bluuebunny/crossref_metadata_2025_split huggingface dataset:
Treatment with Captopril Enhances the Vascular Relaxation Induced by the Angiotensin II Type 2 Receptor (AT2R) Agonist CGP42112 in Diabetic Rats
Authors: Sabra Ramzi 
Published: November 2023

The role of the AT2 receptor (AT2R) in physiological and pathophysiological processes is still controversial. Some studies suggest that expression of AT2R increases in certain diseases like diabetes mellitus. This could have beneficial effects as signaling through the AT2 receptor has been shown to counter the effects of angiotensin II mediated through the AT1 receptor, which are believed to contribute to pathological changes; this is supported by the favorable effects of ACE inhibitors and Angiotensin receptor blockers on the course of diabetes induced complications in renal function. We examined the effect of diabetes and of treatment with captopril on the vascular responses to CGP42112 (CGP), an AT2R selective agonist, in Sprague Dawley rats with streptozotocin (85 mg/kg i.v.) induced diabetes. Rats were followed for either 2, 4 or 12 weeks, at the end of which period thoracic aortas were isolated and used for measurement of relaxant responses to CGP42112 after pre‐contraction with phenylephrine. In a subset of the 4‐week study, osmotic pumps were implanted at week 2 to deliver either captopril (50 mg/kg/day) or losartan (5 mg/kg/day) s.c. for 2 weeks. All aortic ring studies were conducted in the presence of losartan (100 μM) to prevent interactions of CGP with the AT1R. CGP induced a mild (10%) relaxation in aortic rings at 2 weeks, 4 weeks and 12 weeks in the control animals. The response to CGP in aortic rings from diabetic rats was not different from controls. Treatment with losartan for 2 weeks did not alter the responses to CGP observed after 4 weeks. However, treatment with captopril for 2 weeks resulted in a markedly exaggerated relaxant response to CGP reaching up to 50% relaxation. These preliminary results suggest that while diabetes, per se, may not alter responses to CGP, treatment of diabetic rats (but not control rats) with captopril produces a marked increase in the sensitivity of the AT2R. Considering the known anti‐proliferative and anti‐ischemic effects of AT2R activation, these results potentially imply that the use of ACE inhibitors together with AT2R agonists could amplify the protective effect of ACE inhibitors against diabetes induced complications. Thus, the pathophysiological and therapeutic implications of this finding require further study.Support or Funding InformationSupported by funds from the Medical Practice Plan, AUB Faculty of Medicine
Cardiac HMGB1/TLR4 Activation in Pre‐Type 2 Diabetes: A Potential Mechanism Triggering Diabetic Cardiovascular Complications
Authors: Vargas Elizabeth , Li Ying , Carrillo‐Sepulveda Maria Alicia 
Published: November 2023

