for its role in polycyclic aromatic hydrocarbon metabolism, may interfere with endothelial arachidonic acid metabolism, particularly after its induction by drugs and xenobiotics. Furthermore, P450s 4A11 and 4F2 probably contribute to the degradation of lipid mediators of inflammation.
Differential Effects of Angiotensin II and Angiotensin-(1-7) at the Nucleus Tractus Solitarii of Transgenic Rats with Low Brain Angiotensinogen
Authors: Couto Aurea S , Baltatu Ovidiu , Santos Robson A S , Ganten Detlev , Bader Michael , Campagnole-Santos Maria J
Published: July 2021

P42

The potential importance of permanent alteration of the brain renin-angiotensin system on angiotensin (Ang) II and Ang-(1-7) effects at the level of the nucleus tractus solitarii (NTS) was investigated in transgenic rats with a deficit in brain angiotensinogen production TGR(ASrAOGEN) (TGR). Ang II (10 pmol), Ang-(1-7) (10 pmol) or NaCl (0.9%/ 50 nl) were microinjected into the NTS of urethane-anesthetized TGR (n=28) and Sprague-Dawley (SD, n=22) rats. Mean arterial pressure (MAP) and heart rate (HR) were measured via a femoral artery catheter and the baroreflex control of heart rate was evaluated after increases in MAP induced by phenylephrine (baroreflex bradycardia). Ang II microinjections into the NTS of the TGR induced a higher decrease in MAP and HR (-37 ± 5 mmHg and -69 ± 12.5 beats/min, respectively) in comparison with SD rats (-18 ± 1 mmHg and -51 ± 11 beats/min, respectively). In contrast, changes after Ang-(1-7) microinjections into the NTS of TGR (-6 ± 1 mmHg and -13 ± 5 beats/min) were significantly smaller than that induced in SD (-11 ± 2 mmHg and -24 ± 8 beats/min.). The baroreflex sensitivity was accentuated in TGR in comparison to SD rats (0.69 ± 0.06 vs. 0.44 ± 0.03 ms/ mmHg). Ang II microinjection into the NTS produced similar attenuation in the baroreflex bradycardia in both SD (0.28 ± 0.07 vs. 0.5 ± 0.07 ms/ mmHg, before injection) and TGR (0.44 ± 0.1 vs. 0.82 ± 0.1ms/ mmHg, before injection). Ang-(1-7) microinjection elicited a facilitation of the baroreflex bradycardia in SD (0.62 ± 0.1 vs. 0.4 ± 0.03 ms/ mmHg, before injection). However in TGR, baroreflex bradycardia after Ang-(1-7) was not different from saline microinjection. These results indicate that a permanent inhibition of angiotensinogen synthesis in the brain can lead to a functional up-regulation of Ang II receptors. However, the putative Ang-(1-7) receptors seem to be desensitized in the NTS of these transgenic rats. The alterated baroreflex sensitivity, both before and after Ang microinjection, indicates the functionally relevant decrease in brain Ang in TGR and supports differential regulatory mechanisms for the effects of the two Ang peptides.
Cardiac Fibrosis in Tg (mREN2)Rats (TGR) Involves Both At-1 and Endothelin Receptors
Authors: Rossi Gian Paolo P , Seccia Teresa M , Sacchetto Alfredo , Belloni Anna S , Colonna Stefania , Nussdorfer Gastone G , Pessina Achille C , Kreutz Reinhold , Paul Martin
Published: July 2021

