bluuebunny/crossref_metadata_2025_split huggingface dataset:
PS-BPB03-2: VASORELAXANT-MEDIATED ANTIHYPERTENSIVE EFFECT OF THE SEED AQUEOUS EXTRACT OF AFRAMOMUM PRUINOSUM GAGNEP. (ZINGIBERACEAE)
Authors: Mbuyo Elvine Pami Nguelefack , Mekontchou Larissa Orelle , Tchinda Nidele Lonla , Goumtsa Ariane Falone , Atsamo Albert Donatien , Nguelefack Telesphore Benoit 
Published: January 2023

Objective:
Despite the existing therapies, arterial hypertension is still a prevalent and a worldwide major public health concern, prompting the search for new therapeutic alternatives. The present study investigated the antihypertensive and the related mechanisms of action of the seed aqueous extract of Aframomum pruinosum (AEAP), a plant used in Cameroon traditional medicine against cardiovascular ailments.

Design and method:
Male Wistar rats were rendered hypertensive by chronic oral administration of L-NAME (30 mg/kg/day) for three consecutive weeks, and hypertensive rats were selected and treated with L-NAME alone or in combination with AEAP at doses of 75 or 150 mg/kg/day for an additional 3 consecutive weeks. Captopril (25 mg/kg/day, p.o) was used as reference drug. Blood pressure and heart rate were monitored weekly. At the end of the experiment, the heart was harvested, weighed, and sections of cardiac tissue were stained with hematoxylin and eosin for histological analysis. Cumulative concentrations of the aqueous extract (3, 10, 30, 100, and 300 μg/mL) were tested on intact or endothelium-denuded aorta rings precontracted with norepinephrine (NE; 1 μM) or KCl (90 mM) in absence or presence of L-NAME (a nitric oxide synthase inhibitor, 100 μM), indomethacin (a prostaglandin synthesis inhibitor; 10 μM), methylene blue (a guanylate cyclase inhibitor, 1 μM), tetraethylammonium (TEA; a non-specific potassium channel blocker; 5 μM) or glibenclamide (an ATP-sensitive potassium channel blocker; 10 μM). The contractile response to NE, or CaCl2 was also assessed in the presence or absence of the plant extract.

Results:
AEAP completely reversed (p < 0.001) L-NAME-induced elevated systolic and diastolic blood pressure, and increased heart rate. AEAP partially reversed cardiac hypertrophy, leucocyte infiltration, and prevent cardiac necrosis. AEAP induced a concentration-dependent vasorelaxant effect on NE- and KCl-evoked contraction with EC50 values of 13.46 and 49.37 μg/mL, respectively. Removal of the endothelium and the preincubation of aorta rings with L-NAME significantly (p < 0.05) reduced the vasorelaxant effect of the plant extract, while indomethacin and methylene blue had no effect (p > 0.05). Glibenclamide, similar to TEA, significantly (p < 0.001) inhibited the vasorelaxant response to AEAP. Also, the extract significantly (p < 0.01 and p < 0.001) inhibited NE- and CaCl2-induced contraction.

Conclusion:
AEAP possesses antihypertensive effects that are at least partially mediated by its vasorelaxant activity. Nitric oxide release, opening of KATP channels, blockade of alpha-1 receptors, and inhibition of Ca2+ influx account for this vasorelaxant activity.
PS-BPB04-7: PRO-ANGIOGENIC EFFECTS OF LSKL, A THROMBOSPONDIN-1 INHIBITOR, IN HUMAN AND EXPERIMENTAL MODELS OF PREECLAMPSIA
Authors: Marc Casandra , Achille Allahnah , Fregeau Genevieve , Hannou Lydia , Vachon Isabelle , Vaillancourt Cathy , Lavoie Julie , Bertagnolli Mariane 
Published: January 2023

Introduction:
Preeclampsia (PE) is a hypertensive disorder of pregnancy characterized by blood pressure above 140/90 mmHg. It accounts for 10% of all pregnancies and is responsible for 25% of preterm births and low birth weight. To date, no medications can treat or prevent PE. We identified an activation of the anti-angiogenic protein thrombospondin-1 (THBS1) in human and experimental preeclampsia. THBS1 is also a main activator of TGF-β. We postulate that THBS1-mediated TGF-β activation may contribute to impair placental angiogenesis during PE.

Objective:
We aimed to test the effects of LSKL, an inhibitor of THBS1-mediated TGF-β activation, on angiogenesis in placentas of a mouse model of PE and in human placental endothelial cells.