The prevalence of type 2 diabetes mellitus (T2DM) continues to increase at an alarming rate, to the point of epidemic standards, within the United States. One of the major, potentially lethal, complications of T2DM is the development of cardiovascular disease. Toll‐like receptor 4 (TLR4), a key component of innate immunity, has been implicated in diabetic complications due to its resultant increase in inflammation and oxidative stress. While previous findings from our lab have identified that vascular TLR4 plays a major role in DM vascular complications, the activation of TLR4 in the heart from pre‐T2DM has yet to be explored. We hypothesize that TLR4 signaling is activated in the heart of pre‐diabetic rats and is linked to an early inflammatory state. Male Goto‐Kakizaki (GK) rats, a non‐obese model of T2DM, were utilized in this study, with eight and sixteen week old animals representing the pre‐T2DM and T2DM groups, respectively. Wistar rats served as the non‐T2DM (control) group. As expected, blood glucose levels (mg/dL) were elevated in the T2DM group (162.75± 11.32 vs. 85.83 ± 8.25 control group, p<0.001, n=6). A slight increase in blood glucose levels were observed in the pre‐T2DM group (121 ± 7.75 vs. 85.83 ±8.25 control group, p<0.05). T2DM rats exhibited increased systolic blood pressure (SBP) (149± 12mmHg), an expected cardiovascular complication in T2DM, while pre‐T2DM rats displayed normal SBP (112 ± 7 mmHg). To determine whether pre‐T2DM conditions modulate TLR4 signaling in cardiac tissue, western blot analysis was performed. Cardiac TLR4 protein expression was markedly elevated in the pre‐T2DM group (3.1±0.1 fold increase vs. T2DM, p<0.01, n=4), paralleled by increased NFKβ expression (2.4 ± 0.2 fold increase vs. T2DM, p<0.05, n=4), an inflammatory marker and downstream target of TLR4 signaling. MyD88, an adaptor protein of TLR4 signaling, remained unaltered in cardiac tissue of pre‐T2DM subjects, suggesting that cardiac TLR4 activation in pre‐T2DM may occur via an MyD88‐independent mechanism. Of note, we found that high‐mobility group box 1 protein (HMGB1) expression was markedly increased in pre‐T2DM hearts (1.3 fold increase vs. control, p<0.01, n=4). HMGB1 is a damage‐associated molecular pattern (DAMP) molecule that has been suggested to be an endogenous TLR4 ligand. This finding implies HMGB1 may be a sterile ligand candidate in the activation of TLR4‐mediated inflammation in pre‐diabetic milieu. In conclusion, our results suggest that HMGB1/TLR4/NFKβ signaling pathway is activated in pre‐T2DM hearts. Therefore, strategies to target either HMGB1 or TLR4 pose a potential alternative therapy in the prevention of diabetic cardiovascular complications.Support or Funding InformationNIH
Downregulation of Histone Deacetylase 9 (HDAC9) is Associated with Vascular Calcification in Diabetic Mice.
Authors: Benerofe Sara Arielle , Carrillo‐Sepulveda Maria Alicia 
Published: November 2023

Vascular complications, including calcification, are some of the major problems faced by type 2 diabetic (T2DM) patients. In fact, the majority of these diabetic patients will die due to complications of vasculopathy. The current available anti‐diabetic treatment does not prevent the progression of vascular calcification; thus, new therapeutic approaches are urgently needed. Evidence has indicated that HDACs play a central role in the epigenetic mechanisms associated with diabetic complications. Recently, it was identified that a human polymorphism of HDAC9 is linked with acute coronary disease; however, it is still unknown whether HDAC9 is involved in vascular complications in diabetes. We hypothesize that diabetic conditions modulate HDAC9 expression, and this expression is associated with increased markers of vascular calcification. To address our hypothesis, we utilized aortas from fourteen‐week‐old male db/db mice, an experimental model of T2DM, and age‐matched heterozygous nondiabetic (ND) mice (db/m+). Aortas from db/db mice displayed significant increase in osteopontin (OPN) (2.0 fold increase, p<0.01, n=5) and bone morphogenic protein 4 (BMP4) (2.7 fold increase, p<0.05, n=5) compared with the control group, both classical markers of vascular calcification. Most importantly, these findings were correlated with significant downregulation of HDAC9 (1.1 fold reduction vs. control, p<0.05, n=4). Intriguingly, these results differ from previous reports that claim HDAC9 was increased in vascular complications, specifically atherosclerosis and coronary heart disease. Our results suggest that HDAC9 may be regulated differently in diabetic conditions and its downregulation may mediate the progression of vascular diabetic calcification. To further this investigation, we will be utilizing molecular approaches to overexpress HDAC9 in the vasculature to confirm the role of HDAC9 in the diabetic vascular complications.Support or Funding InformationNIH
ANTITHROMBOTIC ACTIVITY OF NEW POTASSIUM SALT 2‐[3‐BROMINE‐1‐(THIETANIL‐3)‐1,2,4‐TRIAZOLIL‐5‐THIO]ACETIC ACID
Authors: Samorodov Aleksandr Vladimirovich , Kamilov Felix Khusainovich , Klen Elena Edmundovna , Khaliullin Ferkat Adelzyanovich , Khalimov Almaz Radikovich 
Published: November 2023

