Adjuvant-induced arthritis promotes vascular hyporesponsiveness to phenylephrine through a nitric oxide-related mechanism.

Arthritis has important cardiovascular repercussions. Phenylephrine-induced vasoconstriction is impaired in rat aortas in the early phase of the adjuvant-induced arthritis (AIA), around the 15th day post-induction. Therefore, the present study aimed to verify the effects of AIA on hyporesponsiveness to phenylephrine in rat aortas. AIA was induced by intradermal injection of Mycobacterium tuberculosis (3.8 mg/dL) in the right hind paw of male Wistar rats (n=27). Functional experiments in isolated aortas were carried out 15 days after AIA induction. Morphometric and stereological analyses of the aortas were also performed 36 days after the induction of AIA. AIA did not promote structural modifications in the aortas at any of the time points studied. AIA reduced phenylephrine-induced contraction in endothelium-intact aortas, but not in endothelium-denuded aortas. However, AIA did not change KCl-induced contraction in either endothelium-intact or denuded aortas. L-NAME (non-selective NOS inhibitor), 1400W (selective iNOS inhibitor), and ODQ (guanylyl cyclase inhibitor) reversed AIA-induced hyporesponsiveness to phenylephrine in intact aortas. 7-NI (selective nNOS inhibitor) increased the contraction induced by phenylephrine in aortas from AIA rats. In summary, the hyporesponsiveness to phenylephrine induced by AIA was endothelium-dependent and mediated by iNOS-derived NO through activation of the NO-guanylyl cyclase pathway.

Ginsenoside Rk1 improves endothelial function in diabetes through activating peroxisome proliferator-activated receptors.

Ginsenoside Rk1, one kind of ginsenoside, is a minor ginsenoside found in Panax ginseng and used as traditional Chinese medicine for centuries. It exhibits anti-tumor and anti-aggregation effects. However, little research has been done on its effect on endothelial function. This study investigated whether ginsenoside Rk1 improved endothelial dysfunction in diabetes and the underlying mechanisms in vivo and in vitro. Male C57BL/6 mice were fed with a 12 week high-fat diet (60% kcal % fat), whereas treatment groups were orally administered with ginsenoside Rk1 (10 and 20 mg per kg per day) in the last 4 weeks. Aortas isolated from C57BL/6 mice were induced by high glucose (HG; 30 mM) and co-treated with or without ginsenoside Rk1 (1 and 10 µM) for 48 h ex vivo. Moreover, primary rat aortic endothelial cells (RAECs) were cultured and stimulated by HG (44 mM) to mimic hyperglycemia, with or without the co-treatment of ginsenoside Rk1 (10 µM) for 48 h. Endothelium-dependent relaxations of mouse aortas were damaged with elevated oxidative stress and downregulation of three isoforms of peroxisome proliferator-activated receptors (PPARs), PPAR-α, PPAR-ß/δ, and PPAR-γ, as well as endothelial nitric oxide synthase (eNOS) phosphorylation due to HG or high-fat diet stimulation, which also existed in RAECs. However, after the treatment with ginsenoside Rk1, these impairments were all ameliorated significantly. Moreover, the vaso-protective and anti-oxidative effects of ginsenoside Rk1 were abolished by PPAR antagonists (GSK0660, GW9662 or GW6471). In conclusion, this study reveals that ginsenoside Rk1 ameliorates endothelial dysfunction and suppresses oxidative stress in diabetic vasculature through activating the PPAR/eNOS pathway.

IL-1ß Inhibition Partially Negates the Beneficial Effects of Diet-Induced Atherosclerosis Regression in Mice.

BACKGROUND: Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions is the global leading cause of death. The most common and effective means to reduce these major adverse cardiovascular events, including myocardial infarction and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, we know little regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. METHODS: Smooth muscle cell lineage-tracing Apoe-/- mice were fed a high-cholesterol Western diet for 18 weeks and then a zero-cholesterol standard laboratory diet for 12 weeks before treating them with an IL (interleukin)-1ß or control antibody for 8 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of smooth muscle cell and other lesion cells by smooth muscle cell lineage tracing combined with single-cell RNA sequencing, cytometry by time-of-flight, and immunostaining plus high-resolution confocal microscopic z-stack analysis. RESULTS: Lipid lowering by switching Apoe-/- mice from a Western diet to a standard laboratory diet reduced LDL cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden, as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1ß antibody treatment after diet-induced reductions in lipids resulted in multiple detrimental changes including increased plaque burden and brachiocephalic artery lesion size, as well as increasedintraplaque hemorrhage, necrotic core area, and senescence as compared with IgG control antibody-treated mice. Furthermore, IL-1ß antibody treatment upregulated neutrophil degranulation pathways but downregulated smooth muscle cell extracellular matrix pathways likely important for the protective fibrous cap. CONCLUSIONS: Taken together, IL-1ß appears to be required for the maintenance of standard laboratory diet-induced reductions in plaque burden and increases in multiple indices of plaque stability.