Methods:
Peptide LSKL or its control peptide SLLK (280 μM in 0.5 ml saline, s.c.) was administered at gestational day (GD) 14.5 in heterozygous transgenic mice overexpressing human renin and human angiotensinogen and controls (C57BL/6). Placentas (n = 12/group) were removed at GD 18.5. Protein expression of TGF-β1, TGF-β2, total and phosphorylated SMAD2 were assessed by western blot of placental lysates. Placental angiogenesis was assessed by CD31+ immunohistochemistry in placental sections. CD31+ endothelial cells were extracted by magnetic sorting from human placentas (n = 4) of healthy pregnancies. Cells (2–5 passages) were treated with LSKL or SLLK (30 μM) under hypoxia (0.5% vs. 8% O2) or thrombin (10 u/mL) exposure (4 hours). THBS1 expression in placental endothelial cells was assessed by immunofluorescence. Cell angiogenesis was quantified by the number of closed tubes formed on matrigel (per 15,000 cells, 4 hours).

Results:
Maternal LSKL treatment significantly reduced the expression of TGF-β2 (P < 0.01) and SMAD2 phosphorylation (P < 0.05) while improving angiogenesis (P < 0.05) in placentas of transgenic preeclamptic mouse compared to controls. In human placental endothelial cells, both thrombin (P < 0.05) and hypoxia (P < 0.05) significantly increased THBS1 expression. Thrombin (P < 0.01) and hypoxia (P < 0.05) significantly impaired cell angiogenesis on matrigel (P < 0.01), which was prevented by LSKL treatment (P < 0.01). In human cells, LSKL similarly reduced TGB-β2 (p < 0.05) and phospho-SMAD2 (P < 0.05) expressions under thrombin and hypoxia cell exposure.

Conclusion:
Our results describe a significant pro-angiogenic effect of LSKL treatment by regulating THBS1 and TGF-β2 mechanisms in human and experimental models of PE.
PS-BPB04-8: ANGIOTENSIN II FORMULATIONS AND THEIR IMPACT ON CARDAIC, VASCULAR AND INFLAMMATORY MECHANISMS OF HYPERTENSION
Authors: Nosalski Ryszard , Booth Emma , Saju Blessy , Alsheikh Eman , Crespo Eva , Maffia Pasquale , Graham Delyth , Guzik Tomasz 
Published: January 2023

Angiotensin II (AngII) is a major effector of the renin-angiotensin system, with a critical role in BP regulation of vascular function, and end-organ damage. AngII infusion, using osmotic minipumps, is the most common animal model of hypertension, recapitulating key vascular and cardiac features of human hypertension. While different formulations of AngII are available, their impact on the key outcomes of the model remains unknown. Accordingly, we compared two most commonly used AngII formulations, and their impact on the key cardiovascular endpoints in a mouse model of hypertension.
Male 12-week old C57BL/6J mice (n = 11–14) underwent Sham, AngII[Val5] or AngII[Ile5] (490 ng/min/kg) treatment for two weeks, using osmotic minipumps. Blood pressure was measured by tail-cuff, vascular function by wire myography, remodelling by picrosirius red staining, mRNA expression using real-time PCR and perivascular (PVAT) inflammation by flow cytometry. Data were analyzed using one-way or RM-two-way ANOVA.
Both AngII formulations caused a significant BP elevation (p < 0.001) in comparison to sham animals, although AngII[Val5] had a more pronounced pressure effect than AngII[Ile5] (171 ± 4 vs 156 ± 6 mmHg, p = 0.03). Similarly, endothelium-dependent relaxation of resistance arteries and aorta was significantly impaired by AngII infusion (p < 0.001), however, a maximum Ach-induced relaxation was significantly lower in AngII[Val5] than in AngII[Ile5]. Both, AngII formulations increased intima-media thickness of the aorta (p < 0.0001), although the effect of AngII[Val5] was ∼2-fold higher than AngII[Ile5] (p = 0.001). While animals infused with AngII[Val5] had enhanced vascular collagen deposition (p < 0.001), this effect was lesser in the AngII[Ile5] group (p = 0.02). The total number of leukocytes infiltrating PVAT was induced by both AngII formulations, however, this effect was less pronounced in AngII[Ile5] (1002 ± 376 vs. 753 ± 242 cell/mg, p = 0.03). The same effect was found among CD11b+ and CD11c+ cells. While a significant increase of CD3+ cells was observed in both AngII formulations. While cardiac hypertrophy was evident in both AngII formulations this effect was significantly lower in AngII[Ile5] (p = 0.03). This was confirmed by cross-sectional area of cardiomyocytes. Histological analysis of heart sections showed a significant effect of both AngII formulation on cardiac fibrosis (p < 0.05). This effect was more pronounced upon AngII[Val5] infusion (p < 0.001) and was associated with higher expression of pro-fibrotic markers.
AngII[Val5] formulation is a more efficient and consistent way to study key cardiovascular endpoints such as BP, inflammation, vascular and cardiac damage compared with AngII[Ile5]. Furthermore, the use of AngII[Val5] leads to a more pronounced phenotype and may be associated with reducing the number of animals needed in experiments.
PS-BPB05-6: TOLVAPTAN INDUCES BODY FLUID LOSS AND ACTIVATION OF RENAL UREA-DRIVEN WATER CONSERVATION IN NORMAL RATS
Authors: Kidoguchi Satoshi , Kitada Kento , Fujisawa Yoshihide , Nakano Daisuke , Yokoo Takashi , Titze Jens , Nishiyama Akira 
Published: January 2023