The previous findings in vitro show high antiaggregation activity of newly synthesized potassium salt of 2‐[3‐bromine‐1‐(thietanil‐3)‐1,2,4‐triazolil‐5‐thio]acetic acid (substance I). The current findings are received in vivo on the model of collagen‐adrenaline thrombosis according to G.S. Di Minno subject to the FASEB Statement of Principles for the use of animals in research and education. Efficacy endpoint is the number of survived animals in comparison with the test group and comparator agents since the injection into the tail vein suspended matter of collagen and adrenalin upto the moment of death or during 14 days of monitoring. The survival analysis was carried out with the help of Kaplan‐Meier method. The difference in survival between the groups was assessed with the help of Wilkonson criterion generalized by Gehan. Intraperinoneal injection of the new secondary 1,2,4‐triazole reduced mortality in lab mice by 1,3 times (p<0,01) in comparison with the camparator agents (table 1). Thus, potassium salt of 2‐[3‐bromine‐1‐(thietanil‐3)‐1,2,4‐triazolil‐5‐thio]acetic acid is a potential antithrombotic agent.
The survival rate in the modeling of the collagen‐adrenaline thrombosis, M±SD

Subject
Control
Pentoxifylline
Aspirin
Substance I

Lifetime, h
2,0±1,8
165,8±157,6*
172,6±102,8*
206,9±135,2*p2<0,001p3<0,005

p1<0,01 ‐ control vs experimental group;
p2 ‐ substance I vs pentoxifylline, p3 ‐ substance I vs aspirin, χ2=33,1.
Polyphenol Rich Extract of Syzygium cumini (L.) Skeels Leaves Decreases Platelet Aggregation and Inhibits Reductase Activity of Protein Disulfide Isomerase.
Authors: da Silva Samira Abdalla , Sousa Hiran Reis , Gaspar Renato , Fontelles João Lucas , Trostchansky Andrés Ezequiel , Paes Antonio Marcus 
Published: November 2023

Platelet agreggation results from conformational changes in αIIbβIII integrin, which are dependent on protein disulfide isomerase (PDI) activity. Recently, PDI inhibition has been proposed as an antithrombotic mechanism, for that polyphenolic compounds have emerged as potential inhibitors. Our group has demonstrated that Syzygium cumini (L.) Skeels (Myrtacea) leaf contains multiple polyphenols, such as: gallic acid, miricetyn‐3‐a‐arabinoside, mirycetin deoxiexoside and quercetin‐3‐glycoside; which enable this species for anti‐platelet activity. Therefore, this study aimed to evaluate the effects of polyphenol rich extract (ERP) from S. cumini leaf on platelet aggregation and PDI reductase activity. Platelet‐rich plasma (PRP) from healthy volunteers (n=5) was incubated with ERP for 25 min at concentrations ranging from 10 to 1000μg/mL, previously to activation with 2.5 or 5μM ADP. ERP at the higher concentration inhibited platelet aggregation in 66 and 33%, respectively. Under thrombin stimulus (0.01 and 0.02 U/mL), ERP (1000 μg/mL) reduced platelet aggregation in ca. 35%. Moreover, ERP also inhibited PMA‐induced platelet aggregation by 29% in its higher concentration. To analyze the ERP effect on αIIbβIII integrin activation, flow citometry protocols were conducted in washed platelets similarly pre‐treated with ERP (10, 100 and 1000μg/mL) before activation with thrombin (0.02U/mL) and tagged with PAC–1 FITC antibody. ERP dose‐dependently reduced the integrin activation in presence of 10 μg/mL (21%), 100 μg/mL (27%) and 1000 μg/mL (33%). Finally, ERP effects on PDI reductase activity were also assessed at concentrations ranging from 0.1 to 100 μg/mL. A maximum inhibitory effect on PDI reductase activity of 77% was found for the higher concentration. Therefore, our data supports that ERP‐containing polyphenols from S. cumini leaf reduce platelet aggregation, probably through PDI inhibition, which demonstrates its prominent anti‐platelet property.Support or Funding InformationFinancial support: FAPEMA; UFMA
Histone Modification is a Novel Epigenetic Mechanism to Up‐Regulate Human Blood‐Brain Barrier Transporters
Authors: You Dahea , Wen Xia , Morris Ayeshia , Richardson Jason R , Aleksunes Lauren M 
Published: November 2023

