Rat Aorta Research Articles

#170 Vasoconstriction Inhibiting Factor: An endogenous inhibitor of vascular calcification as calcimimetic of calcium-sensing receptor

Abstract Background and Aims Patients with chronic kidney disease (CKD), have an increased risk of cardiovascular disease due to the massively accelerated calcification they develop. Vascular calcification is a highly regulated process mediated by different inducers and inhibitors. Recently, the peptide ‘vasoconstriction inhibiting factor’ (VIF) was isolated from bovine adrenal glands and has been described as an inhibitor of the angiotensin II-induced vasoconstriction. Angiotensin II inhibits calcium deposition, but VIF effect on vascular calcification is still unknown. In the present study, VIF has been characterised on vascular calcification assays. Method The effect of VIF was analysed in vitro in human aortic smooth muscle cells (hAoSMC) and ex vivo in rat aortic rings, both cultivated under high phosphate concentrations. VIF was also studied in vivo in rats treated with vitamin D and nicotine (calcification model). Results VIF inhibits calcium deposition in all the models studied. Furthermore, in hAoSMC VIF reduces the production of ROS and the initiation of diverse cascades in the cells, like activation of inflammatory cytokines and MAPK kinases, which in turn trigger the expression of various genes involved in the development of vascular calcification. Furthermore, in presence of VIF the population of apoptotic cells, directly linked to vascular calcification, is decreased. Calcium-sensing receptor (CaSR) has been found as VIF binding partner, pointing to VIF as a new calcimimetic of this receptor, that inhibits vascular calcification by increasing the production of carboxylated Matrix Gla Proteins (cMGP). Conclusion In conclusion, VIF is a new potent endogenous inhibitor of vascular calcification acting as a calcimimetic of CaSR, that leads to an increase cMGP production. This finding represents a basis for a new target for the prevention and therapy of patients with increased vascular calcification and shows an encouraging perspective for the future.

Two methods of isolation of rat aortic smooth muscle cells with high yield.

Vascular smooth muscle cells (VSMCs) are an integral part of blood vessels and are the focus of intensive research in vascular biology, translational research, and cardiovascular diseases. Though immortalized vascular smooth muscle cell lines are available, their use is limited, underscoring the need for primary VSMCs. There are several methods for isolating primary cells from mice. However, the isolation method from rat blood vessels requires optimization, given the differences in the aorta of mice and rats. Here we compare two methods for VSMCs isolation from rats: enzymatic digestion and the "block" method. We observed a significantly higher yield of VSMCs using the enzymatic digestion method. We further confirmed that VSMCs expressed well-established VSMC-specific markers (calponin) with both methods and observed the persistence of this marker up to 9 passages, suggesting a continuation of the secretory phenotype of VSMCs. Overall, this work compares two methods and demonstrates a practical and effective method for isolating VSMCs from rat aorta, providing vascular biologists with a valuable and reliable experimental tool.

Effects of Sacha Inchi (Plukenetia volubilis) Oil on Serum Interleukin 6 Levels and Foam Cell Count in Male Wistar Rats (Rattus norvegicus) Fed a High Fat and Fructose Diet

A high-fat, high-fructose diet induces pro-inflammatory agents such as Interleukin-6 (IL-6) and hyperlipidemia, leading to atherosclerosis. Sacha inchi oil, rich in alpha-linolenic acid (ALA), has anti-inflammatory and lipid-modulating properties. This study aimed to examine the effect of sacha inchi oil on serum IL-6 levels and foam cell count in the aorta of wistar rats on a high-fat, high-fructose diet. This experiment is a true experimental study with a post-test-only control group design, 30 male wistar rats were divided into five groups: normal control (K0), high fat and fructose diet (HFFD) (K1), and three treatment groups receiving HFFD with sacha inchi oil at doses of 0.27 ml/200gBW/day (P1), 0.54 ml/200gBW/day (P2), and 1.08 ml/200gBW/day (P3) for 28 days. On day 29, serum IL-6 levels were measured using ELISA. Mean serum IL-6 levels were K1 (8.465 ng/L), P1 (4.294 ng/L), P2 (3.197 ng/L), P3 (1.379 ng/L), and K0 (1.340 ng/L), with significant differences among groups (p<0.05) except between K0 and P3 (p>0.05). Mean foam cell counts were K1 (55.3 cells/fov), P1 (47.1 cells/fov), P2 (53.2 cells/fov), P3 (52.6 cells/fov), and K0 (44.4 cells/fov), with significant differences between K0 and K1, K1 and P1, K0 and P2, and K0 and P3 (p<0.05). Sacha inchi oil significantly reduced IL-6 levels at all doses compared to the negative control (p<0.05), with the 1.08 ml/200 g/day dose being most effective. The 0.27 ml/200 g/day dose inhibited foam cell formation in HFFD rat. In conclusion, sacha inchi oil reduces serum IL-6 levels and inhibits foam cell formation in male wistar rats on a high-fat, high-fructose diet.