Objective:
We recently reported that body fluid loss models such as high salt-induced natriuresis, impaired urine concentration ability via kidney injury, and enhanced transepidermal water loss due to skin barrier dysfunction exhibit initial water loss and subsequent activation of the water conservation system in multiple organs. This activation of the water conservation system restores or even increases water content at the tissue level, which may be involved in abnormal body fluid distribution in various disease models. The water conservation system consists of renal urea recycling, the energy-intensive nature of hepato-muscular ureagenesis, and suppression of cardiovascular energy expenditure to save energy for ureagenesis. However, it is not clarified whether pharmacological fluid loss by diuretics activates the water conservation system. In this study, we examined the effects of water diuresis by tolvaptan, a selective vasopressin V2 receptor antagonist, on the water conservation system in normal rats.

Design and method:
10-week-old male Sprague Dawley rats were fed with a control diet or a diet containing 0.1% tolvaptan. We measured water intake and urine volume for two weeks and also evaluated time-dependent changes in renal urea recycling and hepato-muscular ureagenesis in these rats. In addition, we also evaluated the effects of tolvaptan on blood pressure, heart rate, and renal sympathetic nerve activity by a radiotelemetry system in separate animals.

Results:
Tolvaptan markedly increased urine volume up to 126.7 ± 25.9 ml on day-1, which gradually decreased as much as one-third (48.1 ± 18.0 ml) on day-7. Tolvaptan significantly decreased body water content and increased plasma albumin concentration, an indicator of oncotic pressure, on day-1, suggesting that tolvaptan decreased body fluid and plasma volume on day-1. These changes in total body water content and plasma albumin concentration were restored on day-7. The restoration of body fluid on day-7 was associated with an increase in urea transporter A1-associated renal urea recycling. On the other hand, tolvaptan did not alter hepato-muscular ureagenesis or cardiovascular energy expenditure throughout the treatment.

Conclusions:
Tolvaptan induces initial body fluid loss by water diuresis and subsequent activation of renal urea recycling to restore the water loss. Activation of the water conservation system in addition to a rise in urine volume may be involved in the unknown distinct effect of the diuretics.
PS-BPB07-1: DEVELOPMENT OF ANTI-THROMBOTIC VACCINE AGAINST HUMAN S100A9
Authors: Shimamura Munehisa , Kaikita Koichi , Hayashi Hiroki , Tsujita Kenichi , Nakagami Hironori , Morishita Ryuichi , Rakugi Hiromi 
Published: January 2023

Background & Purposes:
In post-stroke patients, a decreased adherence to antiplatelet drugs is a major challenge in the prevention of recurrent stroke. Previously, we have reported that a newly designed antiplatelet vaccine against S100A9 could prevent thrombosis in the middle cerebral artery in mice by inhibiting S100A9/CD36 signaling in platelets. However, the difference in amino acid sequences in epitopes between mice and humans might be problematic for clinical use. In the present study, we redesigned the S100A9 vaccine for the common sequence in both humans and monkeys and examined its effects in cynomolgus monkeys.

Methods:
First, we screened several candidate epitopes in mice and the selected epitope in human S100A9, which is common in monkey S100A9, was conjugated to Keyhole limpet hemocyanin. A mixture of the vaccine and Alum adjuvant was subcutaneously injected into 4 cynomolgus monkeys, three times every two weeks. Antibody production against human S100A9 was examined by ELISA or western blotting. Anti-thrombotic effects in collagen-induced thrombosis were examined in a mixture of vaccinated serum and fresh whole blood from another cynomolgus monkey using an automated microchip flow-chamber system. Bleeding time was evaluated in two monkeys.