Removal of endothelial surface-associated von villebrand factor suppresses accelerate datherosclerosis after myocardial infarction.

BACKGROUND: Thromboinflammation involving platelet adhesion to endothelial surface-associated von Willebrand factor (VWF) has been implicated in the accelerated progression of non-culprit plaques after MI. The aim of this study was to use arterial endothelial molecular imaging to mechanistically evaluate endothelial-associated VWF as a therapeutic target for reducing remote plaque activation after myocardial infarction (MI). METHODS: Hyperlipidemic mice deficient for the low-density lipoprotein receptor and Apobec-1 underwent closed-chest MI and were treated chronically with either: (i) recombinant ADAMTS13 which is responsible for proteolytic removal of VWF from the endothelial surface, (ii) N-acetylcysteine (NAC) which removes VWF by disulfide bond reduction, (iii) function-blocking anti-factor XI (FXI) antibody, or (iv) no therapy. Non-ischemic controls were also studied. At day 3 and 21, ultrasound molecular imaging was performed with probes targeted to endothelial-associated VWF A1-domain, platelet GPIbα, P-selectin and vascular cell adhesion molecule-1 (VCAM-1) at lesion-prone sites of the aorta. Histology was performed at day 21. RESULTS: Aortic signal for P-selectin, VCAM-1, VWF, and platelet-GPIbα were all increased several-fold (p < 0.01) in post-MI mice versus sham-treated animals at day 3 and 21. Treatment with NAC and ADAMTS13 significantly attenuated the post-MI increase for all four molecular targets by > 50% (p < 0.05 vs. non-treated at day 3 and 21). On aortic root histology, mice undergoing MI versus controls had 2-4 fold greater plaque size and macrophage content (p < 0.05), approximately 20-fold greater platelet adhesion (p < 0.05), and increased staining for markers of platelet transforming growth factor-ß1 signaling. Accelerated plaque growth and inflammatory activation was almost entirely prevented by ADAMTS13 and NAC. Inhibition of FXI had no significant effect on molecular imaging signal or plaque morphology. CONCLUSIONS: Plaque inflammatory activation in remote arteries after MI is strongly influenced by VWF-mediated platelet adhesion to the endothelium. These findings support investigation into new secondary preventive therapies for reducing non-culprit artery events after MI.

The Effect of an Elevated Dietary Copper Level on the Vascular Contractility and Oxidative Stress in Middle-Aged Rats.

Copper (Cu), being an essential mineral, plays a crucial role in maintaining physiological homeostasis across multiple bodily systems, notably the cardiovascular system. However, an increased Cu level in the body may cause blood vessel dysfunction and oxidative stress, which is unfavorable for the cardiovascular system. Middle-aged (7-8 months old) male Wistar rats (n/group = 12) received a diet supplemented with 6.45 mg Cu/kg (100% of the recommended daily dietary quantity of copper) for 8 weeks (Group A). The experimental group received 12.9 mg Cu/kg of diet (200%-Group B). An ex vivo study revealed that supplementation with 200% Cu decreased the contraction of isolated aortic rings to noradrenaline (0.7-fold) through FP receptor modulation. Vasodilation to sodium nitroprusside (1.10-fold) and acetylcholine (1.13-fold) was potentiated due to the increased net effect of prostacyclin derived from cyclooxygenase-1. Nitric oxide (NO, 2.08-fold), superoxide anion (O2•-, 1.5-fold), and hydrogen peroxide (H2O2, 2.33-fold) measured in the aortic rings increased. Blood serum antioxidant status (TAS, 1.6-fold), Cu (1.2-fold), Zn (1.1-fold), and the Cu/Zn ratio (1.4-fold) increased. An increase in Cu (1.12-fold) and the Cu/Zn ratio (1.09-fold) was also seen in the rats' livers. Meanwhile, cyclooxygenase-1 (0.7-fold), cyclooxygenase-2 (0.4-fold) and glyceraldehyde 3-phosphate dehydrogenase (0.5-fold) decreased. Moreover, a negative correlation between Cu and Zn was found (r = -0.80) in rat serum. Supplementation with 200% Cu did not modify the isolated heart functioning. No significant difference was found in the body weight, fat/lean body ratio, and organ weight for either the heart or liver, spleen, kidney, and brain. Neither Fe nor Se, the Cu/Se ratio, the Se/Zn ratio (in serum and liver), heme oxygenase-1 (HO-1), endothelial nitric oxide synthase (eNOS), or intercellular adhesion molecule-1 (iCAM-1) (in serum) were modified. Supplementation with 200% of Cu potentiated pro-oxidant status and modified vascular contractility in middle-aged rats.

