Effects of cytochalasin D on relaxation process of skinned taenia cecum and carotid artery from guinea pig.

Actin linked regulatory mechanisms are known to contribute contraction/relaxation in smooth muscle. In order to clarify whether modulation of polymerization/depolymerization of actin filaments affects relaxation process, we examined the effects of cytochalasin D on relaxation process by Ca2+ removal after Ca2+-induced contraction of ß-escin skinned (cell membrane permeabilized) taenia cecum and carotid artery preparations from guinea pigs. Cytochalasin D, an inhibitor of actin polymerization, significantly suppressed the force during relaxation both in skinned taenia cecum and carotid artery. The data fitting analysis of the relaxation processes indicates that cytochalasin D accelerates slow (latch-like) bridge dissociation. Cytochalasin D seems to directly disrupts actin filament organization or its length, resulting in modulation of actin filament structure that prevents myosin binding.

Orosomucoid 2 as a biomarker of carotid artery atherosclerosis plaque vulnerability through its generation of reactive oxygen species and lipid accumulation in vascular smooth muscle cells.

BACKGROUND: Orosomucoid (ORM) has been reported as a biomarker of carotid atherosclerosis, but the role of ORM 2, a subtype of ORM, in carotid atherosclerotic plaque formation and the underlying mechanism have not been established. METHODS: Plasma was collected from patients with carotid artery stenosis (CAS) and healthy participants and assessed using mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) technology to identify differentially expressed proteins. The key proteins and related pathways were identified via western blotting, immunohistochemistry, and polymerase chain reaction of carotid artery plaque tissues and in vitro experiments involving vascular smooth muscle cells (VSMCs). RESULTS: We screened 33 differentially expressed proteins out of 535 proteins in the plasma. Seventeen proteins showed increased expressions in the CAS groups relative to the healthy groups, while 16 proteins showed decreased expressions during iTRAQ and bioinformatic analysis. The reactive oxygen species metabolic process was the most common enrichment pathway identified by Gene Ontology analysis, while ORM2, PRDX2, GPX3, HP, HBB, ANXA5, PFN1, CFL1, and S100A11 were key proteins identified by STRING and MCODE analysis. ORM2 showed increased expression in patients with CAS plaques, and ORM2 was accumulated in smooth muscle cells. Oleic acid increased the lipid accumulation and ORM2 and PRDX6 expressions in the VSMCs. The recombinant-ORM2 also increased the lipid accumulation and reactive oxygen species (ROS) in the VSMCs. The expressions of ORM2 and PRDX-6 were correlated, and MJ33 (an inhibitor of PRDX6-PLA2) decreased ROS production and lipid accumulation in VSMCs. CONCLUSION: ORM2 may be a biomarker for CAS; it induced lipid accumulation and ROS production in VSMCs during atherosclerosis plaque formation. However, the relationships between ORM2 and PRDX-6 underlying lipid accumulation-induced plaque vulnerability require further research.

Identification of key pyroptosis-related genes and microimmune environment among peripheral arterial beds in atherosclerotic arteries.

Atherosclerosis is a chronic inflammatory disease characterized with innate and adaptive immunity but also involves pyroptosis. Few studies have explored the role of pyroptosis in advanced atherosclerotic plaques from different vascular beds. Here we try to identify the different underlying function of pyroptosis in the progression of atherosclerosis between carotid arteries and femoral. arteries. We extracted gene expression levels from 55 advanced carotid or femoral atherosclerotic plaques. The pyroptosis score of each sample was calculated by single-sample-gene-set enrichment analysis (ssGSEA). We then divided the samples into two clusters: high pyroptosis scores cluster (PyroptosisScoreH cluster) and low pyroptosis scores cluster (PyroptosisScoreL cluster), and assessed functional enrichment and immune cell infiltration in the two clusters. Key pyroptosis related genes were identified by the intersection between results of Cytoscape and LASSO (Least Absolute Shrinkage and Selection Operator) regression analysis. Finally, all key pyroptosis related genes were validated in vitro. We found all but one of the 29 carotid plaque samples belonged to the PyroptosisScoreH cluster and the majority (19 out of 26) of femoral plaques were part of the PyroptosisScoreL cluster. Atheromatous plaque samples in the PyroptosisScoreL cluster had higher proportions of gamma delta T cells, M2 macrophages, myeloid dendritic cells (DCs), and cytotoxic lymphocytes (CTLs), but lower proportions of endothelial cells (ECs). Immune full-activation pathways (e.g., NOD-like receptor signaling pathway and NF-kappa B signaling pathway) were highly enriched in the PyroptosisScoreH cluster. The key pyroptosis related genes GSDMD, CASP1, NLRC4, AIM2, and IL18 were upregulated in advanced carotid atherosclerotic plaques. We concluded that compared to advanced femoral atheromatous plaques, advanced carotid atheromatous plaques were of higher grade of pyroptosis. GSDMD, CASP1, NLRC4, AIM2, and IL18 were the key pyroptosis related genes, which might provide a new sight in the prevention of fatal strokes in advanced carotid atherosclerosis.