P60

Ang II plays a role in cardiac hypertrophy (CH) and was associated to cardiac fibrosis (CF). However, data are conflicting on the occurrence of CF and little is known on the mechanisms leading to collagen deposition and CF in TGR, a model of CH with an activated tissue renin-angiotensin system (RAS). Thus, we investigated if: 1) CH is associated with excess collagen deposition; 2) the RAS, and/or the endothelin (ET) system contribute to the development of CF. Four-wk old TGR were body weight (BW)- and blood pressure (BP)-matched randomly allocated to receiving the following (oral) treatments for 4 weeks : placebo (P), Bosentan (100 mg/Kg b.w., B) and Irbesartan (50 mg/kg b.w., I). BW and systolic BP were measured weekly. After rats were euthanized, 5 μm thick equatorial sections of the left ventricle (LV) werestained with Sirius Red to visualize fibrillar collagen. A quantitative analysis of percent collagen areas (CA) was performed with a Leica DM photomicroscope coupled to a PC with Qwin Leica Image Software. Development of severe hypertension and CH was prevented in group I (BP: 160±mmHg; normalized LV wt: 2.28±0.06 mg/g bw, p<0.0001 by ANOVA & Bonferroni test), but not inboth groups P (BP:262±; 3.71±.10) and B (275±; 3.38±.11). At variance, collagen deposition was significantly reduced not only in group I (CA % arb. Units: 1.38±.10) , but also in B (1.33±.10), compared to P (2.19±.40, p<0.0001). Thus, hypertension and CH are associated with excess collagen deposition in the LV of TGR rats. Although both hypertension and CH were prevented by selective AT1 but not mixed ETA/ETB receptor blockade, both treatments hindered CF. Thus, our data show a dissociation between the BP changes and the molecular mechanisms underlying fibrosis, which do and do not involve ET-1, respectively.
Association Between TGF-β Gene Polymorphism and Left Ventricular Hypertrophy in Essential Hypertension
Authors: Enjuto Montserrat , Inigo Pablo , Francino Antonio , Gomez-Angelats Elisenda , Campistol Alejandro Josep M , Sierra De la
Published: July 2021

P61

The aim of the study was to evaluate the possible association between two TGF-β1 gene polymorphisms and left ventricular hypertrophy in a group of essential hypertensive patients (EH). Ninety-three non treated EH underwent 24-hour ambulatory blood pressure, two-dimensional guided M-mode echocardiography in order to measure left ventricular mass index (LVMI), and TGF-β1 Leu
10
/Pro and Arg
25
/Pro polymorphism genotyping. As shown in the table, 24-h SBP and DBP were significantly higher in homozygous patients for the Arg
25
allele of the TGF-β1 gene. Likewise, homozygous patients for the Leu
10
allele and homozygous patients for the Arg
25
allele of the TGF-β1 gene had significanlty higher LVMI. We conclude that these TGF-β1 gene polymorphisms are associated with the severity of blood pressure and left ventricular hypertrophy in EH patients.
Doxazosin Inhibits MCP-1 Directed Migration of Human Monocytes
Authors: Kintscher Ulrich , Wakino Shu , Kim Sarah , Kon Dedrick , Hsueh Willa A , Law Ronald E
Published: July 2021

P65

Monocyte chemotactic protein 1 (MCP-1) - directed transendothelial migration of monocytes plays a key role in the early development of atherosclerosis. Migration requires degradation of extracellular matrices, a process that involves matrix metalloproteinases (MMP) and tissue inhibitors of MMPs (TIMP). Recent studies suggest that the alpha1-adrenergic receptor (alpha1-AR) antagonist doxazosin (Dox) might have antiatherosclerotic effects by inhibiting proliferation and migration of vascular cells. The purpose of the present study was to determine the effects of Dox on MCP-1 directed monocyte migration, MMP-9 and TIMP-1 regulation, and to identify signalling pathways mediating these processes. MCP-1 (50ng/ml) stimulated migration of human peripheral blood monocytes (HPBM) by 2.8 +/-0.7 fold and of THP-1 human monocytes by 7 +/- 2.7 - fold (both p<0.05 vs. unstimulated cells). Dox inhibited MCP-1 induced migration in a dose dependent manner, with a maximal reduction at 10μM of 50.8 +/- 1.9% in HPBM and 73.5 +/- 3.2 % in THP-1 cells (both p<0.01 vs. MCP-1 alone). Dox blocked migration even after pretreatment with phenoxybenzamine (PBA), an irreversible alpha1-AR antagonist (THP-1 cells: PBA 1μM + Dox 10μM: 76 +/- 11.6% inhibition, p<0.01 vs. MCP-1 alone), suggesting that the antimigratory activity of Dox is mediated through a novel mechanism unrelated to its blocking the alpha1-AR. Pretreatment of monocytes with SB203580 (10μM), a p38 MAPK inhibitor, also attenuated MCP-1 directed migration ( HPBM: 55.3 +/- 0.3% inhibtion; THP-1 cells: 55.4 +/- 13.9% inhibition, both p< 0.05 vs. MCP-1 alone), indicating that activation of this pathway is required for migration towards MCP-1 by these cells. Dox (10μM) inhibited MMP-9 activity by 62% +/- 3.15% (p<0.05) measured by ELISA, whereas MMP-9 protein levels were not affected. Dox also increased phorbol 12-myristate 13-acetate (PMA) induced TIMP-1 expression, which may account for its inhibition of MMP activity. The present study demonstrates a potential novel antiatherosclerotic action of Dox by blocking MCP-1 directed-monocyte migration, mediated, in part, through an inhibtion of MMP-9 activity and upregulation of TIMP-1 production.
Chronic Angiotensin II Augments Integrin-Dependent Cell Functions Via PKC and Map-Kinase Activation in Rat Vsmcs
Authors: Kappert Kai , Blaschke Florian , Schmidt Gunther , Fleck Eckart , Law Ronald E , Graf Kristof
Published: July 2021