Deficiency of neutral cholesterol ester hydrolase 1 (NCEH1) impairs endothelial function in diet-induced diabetic mice.

BACKGROUND: Neutral cholesterol ester hydrolase 1 (NCEH1) plays a critical role in the regulation of cholesterol ester metabolism. Deficiency of NCHE1 accelerated atherosclerotic lesion formation in mice. Nonetheless, the role of NCEH1 in endothelial dysfunction associated with diabetes has not been explored. The present study sought to investigate whether NCEH1 improved endothelial function in diabetes, and the underlying mechanisms were explored. METHODS: The expression and activity of NCEH1 were determined in obese mice with high-fat diet (HFD) feeding, high glucose (HG)-induced mouse aortae or primary endothelial cells (ECs). Endothelium-dependent relaxation (EDR) in aortae response to acetylcholine (Ach) was measured. RESULTS: Results showed that the expression and activity of NCEH1 were lower in HFD-induced mouse aortae, HG-exposed mouse aortae ex vivo, and HG-incubated primary ECs. HG exposure reduced EDR in mouse aortae, which was exaggerated by endothelial-specific deficiency of NCEH1, whereas NCEH1 overexpression restored the impaired EDR. Similar results were observed in HFD mice. Mechanically, NCEH1 ameliorated the disrupted EDR by dissociating endothelial nitric oxide synthase (eNOS) from caveolin-1 (Cav-1), leading to eNOS activation and nitric oxide (NO) release. Moreover, interaction of NCEH1 with the E3 ubiquitin-protein ligase ZNRF1 led to the degradation of Cav-1 through the ubiquitination pathway. Silencing Cav-1 and upregulating ZNRF1 were sufficient to improve EDR of diabetic aortas, while overexpression of Cav-1 and downregulation of ZNRF1 abolished the effects of NCEH1 on endothelial function in diabetes. Thus, NCEH1 preserves endothelial function through increasing NO bioavailability secondary to the disruption of the Cav-1/eNOS complex in the endothelium of diabetic mice, depending on ZNRF1-induced ubiquitination of Cav-1. CONCLUSIONS: NCEH1 may be a promising candidate for the prevention and treatment of vascular complications of diabetes.

Detoxification and Activating Blood Circulation Decoction Promotes Reendothelialization of Damaged Blood Vessels via VEGF Signaling Pathway Activation by miRNA-126.

The occurrence of in-stent restenosis (ISR) poses a significant challenge for percutaneous coronary intervention (PCI). Thus, the promotion of vascular reendothelialization is essential to inhibit endothelial proliferation. In this study, we clarified the mechanism by which Detoxification and Activating Blood Circulation Decoction (DABCD) promotes vascular reendothelialization to avoid ISR by miRNA-126-mediated modulation of the vascular endothelial growth factor (VEGF) signaling pathway. A rat model of post-PCI restenosis was established by balloon injury. The injured aortic segment was collected 14 and 28 d after model establishment. Our findings indicate that on the 14th and 28th days following balloon injury, DABCD reduced intimal hyperplasia and inflammation and promoted vascular reendothelialization. Additionally, DABCD markedly increased nitric oxide (NO) expression and significantly decreased ET-1 production in rat serum. DABCD also increased the mRNA level of endothelial nitric oxide synthase (eNOS) and the protein expression of VEGF, p-Akt, and p-extracellular signal-regulated kinase (ERK)1/2 in vascular tissue. Unexpectedly, the expression of miR-126a-5p mRNA was significantly lower in the aortic tissue of balloon-injured rats than in the aortic tissue of control rats, and higher miR-126a-5p levels were observed in the DABCD groups. The results of this study indicated that the vascular reendothelialization effect of DABCD on arterial intimal injury is associated with the inhibition of neointimal formation and the enhancement of vascular endothelial activity. More specifically, the effects of DABCD were mediated, at least in part, through miR-126-mediated VEGF signaling pathway activation.

In vitro and in vivo exposure of endothelial cells to dibutyl phthalate promotes monocyte adhesion.