Coronary and carotid artery dysfunction and KV7 overexpression in a mouse model of Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disease caused by expression of progerin, a lamin A variant that is also expressed at low levels in non-HGPS individuals. Although HGPS patients die predominantly from myocardial infarction and stroke, the mechanisms that provoke pathological alterations in the coronary and cerebral arteries in HGPS remain ill defined. Here, we assessed vascular function in the coronary arteries (CorAs) and carotid arteries (CarAs) of progerin-expressing LmnaG609G/G609G mice (G609G), both in resting conditions and after hypoxic stimulus. Wire myography, pharmacological screening, and gene expression studies demonstrated vascular atony and stenosis, as well as other functional alterations in progeroid CorAs and CarAs and aorta. These defects were associated with loss of vascular smooth muscle cells and overexpression of the KV7 family of voltage-dependent potassium channels. Compared with wild-type controls, G609G mice showed reduced median survival upon chronic isoproterenol exposure, a baseline state of chronic cardiac hypoxia characterized by overexpression of hypoxia-inducible factor 1α and 3α genes, and increased cardiac vascularization. Our results shed light on the mechanisms underlying progerin-induced coronary and carotid artery disease and identify KV7 channels as a candidate target for the treatment of HGPS.

Human Plaque Myofibroblasts to Study Mechanisms of Atherosclerosis.

Background Plaque myofibroblasts are critical players in the initiation and advancement of atherosclerotic disease. They are involved in the production of extracellular matrix, the formation of the fibrous cap, and the underlying lipidic core via modulation processes in response to different environmental cues. Despite clear phenotypic differences between myofibroblast cells and healthy vascular smooth muscle cells, smooth muscle cells are still widely used as a cellular model in atherosclerotic research. Methods and Results Here, we present a conditioned outgrowth method to isolate and culture myofibroblast cells from plaques. We obtained these cells from 27 donors (24 carotid and 3 femoral endarterectomies). We show that they keep their proliferative capacity for 8 passages, are transcriptionally stable, retain donor-specific gene expression programs, and express extracellular matrix proteins (FN1, COL1A1, and DCN) and smooth muscle cell markers (ACTA2, MYH11, and CNN1). Single-cell transcriptomics reveals that the cells in culture closely resemble the plaque myofibroblasts. Chromatin immunoprecipitation sequencing shows the presence of histone H3 lysine 4 dimethylation at the MYH11 promoter, pointing to their smooth muscle cell origin. Finally, we demonstrated that plaque myofibroblasts can be efficiently transduced (>97%) and are capable of taking up oxidized low-density lipoprotein and undergoing calcification. Conclusions In conclusion, we present a method to isolate and culture cells that retain plaque myofibroblast phenotypical and functional capabilities, making them a suitable in vitro model for studying selected mechanisms of atherosclerosis.

IL-13 neutralization attenuates carotid artery intimal hyperplasia and increases endothelial cell migration via modulating the JAK-1/STAT-3 signaling pathway.

The aim of this study was to investigate how the concentration of interleukin-13 (IL-13) affects the regulation of endothelial cell migration after injury. The incubation of recombinant human interleukin-13 (rhIL-13) strongly increased the content of reactive oxygen species (ROS) in HUVECs via the JAK-1/STAT-3/NOX-4 signaling pathway. Antagonizing the high intracellular ROS that was induced by rhIL-13 promoted the migration of HUVECs. Furthermore, IL-13 neutralization not only inhibited intimal hyperplasia, but also promoted the migration of endothelial cells (ECs) after injury. The results suggest that IL-13 inhibition is a potential means of stimulating endothelial cells recovery after injury. Therefore, the attenuation of IL-13 activation may have therapeutic value for vascular disease.