P78

The activation of local and systemic renin-angiotensin-system is directly and indirectly involved in the mechanisms of vascular remodeling during chronic hypertension. The following study investigated the effect of chronic angiotensin II (AII) admininstration on cell- matrix interaction of VSMCs in vitro. Adhesion to vitronectin (VN) and collagen I (ColI) as well as migration of rat vascular smooth muscle cells (VSMCs) were significantly augmented by chronic treatment with AII for 48 hours and 72 hours. These changes were not due to an increase of the integrins on the cells surface known to be capable in binding vitronectin and collagen I: alpha1, alpha5, beta1 and beta5, as demonstrated by flow cytometry. Also the phosporylation of the focal adhesion kinase (FAK), known to play an important role in adhesion and spreading processes, was not affected after chronic stimulation with Ang II. Downregulation of PKC activity by phorbol-myristate acetate (PMA, 0.1μmol/L), or treatment with the PKC inhibitor calphostin C before treatment with AII significantly reduced the effect of AII on adhesion to VN and ColI (each p<0.05) and also inhibited the effect on VSMC migration (p<0.01). Inhibition of MAP-kinase activation with PD 98059 before treatment with AII demonstrated comparable effects. PD 98059 significantly reduced the increase in adhesion on both, VN and ColI and diminished the effect on PDGF-directed migration of rVSMCs. Theses data indicate that regulation of these functional alterations in integrin-dependent cellular behaviour after chronic AII treatment involved protein kinase C (PKC) and mitogen-activated protein kinase (MAPK)-activation. These data demonstrate that Ang II plays a critical role in augmenting cellular functions in VSMCs, which are important molecular events for the development of vascular hyperplasia .
Impaired Function and Expression of Endothelial Small Ca
2+
-Activated K Channels (SK3) in Reendothelialized Carotid Arteries After Balloon Catheter Injury (BCI)
Authors: Koehler Ralf , Real Robert , Degenhardt Christiane , Kuehn Meike , Orzechowski Hans-Dieter , Paul Martin , Hoyer Joachim
Published: July 2021

P80

Ca
2+
-activated K channels (K
Ca
) play an important role in endothelial function by controlling the electrochemical driving force for Ca
2+
influx and subsequent formation of vasoactive factors like NO and EDHF. An altered endothelial function have been suggested to be present in reendothelialized arteries after balloon catheter injury (BCI). We compared expression and function of endothelial rSK3 in reendothelialized carotid artery (CA) after BCI and not injured contralateral CA (CON) by using single-cell RT-PCR and patch-clamp (PC) techniques. Male Sprague-Dawley rats underwent BCI of the right CA. After 6 weeks, neointima formation and reendothelialization were confirmed by morphological analysis. Single endothelial cells (EC) were directly harvested from the luminal wall with the patch pipette. cDNA of rSK3 was amplified along with cDNA of endothelial nitric oxide synthase (rNOSIII), an EC marker. The percentage of rNOSIII
+
-samples was not different between CON (84% ± 10) and injured CA (84% ± 6). Expression of rSK3 was detected in 50% ± 10 of rNOSIII
+
-EC from CON. From injured CA, expression of rSK3 was detected in only 3% ± 3 of NOSIII
+
-EC (P<0.05). In whole-cell PC experiments in EC from CON, cell dialysis with Ca
2+
induced K currents, which were sensitive to apamin, a blocker of SK3. In contrast, in EC from injured CA, cell dialysis with Ca
2+
did not activate K currents. In current-clamp PC measurements of EC membrane potential (Vm) of CON, acetylcholine (ACh) induced a sustained and apamin-sensitive hyperpolarization from a resting Vm of -19 ± 5 mV to 40 ± 13 mV (ΔVm -19 ± 7 mV). In injured CA, an ACh-induced hyperpolarization was not observed (-22 ± 7 mV, ΔVm 0 ± 1 mV). Resting Vm (22 ± 6 mV) was not different. In conclusion, the deficient expression of K
Ca
in reendothelialized injured CA abolishes endothelial hyperpolarization in response to humoral stimulation. The defective hyperpolarization presumably leads to a decreased Ca
2+
influx and subsequent synthesis of vasoactive factors. This might indicate a diminished function of the newly formed endothelium in CA after BCI.
Increased 2-Methoxyestradiol Production in Human Coronary Artery Versus Aortic Smooth Muscle Cells: Potential Role in Mediating the Cardioprotective Effects of Estradiol
Authors: Zacharia Lefteris C , Jackson Edwin K , Gillespie Delbert G , Dubey Raghvendra K
Published: July 2021