The effect of endothelial cells' exposure to dibutyl phthalate (DBP) on monocyte adhesion is largely unknown. We evaluated monocyte adhesion to DBP-exposed endothelial cells by combining three approaches: short-term exposure (24 h) of EA.hy926 cells to 10-6, 10-5, and 10-4 M DBP, long-term exposure (12 weeks) of EA.hy926 cells to 10-9, 10-8, and 10-7 M DBP, and exposure of rats (28 and 90 days) to 100, 500, and 5000 mg DBP/kg food. Monocyte adhesion to human EA.hy926 and rat aortic endothelial cells, expression of selected cellular adhesion molecules and chemokines, and the involvement of extracellular signal-regulated kinase 1/2 (ERK1/2) were analyzed. We observed increased monocyte adhesion to DBP-exposed EA.hy926 cells in vitro and to rat aortic endothelium ex vivo. ERK1/2 inhibitor prevented monocyte adhesion to DBP-exposed EA.hy926 cells in short-term exposure experiments. Increased ERK1/2 phosphorylation in rat aortic endothelium and transient decrease in ERK1/2 activation following long-term exposure of EA.hy926 cells to DBP were also observed. In summary, exposure of endothelial cells to DBP promotes monocyte adhesion, thus suggesting a possible role for this phthalate in the development of atherosclerosis. ERK1/2 signaling could be the mediator of monocyte adhesion to DBP-exposed endothelial cells, but only after short-term high-level exposure.

Recombinant human proteoglycan 4 lowers inflammation and atherosclerosis susceptibility in female low-density lipoprotein receptor knockout mice.

Recombinant human proteoglycan 4 (rhPRG4) is a macromolecular mucin-like glycoprotein that is classically studied as a lubricant within eyes and joints. Given that endogenously produced PRG4 is present within atherosclerotic lesions and genetic PRG4 deficiency increases atherosclerosis susceptibility in mice, in the current study we investigated the anti-atherogenic potential of chronic rhPRG4 treatment. Female low-density lipoprotein receptor knockout mice were fed an atherogenic Western-type diet for 6 weeks and injected three times per week intraperitoneally with 0.5 mg rhPRG4 or PBS as control. Treatment with rhPRG4 was associated with a small decrease in plasma-free cholesterol levels, without a change in cholesteryl ester levels. A marked increase in the number of peritoneal foam cells was detected in response to the peritoneal rhPRG4 administration, which could be attributed to elevated peritoneal leukocyte MSR1 expression levels. However, rhPRG4-treated mice exhibited significantly smaller aortic root lesions of 278 ± 21 × 103 µm2 compared with 339 ± 15 × 103 µm2 in the aortic root of control mice. The overall decreased atherosclerosis susceptibility coincided with a shift in the monocyte and macrophage polarization states towards the patrolling and anti-inflammatory M2-like phenotypes, respectively. Furthermore, rhPRG4 treatment significantly reduced macrophage gene expression levels as well as plasma protein levels of the pro-inflammatory/pro-atherogenic cytokine TNF-alpha. In conclusion, we have shown that peritoneal administration and subsequent systemic exposure to rhPRG4 beneficially impacts the inflammatory state and reduces atherosclerosis susceptibility in mice. Our findings highlight that PRG4 is not only a lubricant but also acts as an anti-inflammatory agent. KEY POINTS: Endogenously produced proteoglycan 4 is found in atherosclerotic lesions and its genetic deficiency in mice is associated with enhanced atherosclerosis susceptibility. In this study we investigated the anti-atherogenic potential of chronic treatment with recombinant human PRG4 in hypercholesterolaemic female low-density lipoprotein receptor knockout mice. We show that recombinant human PRG4 stimulates macrophage foam cell formation, but also dampens the pro-inflammatory state of monocyte/macrophages, eventually leading to a significant reduction in plasma TNF-alpha levels and a lowered atherosclerosis susceptibility. Our findings highlight that peritoneal recombinant human PRG4 treatment can execute effects both locally and systemically and suggest that it will be of interest to study whether rhPRG4 treatment is also able to inhibit the progression and/or induce regression of previously established atherosclerotic lesions.

Fisetin Suppresses Atherosclerosis by Inhibiting Ferroptosis-Related Oxidative Stress in Apolipoprotein E Knockout Mice.

INTRODUCTION: Fisetin has been demonstrated to inhibit the occurrence of atherosclerosis; however, the mechanism of fisetin suppressing atherosclerosis remains elusive. METHODS: The function of fisetin in the inhibition of atherosclerosis was evaluated by hematoxylin and eosin and Oil Red O staining in ApoE-/- mice. Molecular biomarkers of atherosclerosis progression were detected by Western blot and qPCR. Moreover, the inhibition of atherosclerosis on oxidative stress and ferroptosis was evaluated by immunofluorescence staining, qPCR, and Western blot assays. RESULTS: The obtained results showed that serum lipid was attenuated and consequentially the formation of atherosclerosis was also suppressed by fisetin in ApoE-/- mice. Exploration of the mechanism revealed that molecular biomarkers of atherosclerosis were decreased under fisetin treatment. The level of reactive oxygen species and malondialdehyde declined, while the activity of superoxide dismutases and glutathione peroxidase was increased under the fisetin treatment. Additionally, the suppressor of ferroptosis, glutathione peroxidase 4 proteins, was elevated. The ferritin was decreased in the aortic tissues treated with fisetin. CONCLUSIONS: In summary, fisetin attenuated the formation of atherosclerosis through the inhibition of oxidative stress and ferroptosis in the aortic tissues of ApoE-/- mice.