Bioinformatics analysis of carotid vulnerable plaques associated with the SARS-CoV-2 pattern.

The rupture of carotid artery vulnerable plaque plays a critical role in ischemic stroke, and the widely spread new coronavirus in recent years plays a certain role in the development of human carotid artery vulnerable plaque, we screened out 27 differential expression genes (DEGs) of stable plaque and vulnerable plaque associated with the new coronavirus. Through the construction of the protein-protein interaction (PPI) network, the Cathepsin B (CTSB) and Niemann-Pick Disease Type 2 (NPC2) were identified as crucial expression genes, and further, we confirmed the validity of core gene expression in two validation sets. Additionally, we discovered a significant connection between CTSB, NPC2 and 28 different kinds of immune cells in carotid plaque tissue. We screened out 65 target interacting drugs based on 10 differentially expressed genes through online tools and finally verified the high expression of 2 core genes in fragile plaques through clinical sample experiments. These findings imply that two core genes may be novel targets for molecular diagnostics and immunotherapy of vulnerable plaques.

Phosphorylation of USP20 on Ser334 by IRAK1 promotes IL-1ß-evoked signaling in vascular smooth muscle cells and vascular inflammation.

Reversible lysine-63 (K63) polyubiquitination regulates proinflammatory signaling in vascular smooth muscle cells (SMCs) and plays an integral role in atherosclerosis. Ubiquitin-specific peptidase 20 (USP20) reduces NFκB activation triggered by proinflammatory stimuli, and USP20 activity attenuates atherosclerosis in mice. The association of USP20 with its substrates triggers deubiquitinase activity; this association is regulated by phosphorylation of USP20 on Ser334 (mouse) or Ser333 (human). USP20 Ser333 phosphorylation was greater in SMCs of atherosclerotic segments of human arteries as compared with nonatherosclerotic segments. To determine whether USP20 Ser334 phosphorylation regulates proinflammatory signaling, we created USP20-S334A mice using CRISPR/Cas9-mediated gene editing. USP20-S334A mice developed ∼50% less neointimal hyperplasia than congenic WT mice after carotid endothelial denudation. WT carotid SMCs showed substantial phosphorylation of USP20 Ser334, and WT carotids demonstrated greater NFκB activation, VCAM-1 expression, and SMC proliferation than USP20-S334A carotids. Concordantly, USP20-S334A primary SMCs in vitro proliferated and migrated less than WT SMCs in response to IL-1ß. An active site ubiquitin probe bound to USP20-S334A and USP20-WT equivalently, but USP20-S334A associated more avidly with TRAF6 than USP20-WT. IL-1ß induced less K63-linked polyubiquitination of TRAF6 and less downstream NFκB activity in USP20-S334A than in WT SMCs. Using in vitro phosphorylation with purified IRAK1 and siRNA-mediated gene silencing of IRAK1 in SMCs, we identified IRAK1 as a novel kinase for IL-1ß-induced USP20 Ser334 phosphorylation. Our findings reveal novel mechanisms regulating IL-1ß-induced proinflammatory signaling: by phosphorylating USP20 Ser334, IRAK1 diminishes the association of USP20 with TRAF6 and thus augments NFκB activation, SMC inflammation, and neointimal hyperplasia.

Gut microbiota, circulating inflammatory markers and metabolites, and carotid artery atherosclerosis in HIV infection.