Results:
Based on results of computed epitope prediction, we first assessed whether 2 to 11, 97–106, or 104–113 amino acids, where the amino acid sequence is approximately similar between humans and monkeys, could raise antibodies against S100A9 in mice. Compared with the vaccine for the 2–11 amino acids epitopes, 97–106 amino acids and 104–113 amino acids demonstrated higher antigenicity, indicating that epitopes in the C-terminus are stronger than those in the N-terminus. Based on this result, we selected 102 to 112 amino acids as the epitope for the human S100A9 vaccine. When the monkeys were vaccinated, antibody titers against human S100A9 were significantly increased. Western blotting analysis showed that the produced antibody recognized human S100A9. Although the addition of pre-vaccinated serum into fresh whole blood did not affect thrombus formation, post-vaccination serum inhibited collagen-induced thrombosis formation in two monkeys. Anti-thrombotic effects were partially inhibited by the epitope peptide, indicating the feasibility of neutralizing anti-thrombotic effects by vaccination. Notably, prolongation of bleeding time was not observed in vaccinated monkeys.

Conclusions:
Anti-human S100A9 vaccine revealed anti-thrombotic effects in some monkeys without affecting bleeding time. Although further studies are necessary to increase the effects and warrant the safety aspects, this vaccine could clarify the use of an anti-thrombotic vaccine in clinical settings.
PS-BPB08-2: DEVELOPMENT OF NON-PEPTIDE TYPE OF ANGIOTENSIN II TYPE 1 RECEPTOR LIGAND AS A BIASED AGONIST
Authors: Matsuo Yoshino , Suematsu Yasunori , Morita Hidetaka , Miura Shinichiro 
Published: January 2023

Objective:
Peptide type of angiotensin II (Ang II) type 1 (AT1) receptor ligand as a biased agonist antagonized Ang II-stimulated G protein signaling, but stimulated several kinase pathways. Here, we developed non-peptide type of AT1 receptor compound as a biased ligand.

Design and method:
We synthesized three non-peptide type of AT1 receptor ligands (Compound A, B and C) as candidates of biased ligands. These compounds were provided by Daiichi Sankyo Co. LTD. In vitro studies, inositol phosphate (IP) production and extracellular signal-regulated kinase (ERK) 1/2 activation were measured using AT1 receptor overexpressing cell system. We mutated the AT1 receptor (L112A, Q257A, Y292A and N295A receptors) and examined binding modes of receptor-ligand by competition binding study, and subsequently analyzed whether these ligands would induce IP production and ERK1/2 activation.

Results:
Kd of Compound A, B and C to AT1 wild-type receptor by competition binding study were 0.8, 21 and 48 nM, respectively. Kd of Compound A, B and C to L112A receptor were 37, 23 and 31 nM, respectively. Compound B and C decreased and increased IP production, respectively, whereas Compound A did not change IP production. Compound B and C, but not Compound A, activated ERK1/2. L112A had a key role of IP production.

Conclusions:
Compound A, B and C were a neutral antagonist, an inverse agonist, and an agonist with regard to IP production, respectively. On the other hand, Compound B and C, but not Compound A, were agonists to ERK1/2 activation. Thus, we developed non-peptide type of AT1 receptor compound as a biased ligand.
PS-BPB09-2: DEVELOPMENT OF LONG-ACTING HUMAN ADRENOMEDULLIN FC FUSION PROTEINS
Authors: Nagata Sayaka , Yamasaki Motoo , Kuroishi Nobuko , Kitamura Kazuo 
Published: January 2023

Background:
Human adrenomedullin (hAM) is a hypotensive peptide hormone with strong anti-inflammatory effects. AM consists of 52 amino acid residues with an amidated C-terminus and a ring structure formed by intramolecular disulfide bonds. Both of these structures are necessary for binding to receptors and to exert their biological functions. AM has also shown therapeutic effects in various animal disease models, including inflammatory bowel disease, ischemic heart disease, sepsis and stroke. However, the short half-life of native AM in the blood required continuous administration for treatment. To solve this, we have developed four human IgG1 and IgG4 Fc fusion proteins containing full-length human AM or AM(6–52).