Inhibition of vascular calcification by Compound Danshen Dripping Pill through multiple mechanisms.

BACKGROUND: Vascular calcification refers to the abnormal accumulation of calcium in the walls of blood vessels and is a risk factor often overlooked in cardiovascular disease. However, there is currently no specific drug for treating vascular calcification. Compound Danshen Dripping Pill (CDDP) is widely used to treat cardiovascular diseases, but its effect on vascular calcification has not been reported. PURPOSE: We investigated the effects of CDDP on vascular calcification in ApoE-/- mice and in vitro and elucidated its mechanism of action. STUDY DESIGN: Firstly, we found that CDDP has the potential to improve calcification based on network pharmacology analysis. Then, we performed the following experiments: in vivo, ApoE-/- mice were fed a high-fat diet randomly supplemented with CDDP for 16 weeks. Atherosclerosis and vascular calcification were determined. In vitro, human aortic smooth muscle cells (HASMCs), human umbilical vein endothelial cells (HUVECs), and human aortic endothelial cells (HAECs) were used to determine the mechanisms for CDDP-inhibited vascular calcification. RESULTS: In this study, we observed that CDDP reduced intimal calcification in atherosclerotic lesions of ApoE-deficient mice fed a high-fat diet, as well as the calcification in cultured SMCs and ECs. Mechanistically, CDDP inhibited the Wnt/ß-catenin pathway by up-regulating the expression of DKK1 and LRP6, which are upstream inhibitors of Wnt, leading to a reduction in the expression of osteoblastic transition markers (ALP, OPN, BMP2, and RUNX2). Furthermore, CDDP enhanced the secretion of DKK1, which plays a role in mediating EC-SMC crosstalk in calcification. Additionally, VC contributes to vascular aging by inhibiting Sirt1 and increasing senescence parameters (SA-ß-gal, p21, and p16). However, CDDP reversed these changes by activating Sirt1. CDDP also reduced the levels of pro-inflammatory cytokines and the senescence-associated secretory phenotype in vivo and in vitro. CONCLUSIONS: Our study suggests that CDDP reduces vascular calcification by regulating the DKK1/LRP6/ß-catenin signaling pathway in ECs/SMCs and interactions with the crosstalk of ECs and SMCs. It also reduces the senescence of ECs/SMCs, contributing to the Sirt1 activation, indicating CDDP's novel role in ameliorating vascular calcification.

Establishment of a model of LPS-induced inflammatory injury in human aortic endothelial cells.

PURPOSE: We aim to establish an LPS-induced human aortic endothelial cells (HAECs) inflammatory injury model and explore the optimal conditions for inducing its injury. We expect to provide modeling references for the related experiments of vascular inflammatory diseases. METHODS: HAECs were cultured in vitro and treated with different concentrations of lipopolysaccharide (LPS) (0.1, 1, 10, 50, 100 µg/mL) for 6, 12, and 24 h to establish the HAECs inflammatory injury model. The cell viability was determined by CCK-8 assay; the expression levels of inflammatory cytokines in the cells were detected by RT-PCR；the apoptosis rate of the cells was detected by flow cytometry. RESULTS: ① Within 24 h of LPS treatment, the cell viability of the 0.1 and 1 µg/mL groups showed an overall increasing trend with time, while the cell viability of the 10, 50, and 100 µg/mL groups increased first and then decreased with time, and the cell viability of 50 and 100 µg/mL groups was significantly lower than the normal control group at 24 h (P<0.01). ② RT-PCR results showed that after 50 and 100 µg/mL LPS for 24 h, the inflammatory cytokines all showed an apparent upward trend compared with the normal control group (P<0.05), which was more significant in the 100 µg/mL group. ③ After 100 µg/mL LPS for 24 h, the apoptotic necrosis rate of HAECs was higher than the normal control group (P<0.01). CONCLUSIONS: This experiment successfully established a HAECs injury model, indicating that the optimal conditions for inducing injury are an LPS concentration of 100 µg/mL and a treatment time of 24 h.

Gualou-Xiebai herb pair and its active ingredients act against atherosclerosis by suppressing VSMC-derived foam cell formation via regulating P2RY12-mediated lipophagy.