Multidrug Resistance Protein 1 (MDR1, ABCB1) and the Breast Cancer Resistance Protein (BCRP, ABCG2) expressed at the blood‐brain barrier (BBB) are key efflux transporters that extrude chemicals from the BBB and regulate the efficacy and/or toxicity of chemicals in the brain. Prior studies in cancer cells have pointed to the ability of histone deacetylase (HDAC) inhibitors to modulate the expression and function of MDR1 and BCRP. However, whether or not such regulation occurs at the BBB is not known. Here we sought to test whether HDAC inhibitors could potentially alter expression and function of MDR1 and BCRP at the BBB. To test this, we treated immortalized human brain capillary endothelial (hCMEC/D3) cells, a model of the BBB, with six different HDAC inhibitors, valproic acid (VPA), sodium butyrate (NaB), romidepsin, apicidin, suberoylanilide hydroxamic acid (SAHA), and trichostatin A (TSA), and assessed for expression and function of MDR1 and BCRP. HDAC inhibition following treatment was confirmed by increased levels of acetylated histone H3 protein. After 12 h of treatment, VPA, apicidin, SAHA, and TSA up‐regulated MDR1 mRNA levels between 50% and 200%. All six HDAC inhibitors significantly induced BCRP mRNA levels between 100% and 270%. Similarly, the protein expression of MDR1 and BCRP transporters was up‐regulated about two‐fold at 24 h. Enhanced MDR1 expression corresponded with reduced intracellular accumulation of the substrate rhodamine 123. Collectively, these results demonstrate that HDAC inhibitors up‐regulate MDR1 and BCRP transporters at the BBB by modifying histone acetylation. The clinical use of HDAC inhibitors may enhance efflux transporter activity at the BBB and restrict access of xenobiotics to the brain.Support or Funding InformationSupported by R01ES021800, P30ES005022.
11/14‐oxy‐DPA, the 12‐LOX derived metabolites of DPA, inhibit platelet activation through the cGMP‐PKG signaling pathway
Authors: Yeung Jennifer , Edokobi Nnamdi , Hawley Megan , Craven DaQuan , Li Cecilia , Holman Theodore R. , Holinstat Michael 
Published: November 2023

Platelet‐mediated thrombosis is the primary underlying mechanism leading to cardiovascular life‐threatening clinical events. Regulating excessive platelet reactivity is an essential aspect of antithrombotic therapy. Previously, we had shown that lipid metabolites of 12‐lipoxygenase (12‐LOX) such as 12‐HETrE, a 12‐LOX oxylipin of the ω‐6 polyunsaturated fatty acid dihomo‐gamma‐linolenic acid, could function as an inhibitor to platelet activation. Here, we demonstrate for the first time that another ω‐6 PUFA, docosapentaenoic acid (DPA; 22:5n‐6), can be endogenously oxidized by 12‐LOX to generate two distinct oxylipins, 11‐oxy‐DPA and 14‐oxy‐DPA to regulate platelet reactivity. Although both 11‐oxy‐DPA and 14‐oxy‐DPA were able to significantly inhibit platelet activation, we observed differences in the potency of the 12‐LOX‐derived metabolites of DPA,n‐6. 11‐oxy‐DPA was able to significantly inhibit platelet aggregation at a lower concentration than 14‐oxy‐DPA. We also observed calcium mobilization was significantly attenuated in the presence of 11‐oxy‐DPA or 14‐oxy‐DPA. Furthermore, to determine whether 11‐oxy‐DPA and 14‐oxy‐DPA could be regulating platelet function through a Gαs signaling pathway by which it could be behaving similarly to prostacyclin or 12‐HETrE, we measured a downstream effector of cAMP, the phosphorylation of vasodilator‐stimulated phosphoprotein (VASP) at serine 157 by PKA. We observed no differences in VASP phosphorylation at serine 157 in platelets following 11‐oxy‐DPA or 14‐oxy‐DPA treatment compared to vehicle control. However, VASP phosphorylation at serine 239, a substrate of PKG, was enhanced in platelets treated with 11‐oxy‐DPA or 14‐oxy‐DPA treatment. Thus, this suggest that 11‐oxy‐DPA and 14‐oxy‐DPA inhibit platelet activation through a novel cGMP‐PKG‐dependent pathway. This is the first study to demonstrate the novel metabolites of ω‐6 DPA generated by 12‐LOX have role in regulating platelet activation through the cGMP‐PKG signaling pathway and expands potential targets for future therapeutic intervention for the prevention of thrombotic events leading to myocardial infarction and stroke.Support or Funding InformationF31 HL129481GM105671
Tetramethylpyrazine reduces intra‐platelet calcium via suppressing the SGK1/STIM1/Orai1 pathway
Authors: Wang Zhentao , Cang Hongbo , Gao Shuibo , Han Lihua , Wu Hong 
Published: November 2023