Development of a method for quantitative determination of nitric oxide (NO) in rat tissues based on high-performance liquid chromatography and mass spectrometry

A quantitative assessment of nitric oxide (NO) production in body tissues is an urgent problem in pharmacology and biochemistry. The study of physiological processes occurring with the participation of NO, as well as the metabolism and pharmacodynamics of pharmacological agents from the group of NO donors, requires the introduction of accurate and reproducible methods for the quantitative determination of this metabolite in biological media.The aim of the study was to develop the HPLC-MS/MS methods for the quantitative determination of NO in various tissues of rats.Materials and methods. The indirect NO quantification was based on estimation of the level of more stable metabolites: nitrites and nitrates extracted from rat tissues by homogenization with water. The reduction of nitrates to nitrites was carried out using nitrate reductase. The derivatization of nitrites was based on a reaction with Griess reagent. The resulting azo dye was determined by HPLC-MS/MS using an Agilent InfinityLab Poroshell 120 EC-C18 4.6×100 mm, 2.7 μm analytical column. The total chromatographic analysis time was 12 minutes, and the analyte retention time was 6.1 minutes. The analytical range of the method was 0.1–100.0 nmol (in terms of nitrite) per 1 ml of plasma or tissue homogenate.Results. The developed a bioanalytical method was validated according to the following parameters: a selectivity, a matrix effect, a recovery degree, a sample transfer, an analytical range linearity, a lower limit of quantification (LLOQ), an intra- and inter-assay accuracy and precision, and a stability at all the stages of the analysis. To test the method, the NO content in the plasma, brain, heart, aorta and lungs of rats was determined.Conclusion. The developed bioanalytical HPLC-MS/MS methods fully meets the validation requirements. The metrological characteristics of the technique make it possible to highly accurately estimate the NO production in various tissues of rats, which is undoubtedly relevant and in demand in the study of pathological processes as well as the mechanism of action of pharmacological agents from the group of NO donors.

Evaluation of Anti-angiogenic Activity of Iraqi Mesembryanthemum cordifolium using rat aorta ring assay

New blood vessels are produced during angiogenesis, which is necessary in both normal and abnormal physiological and pathologic states. The use of non-toxic plant extracts that inhibit angiogenesis might lead to more focus on the use of natural sources as a pivotal therapeutic agent in the treatment of many diseases and malignancies. Mesembryanthemum cordifolium is belongs to the Aizoaceae family and commonly known as the Heartleaf Ice Plant or Tiny Sun Rose. The plant was cultivated in the city of al Diwaniyah. No previous research has been conducted on this plant and its antiangiogenic properties. Hence, this research aimed to assess the antiangiogenic capacity of the Iraqi M. cordifolium. For this study, male albino rats aged 12 to 14 weeks were used. With a Soxhlet apparatus, the defatted plant components were extracted in 85 % methanol for 12 h. Subsequently, each fraction was fractionated using petroleum ether, chloroform and ethyl acetate and each fraction underwent phytochemical analysis. Rat aortic rings were infused with 100 ?g/mL of each plant component and placed in a growth medium. In addition, the dose-response relationship was assessed for the most active fraction at 6 different concentrations ranging from 6.25 ?g/mL to 100 ?g/mL. Compared to the negative control (1 % DMSO), the results demonstrated that fractions of M. cordifolium inhibit the growth of blood vessels in a dose-dependent manner; chloroform demonstrates the most potent effect. The results indicate that the chloroform fraction derived from M. cordifolium has a good ability to limit blood vessel development. This makes it a viable option for future investigation as an angiogenesis inhibitor.