BACKGROUND: Atherosclerosis (AS) is a chronic disease characterized by lipid accumulation in the aortic wall and the formation of foam cells overloaded with large lipids inclusions. Currently, Western medicine is primarily used to improve lipid metabolism disorders and reduce inflammatory reactions to delay AS progression, but these medicines come with serious side effects and drug resistance. Gualou-Xiebai (GLXB) is a renowned herb pair that has been proven effective against AS. However, the potential molecular mechanism through which GLXB exerts the anti-atherosclerotic effects of increasing lipophagy in vascular smooth muscle cells (VSMCs) remains unknown. PURPOSE: This study aims to explore the role of lipophagy and the therapeutic mechanism of GLXB in AS. METHODS: UPLC-Q-TOF-MS for the determination of the main components of GLXB-containing serum. An AS mouse model was established by feeding a high-fat diet (HFD) to ApoE-/- mice for 12 weeks. Ultrasonography monitoring was used to confirm the successful establishment of the AS model. Plaque areas and lipid deposition were evaluated using HE staining and aorta imagingafter GLXB treatment. Immunofluorescence staining and Western blotting were utilized to observe the P2RY12 and lipophagy levels in AS mice. VSMCs were stimulated with oxidized low-density lipoprotein (ox-LDL) to induce foam cell formation. The degree of lipophagy and the related molecular mechanisms were assessed after treating the VSMCs with GLXB-containing serum or si-P2RY12 transfection. The active components of GLXB-containing serum that act on P2RY12 were screened and verified by molecular docking and dual-luciferase reporter assays. RESULTS: Seventeen components of GLXB were identified in rat serum by UPLC-Q-TOF-MS. GLXB significantly reduced lipid deposition in HFD-fed ApoE-/- mice and ox-LDL-induced VSMCs. GLXB strikingly increased lipophagy levels by downregulating P2RY12, p62, and plin2, upregulating LC3Ⅱ protein expression, and increasing the number of autophagosomes. Notably, the lipophagy inhibitor CQ and the P2RY12 receptor agonist ADPß abolished the GLXB-induced increase in lipophagy. Last, we confirmed that albiflorin, apigenin, luteolin, kaempferol, 7,8-dihydroxyflavone, and hesperetin from GLXB significantly inhibited P2RY12. CONCLUSION: GLXB activates lipophagy and inhibits lipid accumulation-associated VSMC-derived foam cell formation through suppressing P2RY12 activation, resulting in anti-atherosclerotic effects. The GLXB components albiflorin, apigenin, luteolin, kaempferol, 7,8-dihydroxyflavone, and hesperetin are the potential active effectors against P2RY12.

Hydrogen sulfide dysfunction in metabolic syndrome-associated vascular complications involves cGMP regulation through soluble guanylyl cyclase persulfidation.

Here, by using in vitro and ex vivo approaches, we elucidate the impairment of the hydrogen sulfide (H2S) pathway in vascular complications associated with metabolic syndrome (MetS). In the in vitro model simulating hyperlipidemic/hyperglycemic conditions, we observe significant hallmarks of endothelial dysfunction, including eNOS/NO signaling impairment, ROS overproduction, and a reduction in CSE-derived H2S. Transitioning to an ex vivo model using db/db mice, a genetic MetS model, we identify a downregulation of CBS and CSE expression in aorta, coupled with a diminished L-cysteine-induced vasorelaxation. Molecular mechanisms of eNOS/NO signaling impairment, dissected using pharmacological and molecular approaches, indicate an altered eNOS/Cav-1 ratio, along with reduced Ach- and Iso-induced vasorelaxation and increased L-NIO-induced contraction. In vivo treatment with the H2S donor Erucin ameliorates vascular dysfunction observed in db/db mice without impacting eNOS, further highlighting a specific action on smooth muscle component rather than the endothelium. Analyzing the NO signaling pathway in db/db mice aortas, reduced cGMP levels were detected, implicating a defective sGC/cGMP signaling. In vivo Erucin administration restores cGMP content. This beneficial effect involves an increased sGC activity, due to enzyme persulfidation observed in sGC overexpressed cells, coupled with PDE5 inhibition. In conclusion, our study demonstrates a pivotal role of reduced cGMP levels in impaired vasorelaxation in a murine model of MetS involving an impairment of both H2S and NO signaling. Exogenous H2S supplementation through Erucin represents a promising alternative in MetS therapy, targeting smooth muscle cells and supporting the importance of lifestyle and nutrition in managing MetS.

Preparation and Preliminary Structure-Activity Relationship Studies of Schwarzinicine A Analogs as Vasorelaxant Agents.