P86

Estradiol (E) protects premenopausal women against coronary artery disease (CAD), however, the mechanisms involved remain unclear. Recent findings that E protects neointima formation in α and β estrogen receptor(ER) knockout mice suggest that the effects may be mediated via an ER independent mechanism. Because 2-methoxyestradiol(2MeOE),an endogenous metabolite of E with no affinity for ER, is more potent than E in inhibiting vascular smooth muscle cell (SMC) growth, it is feasible that 2MeOE mediates the protective effects of E. Since formation of 2MeOE involves the methylation of 2-hydroxyestradiol (2OHE; major estradiol metabolite)by COMT, the protective effects of estradiol on the coronary circulation may be due in part to the efficient conversion of E and 2OHE to 2MeOE in coronary vascular SMCs. To test this hypothesis, we compared the kinetics of 2MeOE synthesis in human aortic (A) versus coronary artery (CA) SMCs. Incubation of human ASMCs and CASMCs with 10μM 2OHE resulted in a time-dependent formation of 2MeOE. As compared to ASMCs, CASMCs were twice as effective in metabolizing 2OHE to 2MeOE and the Vmax and Km were 15.7±.7 vs 38.3±.9 pmol 2MeOE/10
6
cells/min and .35±.07 vs .12±.02 μM, respectively. Rat ASMCs and porcine aortic endothelial cells (ECs) also metabolized 2OHE to 2MeOE, the Vmax being 17.1±.8 and 14.22±.3 pmol/10
6
cells/min, respectively, and Km of 0.48±.1 and 0.32±.04μM,respectively. Our findings indicate that vascular SMCs and ECs effectively metabolize 2OHE to 2MeOE and that as compared to ASMCs, CASMCs have increased COMT activity and 2MeOE formation.Thus, local metabolism of 2OHE to 2MeOE may mediate the anti-vasoocclusive effects of E via an ER-independent mechanism. Moreover, increased COMT activity may selectively protect the coronary artery by generating more 2MeOE and this may be responsible for the decreased incidence of CAD in premenopausal women.
Effects of Angiotensin II Type 2 Receptor Stimulation on Collagen Synthesis in Vascular Smooth Muscle Cells
Authors: Mifune Mizuo , Sasamura Hiroyuki , Nakaya Hideaki , Shimizu-Hirota Ryoko , Hayashi Matsuhiko , Saruta Takao
Published: July 2021