Effect of Sodium Fluoride on Oxidative Stress and Antioxidant System in Male Wistar Rats

This research aimed to examine the effect of sodium fluoride (NaF) on oxidative stress and antioxidant systems in the serum, aorta, and cardiac tissues of male Wistar rats. The increased attention to sodium fluoride and its detrimental effects on human health in recent years has led to concerns. Excessive ingestion and accumulation of sodium fluoride from water, toothpaste, and pesticides could initiate oxidative stress in the blood, aorta, and heart. This heightened oxidative stress and disturbance in the antioxidant system pose a significant risk and may predispose the body to severe vascular and cardiovascular-related diseases, which are leading causes of global mortality. In this study, ten male Wistar rats weighing 150-180g were grouped into two (n=5). Group I Control received distilled water, and Group 2 received 10mg/kg NaF orally for 21 days. Markers of oxidative stress malondialdehyde (MDA) and reduced glutathione (GSH), antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and total antioxidant capacity (TAC) were measured in the serum, aorta, and heart. Data were analyzed using descriptive statistics of student t-test at α0.05. The data were presented as mean ± SEM. The oral administration of sodium fluoride resulted in an elevation of MDA levels in the serum, aorta, and heart of the rats exposed to NaF. Additionally, the oral administration of sodium fluoride led to a reduction in superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) levels in the serum, aorta, and heart of rats exposed to NaF. Furthermore, the oral administration of sodium fluoride resulted in a decrease in the total antioxidant capacity (TAC) in the serum, aorta, and heart of rats exposed to sodium fluoride. This study demonstrated that sodium fluoride exposure diminishes the antioxidant capacity in the serum, aorta, and heart, indicating that sodium fluoride exposure induces oxidative stress, potentially affecting the functions of the aorta and heart. Therefore, the continuous exposure to fluoride in populations residing in regions with endemic fluorosis worldwide should be a topic of considerable concern. This research work was self-funded. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Kidney Stem Cell Reprogramming Drives Spontaneous Hypertension via Exosomes

The spontaneously hypertensive rat (SHR) is a commonly used animal model for studying primary hypertension. However, the precise mechanism of these rats developing hypertension remains to be defined. Here, we have first demonstrated that kidney stem cell reprogramming is a driver for the development of hypertension in SHR rats. In this study, we treated 5-week-old SHR rats with WKY-kidney stem cell-derived exosomes (WKY-Exo) and 5-week-old WKY rats with SHR-Exo, respectively, via i.p. injections. Notably, WKY-Exo effectively blocked the development of hypertension and arterial stiffening in SHRs. It is fascinating that WKY rats developed hypertension and arterial stiffening due to treatment of SHR-Exo. We confirmed that both WKY- and SHR-Exo were up taken into aortic endothelial and smooth muscle cells after i.p. delivery of CD63/GFP-labelled exosomes. We demonstrated that WKY-Exo can improve vascular functions and arterial compliance of SHR rats as evidenced by preventing the development of arterial stiffness and restoring renal artery endothelial function. We also demonstrated that the vascular functions were impaired in WKY rats after SHR-Exo treatment. Furthermore, our data show that expression of Elastin, Emilin1, and Myh11 was significantly downregulated in the aorta of SHR rats, which can be repaired by WKY-Exo treatment. SHR-Exo decreased expression of Elastin and Emilin1 without affecting Myh11 in the aorta of WKY rats, suggesting that Elastin and Emilin1 deficiency could contribute to arterial stiffening leading to the development of spontaneous hypertension. Proteomic analysis reveals the enrichment of protein-protein interaction network for elastic fiber assembling, blood vessel development, and regulation of angiogenesis in WKY-Exo vs SHR-Exo. In conclusion, our data first demonstrates that kidney stem cell reprograming is a driver for the development of spontaneous hypertension in SHR rats. WKY-KSCs-derived exosome contains a proteome profile promoting arterial repair, which may provide a therapeutic means to prevent and treat primary hypertension. NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Protection from hypertension phenotype by a mitochondrial division inhibitor in mice infused with angiotensin II