Schwarzinicines A-D, a series of alkaloids recently discovered from Ficus schwarzii, exhibit pronounced vasorelaxant activity in rat isolated aorta. Building on this finding, a concise synthesis of schwarzinicines A and B has been reported, allowing further investigations into their biological properties. Herein, a preliminary exploration of the chemical space surrounding the structure of schwarzinicine A (1) was carried out aiming to identify structural features that are essential for vasorelaxant activity. A total of 57 analogs were synthesized and tested for vasorelaxant activity in rat isolated aorta. Both efficacy (Emax) and potency (EC50) of these analogs were compared. In addition to identifying structural features that are required for activity or associated with potency enhancement effect, four analogs showed significant potency improvements of up to 40.2-fold when compared to 1. Molecular dynamics simulation of a tetrameric 44-bound transient receptor potential canonical-6 (TRPC6) protein indicated that 44 could potentially form important interactions with the residues Glu509, Asp530, Lys748, Arg758, and Tyr521. These results may serve as a foundation for guiding further structural optimization of the schwarzinicine A scaffold, aiming to discover even more potent analogs.

Structural Characterization and Rat Aortic Vascular Reactivity of Bradykinin-Potentiating Peptides (BPPs) from the Snake Venom of Bothrops moojeni from Delta do Parnaíba Region, Brazil.

Snake venoms contain various bradykinin-potentiating peptides (BPPs). First studied for their vasorelaxant properties due to angiotensin converting enzyme (ACE) inhibition, these molecules present a range of binding partners, among them the argininosuccinate synthase (AsS) enzyme. This has renewed interest in their characterization from biological sources and the evaluation of their pharmacological activities. In the present work, the low molecular weight fraction of Bothrops moojeni venom was obtained and BPPs were characterized by mass spectrometry. Eleven BPPs or related peptides were sequenced, and one of them, BPP-Bm01, was new. Interestingly, some oxidized BPPs were detected. The three most abundant peptides were BPP-Bm01, BPP-Bax12, and BPP-13a, and their putative interactions with the AsS enzyme were investigated in silico. A binding cavity for these molecules was predicted, and docking studies allowed their ranking. Three peptides were synthesized and submitted to vasorelaxation assays using rat aortic rings. While all BPPs were active, BPP-Bm01 showed the highest potency in this assay. This work adds further diversity to BPPs from snake venoms and suggests, for the first time, a putative binding pocket for these molecules in the AsS enzyme. This can guide the design of new and more potent AsS activators.

Mitochondrial KATP channel-mediated autophagy contributes to angiotensin II-induced vascular dysfunction in mice.

BACKGROUND AND AIM: The present study aimed to investigate whether the mitochondrial KATP channel contributes to angiotensin II (Ang II)-induced vascular dysfunction, the development of hypertension, and atherosclerosis. METHODS AND RESULTS: ApoE (-/-) mice fed a high-fat diet were chronically infused with Ang II for eight weeks and concomitantly treated with losartan (ARB), apocynin, or 5-hydroxy decanoate (5-HD), or 3-methyladenine (3-MA). Systolic blood pressure was measured, and pathological changes of aortic or liver tissue were observed. Nitric oxide (NO), superoxide dismutase 2 (SOD2) levels and vasorelaxation rate were measured, and protein and mRNA expressions were examined by western blot and RT-PCR. Ang II-induced development of hypertension was suppressed not only by ARB, and apocynin but also by 5-HD or 3-MA. Ang II infusion decreased aortic NO production and relaxation, as well as SOD2 activity in liver, which were improved by all treatments. In addition, Ang II-induced activation of autophagy was suppressed by 5-HD in aortic tissue, furthermore, Ang II increases the atherosclerotic index in plasma and exacerbates the development of atherosclerosis by increases of fat deposition in the aorta and liver. Lipid metabolism-related mRNA expressions (LXR-α, LDLR, SRBI, Acca, and FASN) were changed by Ang II. Similarly, not only ARB, and apocynin, but also 5-HD and 3-MA suppressed Ang II-induced these changes. CONCLUSIONS: Our present findings evidence that mitochondrial KATP channel-mediated autophagy contributes to Ang II-induced vascular dysfunction, development of hypertension, and atherosclerosis.

A UHPLC-Q-TOF-MS-based serum and urine metabolomics approach reveals the mechanism of Gualou-Xiebai herb pair intervention against atherosclerosis process in ApoE-/- mice.