P84

Previously, we and others have shown that angiotensin II enhances vascular smooth muscle cell extracellular matrix synthesis via stimulation of the type 1 angiotensin (AT1) receptor. Recently, expression of the type 2 (AT2) receptor has been confirmed in the adult vasculature, but its role in vascular remodeling has not yet been fully defined. In particular, conflicting data from in vivo studies have reported that AT2 receptor inhibition may either attenuate or enhance vascular hypertrophy and fibrosis. The aim of this study was to clarify the effects of direct stimulation of AT2 receptors on collagen synthesis in vascular smooth muscle cells in vitro. Firstly, retroviral gene transfer was used to supplement adult vascular smooth muscle cells with AT2 receptors to mimic the vasculature in vivo. Treatment of these cells with the AT2 receptor agonist CGP42212A (10-7 mol/L) alone did not cause a significant change in p42/p44 MAP kinase activity, but caused a modest (33%) decrease in protein tyrosine phosphatase activity. Treatment with CGP42112A also caused a dose- and time-dependent increase in both cell-associated and secretory collagen synthesis (148+17% of control at 48 h, p<0.05) which was completely inhibited by the AT2 receptor antagonist PD123319, but unaffected by the AT1 receptor antagonist losartan. The AT2 receptor-mediated stimulation of collagen synthesis was unaffected by tyrosine phosphatase inhibitors sodium orthovanadate and okadaic acid, but attenuated by pretreatment with pertussis toxin or Galphai antisense oligonuclotides. These results suggest that direct AT2 receptor stimulation can increase rather than decrease collagen synthesis in vascular smooth muscle cells, and suggest a role for Galphai in the AT2 receptor-mediated effects.
Effect of Estrogen Replacement Therapy on Endothelial Function of Peripheral Resistance Artery in Postmenopausal Women - Comparison of Normotensive Subjects and Hypertensive Patients -
Authors: Higashi Yukihito , Sanada Mitsuhiro , Sasaki Shota , Nakagawa Keigo , Ohama Koso , Matsuura Hideo , Kajiyama Goro , Oshima Tetsuya
Published: July 2021

P112

To determine whether endothelial dysfunction is demonstrable in the forearm circulation of hypertensive postmenopausal women (HPW) compared with normotensive postmenopausal women (NPW), and to evaluate the effects of long-term estrogen replacement therapy (ERT) on endothelial function in the HPW and NPW, we randomized both HPW and NPW into groups with ERT for 12 weeks (n=26 and 10) or with placebo (n=8 and 6), respectively. Forearm blood flow was measured using strain-gauge plethysmography during reactive hyperemia to test endothelium-dependent vasodilation, and after sublingual nitroglycerin administration to test endothelium-independent vasodilation. Basal forearm blood flow was similar in NPW and HPW. Forearm blood flow in HPW during reactive hyperemia was significantly less than that in NPW. Increases in forearm blood flow after nitroglycerin were similar in the two groups. ERT lowered LDL cholesterol and increased estradiol and HDL cholesterol; no change occurred in the placebo group. Changes in these parameters evoked by ERT were similar in HPW and NPW. Basal blood pressures, heart rate, forearm blood flow, or body weight was not changed by ERT. After 12 weeks of ERT, maximal forearm blood flow response during reactive hyperemia increased significantly from 18.4 ± 2.6 to 28.6 ± 3.4 mL/min/100 mL tissue (p<.05) in HPW and from 26.5 ± 1.9 to 30.9 ± 3.9 mL/min/100 mL tissue (p<.05) in NPW, but it was not changed by placebo. The improvement of reactive hyperemia after ERT was significantly greater in HPW than in NPW (56 ± 8 vs. 17 ± 3%, P<.05). Changes in forearm blood flow after sublingual nitroglycerin administration were similar before and after 12 weeks of ERT. These findings suggest that continued ERT improves endothelial dysfunction in postmenopausal women, and that HPW show endothelial dysfunction which can be improved by ERT via the mechanism other than beneficial effects on lipid metabolism.
6-[
18
f]Fluorodopamine Positron Emission Tomographic (PET) Scanning in the Diagnostic Localization of Pheochromocytoma
Authors: Goldstein David S , Eisenhofer Graeme , Li Sheng-Ting , Pacak Karel
Published: July 2021

P119

The diagnosis and treatment of pheochromocytoma (PHEO) depend on means to localize the tumor. Computed tomography and magnetic resonance imaging have good sensitivity but poor specificity, and nuclear imaging has limited sensitivity. Here we report initial results of 6-[
18
F]Fluorodopamine PET scanning in patients with known or suspected PHEO. Of 23 patients, 13 had the tumor. Ten had normal plasma levels of metanephrines, excluding PHEO. All 13 patients with PHEO had abnormal fluorodopamine PET scans that identified the tumors. The 10 patients without PHEO had negative PET scans. Fluorodopamine PET scanning detects PHEO with high sensitivity.
Microsatellite DNA Polymorphism of Human Adrenomedullin Gene in Normotensive Subjects and Patients with Essential Hypertension
Authors: Ishimitsu Toshihiko , Hosoya Kazuyoshi , Tsukada Kohju , Minami Junichi , Teranishi Megumi , Saitoh Mayumi , Nakamura Miki , Futoh Yasuo , Ono Hidehiko , Matsuoka Hiroaki
Published: July 2021