Tetramethylpyrazine (TMPZ) is frequently administered for cardiovascular diseases. Although TMPZ is thought to inhibit the elevation of intra‐platelet calcium ions (Ca2+), the underlying mechanism remains unknown. Herein, the time‐dependent effect of TMPZ on adenosine diphosphate (ADP)‐induced platelet aggregation was assessed using a turbidimetric approach. Meanwhile, the intracellular Ca2+ concentration following ADP stimulation was determined using a fluo‐4 Ca2+ probe. We then clarified the mechanisms underlying TMPZ intervention by determining the mRNA and protein levels of Ca2+‐related pathway including stromal interaction molecule l (STIM1), Orai1, and serum/glucocorticoid‐regulated protein kinase 1 (SGK1) by real‐time PCR and Western blot, respectively. The results showed that TMPZ significantly reduced platelet aggregation in a dose‐dependent manner. In addition, TMPZ markedly decreased intracellular Ca2+ concentration following ADP stimulation. Treatment with 2‐mM TMPZ inhibited ADP‐induced expression of STIM1 and Orai1 in platelets at both the mRNA and protein levels, and also reduced the expression of the SGK1 protein. In summary, TMPZ reduces the intra‐platelet Ca2+, which then inhibits platelet activation and thrombosis. The underlying mechanism is presumed to be through the suppression of the SGK1/STIM1/Orai1 pathway.Support or Funding InformationThis work was supported by grants from the Scientific Research Foundation for the Returned Oversea Chinese Scholars, State Ministry of Education of China and Collaborative Innovation Center of Henan University of Traditional Chinese Medicine
Circulating Mitochondrial (mt) DNA Fragments as Precursors of Inflammation in Early Stages of Atherogenesis
Authors: Ruchko Mykhaylo V. , Pastukh Viktor M. , Sukhanov Sergiy , Gorodnya Olena M. , Danchuk Svitlana D. , Gillespie Mark N. 
Published: November 2023