Mitochondrial dysfunction has been implicated in various types of cardiovascular diseases including hypertension. Mitochondrial fission fusion balance is critical for mitochondrial quality control, whereas enhanced fission has been reported in several models of cardiovascular diseases. However, limited information is available regarding the contribution of mitochondrial fission in hypertension. Here, we have tested our hypothesis that inhibition of mitochondrial fission attenuates development of hypertension and associated vascular remodeling. C57BL6 mice were infused with angiotensin II for 2 weeks with or without treatment of mitochondrial fission inhibitor mdivi1. Cultured vascular endothelial cells and adventitial fibroblasts from rat aortas were stimulated with angiotensin II in the presence or absence of mdivi1. In C57BL6 mice infused with angiotensin II for 2 weeks, co-treatment of mitochondrial fission inhibitor mdivi1 significantly inhibited angiotensin II-induced development of hypertension assessed by telemetry. Histological assessment of hearts and aortas demonstrated that mdivi1 inhibited vessel fibrosis and hypertrophy induced by angiotensin II. This was associated with attenuation of angiotensin II-induced decline in mitochondrial aspect ratio seen in both endothelium and media of aortas. Mdivi1 also mitigated angiotensin II-induced cardiac hypertrophy assessed by heart weight body weight ratio as well as by echocardiogram. Proteomic analysis on endothelial cell culture media with angiotensin II and/or mdivi1 treatment revealed that mdivi1 inhibited endothelial cell hypersecretory phenotype induced by angiotensin II. In addition, mdivi1 attenuated angiotensin II-induced protein induction of periostin, a myofibroblast marker in cultured vascular fibroblasts. In conclusion, these data suggest that mdivi1 prevented angiotensin II-induced hypertension and cardiovascular remodeling via multicellular mechanisms in the vasculature. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 3136: Protection From Hypertension Phenotype By A Mitochondrial Division Inhibitor In Mice Infused With Angiotensin II

Mitochondrial dysfunction has been implicated in various types of cardiovascular diseases including hypertension. Mitochondrial fission fusion balance is critical for mitochondrial quality control, whereas enhanced fission has been reported in several models of cardiovascular diseases. However, limited information is available regarding the contribution of mitochondrial fission in hypertension. The goal of this study is to explore the role of mitochondrial fission in hypertension and associated vascular pathology. Accordingly, we have tested our hypothesis that inhibition of mitochondrial fission attenuates development of hypertension and associated vascular remodeling. C57BL6 mice were infused with 1000 ng/kg/min angiotensin II for 2 weeks with or without treatment of mitochondrial fission inhibitor mdivi1 (25 mg/kg/day). Cultured vascular endothelial cells and adventitial fibroblasts from rat aortas were stimulated with 100 nM angiotensin II in the presence or absence of mdivi1 (20 uM). In C57BL6 mice infused with angiotensin II for 2 weeks, co-treatment of mitochondrial fission inhibitor mdivi1 significantly inhibited angiotensin II-induced development of hypertension assessed by telemetry (mean 149 vs 119 mmHg, day 9 p<0.01). Histological assessment of hearts and aortas demonstrated that mdivi1 inhibited vessel fibrosis and hypertrophy induced by angiotensin II. This was associated with attenuation of angiotensin II-induced decline in mitochondrial aspect ratio seen in both endothelium and media of aortas (aortas 1.620 vs 2.168, p<0.001). Mdivi1 also mitigated angiotensin II-induced cardiac hypertrophy assessed by heart weight body weight ratio (7.823 vs 6.336 mg/g, p<0.001) as well as by echocardiogram. Proteomic analysis on endothelial cell culture media with angiotensin II and/or mdivi1 treatment revealed that mdivi1 inhibited endothelial cell hypersecretory phenotype induced by angiotensin II. In addition, mdivi1 attenuated angiotensin II-induced protein induction of periostin, a myofibroblast marker in cultured vascular fibroblasts. In conclusion, these data suggest that mdivi1 prevented angiotensin II-induced hypertension and cardiovascular remodeling via multicellular mechanisms in the vasculature.

Abstract 2031: Supramolecularly Fabricated Non-thrombogenic Small Diameter Vascular Graft

Background: Small diameter (<6mm) arterial reconstruction often requires the use of autologous and synthetic vascular substitutes. While autologous conduits are the gold standard, because of their limited availability synthetic conduits are often used. However, synthetic conduits can lead to poor outcomes due to thrombosis and lumen occlusion occluding the lumen and result in graft failure. By modulating the biopolymer properties of the synthetic conduits, graft failure can be prevented. In this study, we present a thromboresistant synthetic vascular graft (zetagraft) fabricated with tunable biopolymers for small diameter arterial reconstructions. Methods: Zetagrafts were fabricated by the solvent extraction driven self-assembly using a concentric tubular assembly device using polyvinyl alcohol dissolved in dimethyl sulfoxide (15% w/v). We characterized the physicochemical and mechanical properties of zetagraft by fourier transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD), optical and scanning electron microscopy, and tensiometry. In-vitro studies were performed on the zetagraft using fibroblast, HUVEC and MVEC cell lines. Ex vivo hemocompatibility analysis was performed with fresh porcine blood. The zetagrafts were implanted in a rat aorta replacement model to evaluate its thromboresistance. Results: The zetagraft was uniform, highly stretchable, and self-healing when punctured with a needle. Blood circulation followed by FT-IR and XRD analysis showed no protein adsorption on the luminal surface. Immuno fluorescence staining revealed the absence of platelet and blood cell adsorption on the zetagraft lumen, demonstrating its thromboresistance. In vivo implantation of the zetagraft in rat abdominal aorta replacement model revealed the absence of thrombosis and other blood component adsorption on the zetagraft lumen. Conclusions: Advances in material science allow for fabrication of more tunable biocompatible small vascular grafts. In this study we demonstrated the development of a thromboresistant zetagraft. Our early results are very promising and zetagraft will provide a novel small vascular graft and thus fulfil an important unmet clinical need.