P121

Objective: Adrenomedullin (AM) is a hypotensive peptide widely produced in the cardiovascular organs and tissues such as the heart, kidney and the vascular cells. We investigated the association between DNA variations in AM gene and the predisposition to essential hypertension. Methods: We have cloned and sequenced the genomic DNA encoding human AM gene, and determined that the gene is located in the short arm of chromosome 11. The 3’-end of the gene is flanked by the microsatellite marker of CA repeats. Genomic DNA was obtained from the peripheral leukocytes of healthy normotensive subjects (NT; 179 men and 100 women, 57±6 years) aging 50 years or more and patients with essential hypertension (EH; 162 men and 98 women, 53±11 years) who had developed hypertension before the age of 50 years. The genomic DNA was subject to PCR using a fluorescence-labeled primer, and the number of CA repeats were determined by poly-acrylamide gel electrophoresis. Plasma AM concentration was measured by RIA and compared with respect to the number of CA repeats adjacent to the AM gene. Results: The averaged blood pressure was 117±13/73±9 mmHg in NT and 170±23/104±12 mmHg in EH when the patients were not treated. In Japanese, there existed four types of allelles with different CA-repeat number; 11, 13, 14 and 19. The frequencies of these alleles were 11: 28.7%, 13: 33.6%, 14: 34.7% and 19: 3.0% in NT and 11: 30.5%, 13: 28.4%, 14:34.3% and 19: 6.8% in EH. Thus, the frequency of 19 CA repeat allele was higher in EH than in NT (χ
2
=10.25, p<0.02). Namely, 13.5% of EH carried the 19-repeat allele, while the frequency was 6.1% in NT (χ
2
=8.43, p<0.004). In NT, plasma concentrations of AM in 21 homozygotes of 11-repeat allele, 27 homozygotes of 13-repeat allele, 30 homozygotes of 14-repeat allele and 17 heterozygotes carrying 19-repeat allele were 7.5±1.0, 7.0±1.3, 7.2±1.3 and 7.3±1.7 fmol/ml, respectively, and the values were not significantly different between the genotypes. Conclusion: Microsatellite DNA polymorphism of AM gene may be associated with the genetic predisposition to EH, although the gene expression is not likely to be affected by the genotypes.
Genome-Wide Linkage Analysis of Pulse Pressure in Mexican Americans
Authors: Atwood Larry D , Samollow Paul B , Fdn Southwest , Stern Michael P
Published: July 2021

P125

Pulse pressure, a measure of aortic stiffness, has recently been shown to be a strong predictor of cardiovascular mortality in both normotensives and hypertensives. It is also an important predictor of left ventricular hypertrophy. Unfortunately, little is known about the genetic etiology of pulse pressure. To address this problem we performed genetic analysis on 46 randomly ascertained families (1306 individuals) in the San Antonio Family Heart Study. Pulse pressure (PP) was defined as the difference between systolic and diastolic blood pressure. Likelihood methods were used to construct a model that had both single-locus and polygenic components. The single-locus component included sex-specific and genotype-specific effects of both age and body mass index (BMI). Using this model we then performed two-point linkage analysis of 10 large families (440 individuals), which were a randomly chosen subset of the initial 46 families, that have been genotyped for 396 polymorphic markers. Results showed that when the model contained only the polygenic component and simple effects of the covariates then the heritability of PP is .21. Adding the single-locus component was highly significant (p<.0001). In addition, within the single-locus component, the sex-specific and genotype-specific effects of age and BMI were also highly significant (p<.002). This full model accounted for 73% of the total variation of PP. Linkage analysis of this model with each marker showed four markers with lodscores > 1.9, which is the Lander-Kruglyak standard for suggestive linkage.
D21S1439
had a lodscore of 2.78 with a recombination fraction (θ) of 0.02.
D7S1799
had a lodscore of 2.04 (θ=0.01).
D8S1100
had a lodscore of 1.98 (θ=0.08) and
D18S844
had a lodscore of 1.95 (θ=0.11). We conjecture that pulse pressure is affected by several major genes and that together these genes account for the highly significant genotype-specific single-locus effect observed here. Supported by HL54707 and HL45522.