Atherosclerosis and its vascular complications are the leading cause of morbidity and mortality among Western populations. It is widely accepted that inflammation plays a major role in all stages of atherogenesis, including its initial phases. However, the mechanisms of initiation of inflammatory response predisposing to the development of atherosclerosis are not well understood. Mitochondrial dysfunction underlies many key processes in the development of atherosclerosis, including generation of reactive oxygen species, apoptosis, and inflammation. There is growing evidence that oxidative mtDNA damage can directly promote atherosclerosis. The mechanism linking mitochondrial damage to atherogenesis remains unclear, but may include initiation of inflammation by circulating mtDNA fragments known as DAMPs (Damage Associated Molecular Patterns). Previously, we have demonstrated that mice deficient in DNA repair glycosylase Ogg1 and fed a high fat diet developed larger fatty streaks in the aortic valve as a sign of early atherogenesis that was correlated with higher mtDNA oxidative damage in cardiac tissue and elevated content of plasma mtDNA fragments in comparison to wild type animals. In the present study we tested the hypothesis that development of atherosclerosis in animal model is associated with increased abundance of circulating pro‐inflammatory mtDNA fragments. ApoE‐null and wild type mice of increasing age were analyzed for atherosclerotic burden within the aortic valve and for abundance of mtDNA fragments in plasma. ApoE‐null mice develop atherosclerotic plaque in the aortic valve either spontaneously with age or after feeding a high fat diet. Early lesions were detected in ApoE‐null mice by 3 months of age whereas in 5‐month old animals they became prominent. Atherosclerotic burden reached 30% of valve area in 10‐month old animals and remained at that level in 12‐month old mice. Wild type animals did not develop lesions within the aortic valve at any age. The abundance of circulating mtDNA fragments was higher in ApoE‐null mice in all age groups when compared to wild type animals. Importantly, the largest relative increase in mtDNA fragments (5 to 6.5 fold vs. age‐matched controls) was observed in ApoE‐null mice at 3 and 5 months. These results indicate that circulating mtDNA fragments could be precursors of inflammation development in early stages of atherogenesis and suggest that efficient maintenance of mtDNA integrity could be an important target for atheroprotection.Support or Funding InformationNIH R01 HL113614
Functional Analyses of Reticulated Platelets
Authors: Hayman Melissa , Armstrong Paul , Chan Melissa , Warner Timothy 
Published: November 2023

ObjectiveTo isolate reticulated platelets from platelet rich plasma and consequently assess functional reactivity and mRNA content.IntroductionThe platelet population is a heterogeneous mix of various sub‐populations, potentially each with distinct biological capabilities. Of particular interest is the newly formed reticulated sub‐population of platelets, so called because of the presence of RNA in their cytoplasm. This subpopulation is estimated to account for roughly 10% of the total platelet population and has been reported to have a higher thrombotic potential than the non‐reticulated population.MethodsFlow cytometry was used to identify reticulated platelets within platelet rich plasma following staining with thiazole orange. The top 10% of thiazole orange positive cells was selected as the ‘reticulated population’, the middle 50% as the ‘intermediate population’ and the bottom 30% as the ‘non‐reticulated population’. mRNA content in each of the subpopulations was determined using qRT‐PCR. Platelet aggregation and P‐selectin expression, in response to various agonists, were also assessed using flow cytometry. Imaging flow cytometry permitted the localisation and relative assessment of the contribution of both reticulated and non‐reticulated platelets to the formation of aggregates.ResultsReticulated platelets were confirmed to contain more mRNA than either the intermediate or non‐reticulated populations (average Ct value: PF4, 23.9 vs 27.9 vs 29.9; TUBB1, 25.7 vs 30.4 vs 32.8; ITGA2B, 27.0 vs 31.9 vs 33.9). Following exposure to platelet agonists the percentages of reticulated platelets within the single platelet population were significantly reduced compared to vehicle (AA 1 mM, 2.4% vs 9.4%; ADP 20 μM, 6.2% vs 9.6%; collagen 1 μg/mL, 6.0% vs 9.1%; TRAP‐6 25 μM, 1.3% vs 9.1%). Image analysis also indicated that reticulated platelets were more often present at the centre of platelet aggregates. Of all formed aggregates, reticulated platelets were present in 95±2% and non‐reticulated platelets in 42±6%. In addition, reticulated platelets had a significantly higher fold change in the expression of cell surface P‐selectin (CD62P) than non‐reticulated platelets following exposure to TRAP‐6 (2.5 μM, 2.6±0.4 fold greater; 25 μM, 2.7±0.4 fold greater; p=<0.05).ConclusionUpon activation reticulated platelets, i.e. those containing more mRNA, are disproportionally recruited to the centre of forming aggregates demonstrating that this platelet sub‐population are important to the early stages of aggregate formation. Thus reticulated platelets present an interesting target for research as they are more reactive than non‐reticulated platelets and may be early drivers of thrombus formation.Support or Funding InformationWork was generously supported by Barts and the London School of Medicine and Dentistry and The Wellcome Trust (101604/Z/13/Z).