Vasodilation Effects of Ketapang (<i>Terminalia catappa</i> Linn) Fruit on Aortic Rings Rat

Hypertension is an important risk factor for cardiovascular disease. Hypertension requires long-term treatment to keep blood pressure under control. Exploration found that antihypertensive drugs have economic value. East Kalimantan is rich in medicinal plant diversity and needs to be explored to discover new drugs for hypertension. Ketapang fruit (Terminalia catappa Linn) has the potential to have a vasodilation effect so research is needed to explore vasodilation activity in vitro. This research to determine the vasodilation activity of Terminalia catappa fruit using isolated aortic ring bioassay. Twelve Wistar rats were used for bioassays isolated aortic ring with endothelium. The aortic rings is precontracted with phenylephrine. This study used two test groups, Control group and Terminalia catappa fruit ethanol extract group (FTC). The FTC and Control concentrations were given cumulatively in chamber of isolated aortic ring. Log concentration -2 FTC (-11.8 ± 3.5)% and Control (0.0 ± 0.0)%, p = 0,002; log concentration -1,5 FTC (-22.8 ± 2.5)% and Control (0.0 ± 0.0)%, p = 0,002; log concentration -1,0 FTC (-41.5 ± 2.8)% and Control (0.0 ± 0.0)%, p = 0,002; log concentration -0,5 FTC (-58.7 ± 3.1)% and Control (0.0 ± 0.0)%, p = 0,002; log concentration 0 FTC (-79.0 ± 3.4)% and Control (0.0 ± -1.9)%, p = 0,001; log concentration 0,5 FTC (-93.8 ± 3.0)% and Control (0.2 ± 1.5)%, p = 0,002. FTC causes vasodilation in isolated rat aortic rings with endothelium.

Saponins from seeds of Zizyphus jujuba Mill. var. spinosa exhibit vasorelaxant effects on rat isolated aorta

AbstractAimThe vasorelaxant saponins jujuboside A(1), A1(2), B(3), and acetyljujuboside B(4) were isolated from 50% MeOH extracts of seeds of Zizyphus jujuba Mill. var. spinosa Hu ex H.F. Chou (Rhamnaceae), and their effects were studied using isolated rat aortic rings.MethodDried jujube seeds were crushed and extracted with 50% MeOH, and the extracts were concentrated under reduced pressure. Vasorelaxant effects of this extracts were assessed on rat isolated aorta rings. The aorta rings were placed in a well‐oxygenated bath of modified Krebs–Henseleit solution and the mechanical tension was measured isometrically.ResultsThe crude extract was fractionated by chromatographic methods with the guidance of vasorelaxant activity, and four saponins (1~4) were isolated. These compounds were identified using nuclear magnetic resonance (NMR) studies. Compounds 1~4 showed slow relaxation activity against norepinephrine (NE)‐induced contractions of rat aorta with/without endothelium. Treatment with 1~4 did not significantly inhibit NE‐induced vasoconstrictions. Compounds 1~4 inhibited vasoconstriction induced by high‐concentration potassium (60 mM) and NE in the presence of nicardipine but did not affect phorbol 12‐myristate 13‐acetate‐induced vasoconstriction.ConclusionThese results suggest that inhibition of NE‐induced vasoconstriction by 1~4 is due to the inhibition of receptor‐operated Ca2+ channels and voltage‐dependent Ca2+ channels or inhibition of intracellular calcium modulations.