Atherosclerosis (AS) is a metabolic disorder commonly correlated with a high-fat diet (HFD). There are many endogenous metabolic changes associated with AS development. Gualou-Xiebai (GLXB) is a traditional Chinese medicine herb pair that has been used to treat AS. However, the mechanism of GLXB herb pair on the process of AS is still essentially unknown. In this study, aortic histopathological examination and biochemical analyses were used to validate the anti-atherosclerotic effects of GLXB herb pair on ApoE-/- mice during the disease course of AS. The mechanism of GLXB herb pair were performed by metabolomics approach based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). As a result, GLXB herb pair has protective effects on AS lesion development and improves blood lipid levels in ApoE-/- mice. A total of 34, 39, and 49 metabolites were found to be profoundly altered in the 9-week, 14-week, and 19-week model groups compared with the corresponding control groups. Among them, 16, 18, and 18 metabolites showed a trend toward normal levels after pharmacological intervention. Metabolic pathway analysis found that GLXB herb pair mainly affects glycerophospholipid metabolism, pentose and glucuronate interconversions in 9 weeks; linoleic acid metabolism, cysteine and methionine metabolism, and arachidonic acid metabolism in 14 weeks; arachidonic acid metabolism and pentose and glucuronate interconversions in 19 weeks. The results demonstrated that GLXB herb pair mainly played a therapeutic role by regulating glycerophospholipid metabolism and pentose and glucuronate interconversions in the whole process of AS.

GALNT3 protects against phosphate-induced calcification in vascular smooth muscle cells by enhancing active FGF23 and inhibiting the wnt/ß-catenin signaling pathway.

Vascular calcification (VC) acts as a notable risk factor in the cardiovascular system. Disorder of phosphorus (Pi) metabolism promotes VC. Recent findings show that polypeptide N-acetylgalactosaminyltransferase 3(GALNT3) is Pi responsive and with potent effects on Pi homeostasis. However, whether GALNT3 is involved in high Pi-induced VC remains unclear. The present study investigated the potential role of GALNT3 as a novel regulator of VC. In vitro, human aortic smooth muscle cells (HASMCs) calcification was induced by inorganic Pi, while in vivo, C57BL/6 J mice were used to determine the effects of GALNT3 on Vitamin D3-induced medial arterial calcification. Alizarin red staining, Von Kossa staining, calcium and alkaline phosphatase (ALP) activity were performed to test VC. We showed that expression of GALNT3 was increased in the calcified HASMCs and aortas of the calcified mice.In vitro, overexpression of GALNT3 increased the levels of active full-length FGF23, accompanied by suppression of the osteoblast-related factors (Runx2 and BMP2), and further inhibited the formation of calcified nodules. Moreover, the protein levels of Wnt3a and active ß-catenin were determined and it was found that GALNT3 significantly inhibited their expression. LiCl, a Wnt/ß-catenin signaling activator, was observed to reverse the protective effect of GALNT3 overexpression. The opposite results were observed in the GALNT3 knockdown cells. In vivo, overexpression of GALNT3 by adeno-associated virus decreased the serum Pi and slowed the formation of aortic calcification in the calcified mice. In conclusion, our results indicate that GALNT3 counteracts high Pi-induced osteoblastic differentiation of VSMCs and protects against the initiation and progression of VC by inhibiting the Wnt/ß-catenin signaling pathway.

Synergistic anti-inflammatory effects and mechanisms of the combination of resveratrol and curcumin in human vascular endothelial cells and rodent aorta.

Chronic increased pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) play critical roles in the development of endothelial dysfunction and therefore induce cardiovascular disease. Although phytochemicals have the potential ability to reduce the risk of CVD, the big gap between required high concentration in cells and the low bioavailability in the blood of phytochemicals compromise their therapeutic potentials. This study aims to investigate if combined phytochemicals at low levels exert a synergistic anti-inflammatory effect and to define relevant molecular mechanisms. Our results found that combined curcumin (5 µM) and resveratrol (5 µM) synergistically (combination index is 0.78) inhibited TNF-α-induced monocytes adhesion to human endothelial EA.hy 926 cells while the individual chemicals did not have such effect at the selected concentrations. The concentrations of curcumin (5 µM) and resveratrol (5 µM) are very close to the maximum level of curcumin (3.56 µM) and resveratrol (2 µM) in human blood. Dietary supplementation of combined curcumin (500mg/kg) and resveratrol (200mg/kg) synergistically reduced TNF-α-induced vascular inflammation in C57BL/6 mice with a similar pattern in cells. Moreover, the combination ameliorated the TNF-α-induced protein expressions and circulating levels of vascular cell adhesion molecule 1 and monocyte chemotactic protein-1 in EA.hy 926 cells, mice aorta and serum. Furthermore, combined curcumin and resveratrol significantly inhibited TNF-α-induced nuclear factor-kappaB (NF-κB) p65 nuclear protein expression than that by the individual chemical alone in EA.hy 926 cells, indicating that the synergistic effect of the combination may result from that curcumin reduces the required minimum concentration for resveratrol to inhibit the nuclear translocation of NF-κB. In conclusion, the combination of curcumin and resveratrol protects against TNF-α-induced vascular inflammation by suppressing NF-κB signaling in vitro and in vivo models. This study suggests that dietary intake of a combination of curcumin and resveratrol or its foods may be a practical, safe approach to prevent vascular inflammation and therefore prevent/treat vascular diseases in humans.