Methods:
Recombinant Fc-AM derivatives were generated using mammalian cells. After that, we tested the biological activities of Fc-fusion AMs (Fc-AMs) to stimulate the intracellular accumulation of cAMP in HEK-293 cells stably expressing the AM1 receptor. Next, we compared IgG1-AM (6–52) and IgG4-AM (6–52) concentrations in the peripheral blood and tissues of rats after subcutaneous injection. In addition, the antihypertensive effect of Fc-AM was examined on spontaneously hypertensive rats (SHRs) which were given high salt diets.

Results:
We have developed four Fc-AMs, which are long-acting AM derivatives in mammalian cells. Fc-AMs produced in mammalian cells do not require refolding or amidation. Indeed, Fc-AMs could be measured by mAM assay, which recognizes the amidated form, indicating that Fc-AMs produced in mammalian cells were amidated. Fc-AMs, generated in a mammalian cell production system can therefore easily be produced in large amounts with few purification steps. The Fc-AMs stimulated intracellular cAMP production in cultured cells stably expressing the AM type I receptor in vitro. Fc-AM (6–52) induced higher cAMP levels for the receptor than Fc-AM. The pharmacokinetic study was performed using N-terminal deficient AM derivatives, which have strong receptor binding ability. Sufficient concentrations of IgG1-AM (6–52) and IgG4-AM (6–52) were observed in blood 14 days after a single subcutaneous administration. Tissue transfer to the kidney and small intestine was observed after administration of IgG1-AM (6–52) or IgG4-AM (6–52) to rats. In addition, the single subcutaneous administration of IgG4-AM (6–52) suppressed the increase in blood pressure in SHRs which were loaded with high salt diets.

Conclusion:
Fc-AM produced from mammalian cells is easily prepared and seems to be an effective new therapeutic agent for hypertension.
PS-BPB09-8: ANGIOTENSIN RECEPTOR BLOCKER AMELIORATES ELEVATED ARTERIAL BLOOD PRESSURE AND REDUCES BODY MASS IN OLETF RATS FED A HIGH CHOLESTEROL DIET
Authors: Jardines Eira Edith , Rodriguez Ruben , Minas Jacqueline , Nakano Daisuke , Nishiyama Akira , Ortiz Rudy M 
Published: January 2023

Objective:
Cardiovascular disease (CVD) is the leading cause of death in the US (1 in 4) and is a primary outcome of Metabolic Syndrome (MetS). MetS affects nearly 40% of adults over 60 and is defined by a cluster of conditions including elevated arterial pressure and excess abdominal fat. A high cholesterol diet (HCD) is known to promote cluster-factor conditions of MetS. Unfortunately, body mass (BM) reduction is often insufficient to reduce hypertension. The first line of pharmaceutical defense is disruption of the renin-angiotensin aldosterone system (RAS). Inappropriate elevation of RAS can be therapeutically attenuated with angiotensin receptor blocker (ARBs), which has been shown to ameliorate other MetS cluster factor conditions. However, the efficacy of ARB on elevated arterial pressure during a HCD remains unclear.

Design:
A cohort of 5-week-old Otsuka Long Evan Tokushima Fatty (OLETF) rats were fed a HCD (2% + 0.5% cholic acid) or chow ad libitium. After 4 weeks, animals were treated with ARB (10 mg olmesartan /kg/d x 3 wks by oral gavage) and assigned to five groups: 1) lean-strain control Long Tokushima Evans Otsuka (LETO; n = 10), 2) OLETF (OLETF; n = 12), 3) OLETF+HCD (n = 12), 4) OLETF+ARB (n = 12) and 5) OLETF+HCD+ARB (n = 12). Systolic blood pressure was monitored chronically by radio telemetery.

Results:
OLETF SBP was 31% (p < 0.05) greater than LETO. HCD did not exacerbate elevated SBP in OLETF. ARB reduced SBP in OLETF + HCD by 11% (p < 0.05) over 30 days and 13% (p < 0.05) overall compared to OLETF. ARB reduced BM by 3% (p < 0.05) in chow-fed and 4% (p < 0.05) in HCD-fed OLETF compared to OLETF. However, there was no difference in BM between OLETF+ARB and OLETF+HCD+ARB. Neither, HCD nor ARB alone reduced abdominal fat in OLETF rats. ARB in OLETF+HCD induced visceral fat loss (37%; p < 0.05) compared to OLETF. ARB administration did not attenuate plasma angiotensin II levels.

Conclusion:
These findings demonstrate that chronic treatment with ARB can effectively ameliorate the MetS-associated hypertension despite the additional insult of a HCD.