Artificial light at night affects the daily profile of pulse pressure and protein expression in the thoracic aorta of rats

Artificial light at night (ALAN) disrupts 24-h variability of blood pressure, but the molecular mechanisms underlying these effects are unknown. Therefore, we analysed the daily variability of pulse pressure, the maximum value of acceleration rate of aortic pressure (dP/dt(max)) measured by telemetry and protein expression in the thoracic aorta of normotensive male rats exposed to ALAN (1–2 lx) for 3 weeks. Daily, 24-h variability of pulse pressure and dP/dt(max) was observed during a regular light/dark regimen with higher values during the dark compared to the light phase of the day. ALAN suppressed 24-h variability and enhanced ultradian (<12-h) periods of pulse pressure and dP/dt(max) in duration-dependent manners. From beat-to-beat blood pressure variability, ALAN decreased low-frequency bands (a sympathetic marker) and had minimal effects on high-frequency bands. At the molecular level, ALAN decreased angiotensin II receptor type 1 expression and reduced 24-h variability. ALAN caused the appearance of 12-h oscillations in transforming growth factor β1 and fibulin 4. Expression of sarco/endoplasmic reticulum Ca2+-ATPase type 2 was increased in the middle of the light and dark phase of the day, and ALAN did not affect its daily and 12-h variability. In conclusion, ALAN suppressed 24-h variability of pulse pressure and dP/dt(max), decreased the power of low-frequency bands and differentially affected the expression of specific proteins in the rat thoracic aorta. Suppressed 24-h oscillations by ALAN underline the pulsatility of individual endocrine axes with different periods, disrupting the cardiovascular control of central blood pressure.

β,γ-Methylene-ATP and its metabolite medronic acid affect both arterial media calcification and bone mineralization in non-CKD and CKD rats.

Arterial media calcification or pathological deposition of calcium-phosphate crystals in the vessel wall contributes significantly to the high mortality rate observed in patients with CKD. Extracellular nucleotides (ie, ATP or UTP) regulate the arterial calcification process by interacting with (1) purinergic receptors and (2) breakdown via ecto-nucleotidases, such as ectonucleotide pyrophosphatase/phosphodiesterase NPP1 or NPP3, affecting the local levels of calcification inhibitor, pyrophosphate, and stimulator inorganic phosphate (PPi/Pi ratio). Also, it has been shown that ATP analogs (ie, β,γ-methylene-ATP [β,γ-meATP]) inhibit vascular smooth muscle cell calcification in vitro. In the first experiment, daily dosing of β,γ-meATP (2mg/kg) was investigated in rats fed a warfarin diet to trigger the development of non-CKD-related arterial medial calcifications. This study showed that β,γ-meATP significantly lowered the calcium scores in the aorta and peripheral vessels in warfarin-exposed rats. In a second experiment, daily dosing of 4mg/kg β,γ-meATP and its metabolite medronic acid (MDP) was analyzed in rats fed an adenine diet to promote the development of CKD-related arterial medial calcification. Administration of β,γ-meATP and MDP did not significantly decrease aortic calcification scores in this model. Moreover, both compounds induced deleterious effects on physiological bone mineralization, causing an imminent risk for worsening the already compromised bone status in CKD. Due to this, it was not possible to raise the dosage of both compounds to tackle CKD-related arterial calcification. Again, this points out the difficult task of targeting solely ectopic calcifications without negatively affecting physiological bone mineralization. On the other hand, aortic mRNA expression of Enpp1 and Enpp3 was significantly and positively associated with aortic calcification scores, suggesting that normalizing the aortic NPP1/3 activity to control values might be a possible target to treat (CKD-induced) arterial media calcifications.

Resveratrol modulates signalling to inhibit vascular smooth muscle cell proliferation induced by angiotensin II and high glucose

Abstract Objectives The proliferation of vascular smooth muscle cells (VSMCs) induced by hyperglycemia plays a pivotal role in the development of atherosclerosis and restenosis. This study aims to examine the impact of angiotensin II (Ang II) and high glucose on VSMC proliferation and the phosphorylation status of key signalling proteins, specifically ERK1/2, Akt, and STAT3. Furthermore, we assess the inhibitory effects of resveratrol, a polyphenolic compound, on these signalling pathways. Methods Primary vascular smooth muscle cells (VSMCs) isolated from rat aortas were cultured in both standard media (SM: 5.5 mM) and high glucose media (HGM: 25 mM) and then treated with Ang II (100 nM). Proliferation was assessed using the WST-1 assay, and protein analysis was performed through immunoblotting. Results Ang II increased VSMC proliferation by 39 % in standard glucose environments and 17 % in high glucose environments. Resveratrol effectively suppressed Ang II-induced VSMC proliferation in both media. Furthermore, resveratrol inhibited the phosphorylation of ERK1/2 and Akt. Ang II also induced STAT3 phosphorylation by 29 and 18.5 % in SM and HGM, respectively. However, resveratrol treatment reduced STAT3 phosphorylation to control levels. Conclusions These findings demonstrated that resveratrol reduces VSMC proliferation induced by Ang II and high glucose conditions, exerting its inhibitory effects by suppressing ERK1/2, Akt, and STAT3 phosphorylation. These results provide valuable insights into the cardioprotective properties of resveratrol.

Novel Ruthenium‐based Nitrosyl Complexes: NO Donation and Vasorelaxant Potentials for Cardiovascular Therapeutics

AbstractNitrosyl ruthenium complexes have emerged as promising platforms for the controlled delivery of nitric oxide (NO) and nitroxyl (HNO), both of which possess significant therapeutic implications. Considering this scenario, we synthesized and characterized the metal complex cis‐[RuNO(phen)2(2MIM)]3+ and its precursors, where phen and 2MIM correspond to 1,10‐phenantroline and 2‐methylimidazole, respectively. Comprehensive structural elucidation was undertaken using a combination of spectroscopic and electrochemical methodologies, including XANES/EXAFS experiments. This structural data was further validated through DFT computational analyses. We demonstrated that such compound can release NO and HNO in the presence of thiol‐based substrates. Not only this, but the same metal complex can also delivery nitric oxide under irradiation with visible light (λ=460 nm). The nitrosyl complex and its derivatives displayed marked vasodilatory capabilities as evidenced in assays involving isolated rat aorta rings. They also exhibited commendable antioxidant activity in free radical scavenging assays. Collectively, based on these data, this nitrosyl ruthenium compound is a potential therapeutic candidate, especially in the field of cardiovascular pathology like atherosclerosis, thereby deserving further in‐depth investigations.

Biomimetic approach to the design of artificial small‑diameter blood vessels

Objective: To create 2-mm diameter multilayer porous tubular scaffolds (PTS) with characteristics that resemble small-diameter native blood vessels in terms of characteristics.Materials and methods. PTS made of polycaprolactone (PCL, MM 80000) with a PCL-made sealing coat/layer with gelatin addition (PCL-gelatin) with a diameter of 2 mm were created by electrospinning (NANON-01A). Bioactive coating was applied to the PTS surface by sequential incubation in solutions of bovine serum albumin, heparin (Hp), and platelet lysate (PL). Cytotoxicity was investigated under conditions of direct contact of PTS with a monolayer of NIH/3T3 mouse fibroblasts. Viability of human umbilical vein endothelial cells (EA.hy926) was evaluated using Live/Dead® Viability/Cytotoxicity Kit. Permeability and blood flow parameters of the PTS implanted in the infrarenal section of the rat aorta were recorded using Doppler imaging.Results. A three-layer PTS construct with an inner diameter of 2 mm was developed. Its inner and outer layers were formed from 0.2 mL of PCL solution, and the middle sealing coat/layer was from 0.5 mL of PCL with addition of 30% (by weight of polymer) gelatin. Introduction of the sealing coat/layer reduced surgical porosity (SP) from 56.2 ± 8.7 mL/(cm2·min) for a single-layer PTS made of pure PCL to 8.9 ± 2.6 mL/(cm2·min) for a three-layer PTS. The resulting PTS demonstrated physicomechanical characteristics similar to those of native blood vessels; it also showed no cytotoxicity. Application of a bioactive coating of Hp and PL allowed for increased in vitro adhesion and proliferation of endothelial cells. The technique of implantation of 10 mm long fragments of three-layer PTS into the infrarenal section of a rat aorta was corrected, thus minimizing blood loss and narrowing the anastomosis site. In an acute experiment, it was proven that the prostheses were patent and that blood flow parameters (systolic and diastolic velocity, resistivity index) were close to the corresponding indicators of native rat aorta.Conclusion. The developed three-layer PTS constructs have low SP and physicomechanical properties close to those of native blood vessels. Bioactive coating improves the in vitro matrix properties of PTS relative to human endothelial cells. At short-term implantation into the aorta of experimental animals, PTS showed no early thrombosis, while blood flow parameters were close to those of native rat aorta. Thus, three-layer PTS with bioactive coating can be used as a scaffold for creation of in situ tissue-engineered construct of a small-diameter blood vessel.

Icariin attenuates vascular endothelial dysfunction by inhibiting inflammation through GPER/Sirt1/HMGB1 signaling pathway in type 1 diabetic rats

Icariin, a flavonoid glycoside, is extracted from Epimedium. This study aimed to investigate the vascular protective effects of icariin in type 1 diabetic rats by inhibiting high-mobility group box 1 (HMGB1)-related inflammation and exploring its potential mechanisms. The impact of icariin on vascular dysfunction was assessed in streptozotocin (STZ)-induced diabetic rats through vascular reactivity studies. Western blotting and immunofluorescence assays were performed to measure the expressions of target proteins. The release of HMGB1 and pro-inflammation cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The results revealed that icariin administration enhanced acetylcholine-induced vasodilation in the aortas of diabetic rats. It also notably reduced the release of pro-inflammatory cytokines, including interleukin-8 (IL-8), IL-6, IL-1β, and tumor necrosis factor-alpha (TNF-α) in diabetic rats and high glucose (HG)-induced human umbilical vein endothelial cells (HUVECs). The results also unveiled that the pro-inflammatory cytokines in the culture medium of HUVECs could be increased by rHMGB1. The increased release of HMGB1 and upregulated expressions of HMGB1-related inflammatory factors, including advanced glycation end products (RAGE), Toll-like receptor 4 (TLR4), and phosphorylated p65 (p-p65) in diabetic rats and HG-induced HUVECs, were remarkably suppressed by icariin. Notably, HMGB1 translocation from the nucleus to the cytoplasm in HUVECs under HG was inhibited by icariin. Meanwhile, icariin could activate G protein-coupled estrogen receptor (GPER) and sirt1. To explore the role of GPER and Sirt1 in the inhibitory effect of icariin on HMGB1 release and HMGB-induced inflammation, GPER inhibitor and Sirt1 inhibitor were used in this study. These inhibitors diminished the effects of icariin on HMGB1 release and HMGB1-induced inflammation. Specifically, the GPER inhibitor also negated the activation of Sirt1 by icariin. These findings suggest that icariin activates GPER and increases the expression of Sirt1, which in turn reduces HMGB1 translocation and release, thereby improving vascular endothelial function in type 1 diabetic rats by inhibiting inflammation.

Structural Features of the Wall of the Ascending Aorta of Premature Rats

Structural Features of the Wall of the Ascending Aorta of Premature Rats

Thymoquinone Reverses Homocysteine-Induced Endothelial Dysfunction Via Inhibition of Endoplasmic Reticulum-Stress Induced Oxidative Stress Pathway

Hyperhomocysteinemia causes endoplasmic reticulum (ER) stress, which elevates reactive oxygen species (ROS) and induces endothelial dysfunction, the hallmark of cardiovascular diseases. Nigella sativa seeds contain thymoquinone (TQ), a cardioprotective bioactive component. Nevertheless, research on investigating the effectiveness of TQ in preventing endothelial dysfunction caused by homocysteine (Hcy) is scarce. Therefore, the purpose of this work was to examine the role of TQ in restoring Hcy-induced endothelial dysfunction as well as the mechanisms behind this role. Male Sprague-Dawley (SD) rat aortas were isolated and then co-treated in an organ bath with Hcy and TQ, tauroursodeoxycholic acid (TUDCA), apocynin, or Tempol to examine vascular function. Furthermore, human umbilical vein endothelial cells (HUVECs) were treated with Hcy and TQ, Tempol, apocynin, TUDCA or H2O2 to determine the cell viability via a phase contrast microscope and dye exclusion test. ER stress pathway involvement, ROS and NO bioavailability were investigated using immunoassays and fluorescence staining, respectively. The binding affinity of TQ to GRP78 has been identified using molecular docking. According to our findings, Hcy hindered endothelium-dependent relaxation in an isolated aorta and caused apoptosis in HUVECs. TQ, TUDCA, Tempol, and apocynin were able to counteract these negative effects. In HUVECs, treatment with TQ decreased ROS levels, increased NO bioavailability, and decreased GRP78 and NOX4 protein. According to the molecular docking study outcomes, TQ could attach to GRP78 effectively via a hydrogen bond and a hydrophobic connection to the amino acid at GRP78 ATP binding pocket. Taken together, the findings show that TQ protected endothelial function caused by Hcy via inhibiting ER stress-mediated ROS and eNOS uncoupling.