BACKGROUND: Alterations in gut microbiota have been implicated in HIV infection and cardiovascular disease. However, how gut microbial alterations relate to host inflammation and metabolite profiles, and their relationships with atherosclerosis, have not been well-studied, especially in the context of HIV infection. Here, we examined associations of gut microbial species and functional components measured by shotgun metagenomics with carotid artery plaque assessed by B-mode carotid artery ultrasound in 320 women with or at high risk of HIV (65% HIV +) from the Women's Interagency HIV Study. We further integrated plaque-associated microbial features with serum proteomics (74 inflammatory markers measured by the proximity extension assay) and plasma metabolomics (378 metabolites measured by liquid chromatography tandem mass spectrometry) in relation to carotid artery plaque in up to 433 women. RESULTS: Fusobacterium nucleatum, a potentially pathogenic bacteria, was positively associated with carotid artery plaque, while five microbial species (Roseburia hominis, Roseburia inulinivorans, Johnsonella ignava, Odoribacter splanchnicus, Clostridium saccharolyticum) were inversely associated with plaque. Results were consistent between women with and without HIV. Fusobacterium nucleatum was positively associated with several serum proteomic inflammatory markers (e.g., CXCL9), and the other plaque-related species were inversely associated with proteomic inflammatory markers (e.g., CX3CL1). These microbial-associated proteomic inflammatory markers were also positively associated with plaque. Associations between bacterial species (especially Fusobacterium nucleatum) and plaque were attenuated after further adjustment for proteomic inflammatory markers. Plaque-associated species were correlated with several plasma metabolites, including the microbial metabolite imidazole-propionate (ImP), which was positively associated with plaque and several pro-inflammatory markers. Further analysis identified additional bacterial species and bacterial hutH gene (encoding enzyme histidine ammonia-lyase in ImP production) associated with plasma ImP levels. A gut microbiota score based on these ImP-associated species was positively associated with plaque and several pro-inflammatory markers. CONCLUSION: Among women living with or at risk of HIV, we identified several gut bacterial species and a microbial metabolite ImP associated with carotid artery atherosclerosis, which might be related to host immune activation and inflammation. Video Abstract.

Arterial wall inflammation assessed by 18F-FDG-PET/CT is higher in individuals with Type 1 diabetes and associated with circulating inflammatory proteins.

AIMS: The article investigates whether chronic hyperglycaemia in Type 1 diabetes (T1D) is associated with a proinflammatory immune signature and with arterial wall inflammation, driving the development of atherosclerosis. METHODS AND RESULTS: Patients with T1D (n = 41), and healthy age-, sex-, and body mass index-matched controls (n = 20) were recruited. Arterial wall inflammation and haematopoietic activity were measured with 2'-deoxy-2'-(18F)-fluoro-D-glucose (18F-FDG) positron emission tomography/computed tomography. In addition, flow cytometry of circulating leucocytes was performed as well as targeted proteomics to measure circulating inflammatory markers. 18F-FDG uptake in the wall of the abdominal aorta, carotid arteries, and iliac arteries was higher in T1D compared with that in the healthy controls. Also, 18F-FDG uptake in the bone marrow and spleen was higher in patients with T1D. CCR2 and CD36 expressions on circulating monocytes were higher in patients with T1D, as well as several circulating inflammatory proteins. In addition, several circulating inflammatory markers (osteoprotegerin, transforming growth factor-alpha, CX3CL1, and colony-stimulating factor-1) displayed a positive correlation with FDG uptake. Within T1D, no differences were found between people with a high and low HbA1c. CONCLUSION: These findings strengthen the concept that chronic hyperglycaemia in T1D induces inflammatory changes that fuel arterial wall inflammation leading to atherosclerosis. The degree of hyperglycaemia appears to play a minor role in driving this inflammatory response in patients with T1D.

Genetic Deletion of FXR1 Reduces Intimal Hyperplasia and Induces Senescence in Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMC) play a critical role in the development and pathogenesis of intimal hyperplasia indicative of restenosis and other vascular diseases. Fragile-X related protein-1 (FXR1) is a muscle-enhanced RNA binding protein whose expression is increased in injured arteries. Previous studies suggest that FXR1 negatively regulates inflammation, but its causality in vascular disease is unknown. In the current study, RNA-sequencing of FXR1-depleted VSMC identified many transcripts with decreased abundance, most of which were associated with proliferation and cell division. mRNA abundance and stability of a number of these transcripts were decreased in FXR1-depleted hVSMC, as was proliferation (P < 0.05); however, increases in beta-galactosidase (P < 0.05) and γH2AX (P < 0.01), indicative of senescence, were noted. Further analysis showed increased abundance of senescence-associated genes with FXR1 depletion. A novel SMC-specific conditional knockout mouse (FXR1SMC/SMC) was developed for further analysis. In a carotid artery ligation model of intimal hyperplasia, FXR1SMC/SMC mice had significantly reduced neointima formation (P < 0.001) after ligation, as well as increases in senescence drivers p16, p21, and p53 compared with several controls. These results suggest that in addition to destabilization of inflammatory transcripts, FXR1 stabilized cell cycle-related genes in VSMC, and absence of FXR1 led to induction of a senescent phenotype, supporting the hypothesis that FXR1 may mediate vascular disease by regulating stability of proliferative mRNA in VSMC.

17α-ethinylestradiol modulates endothelial function in ovariectomized rat carotid arteries.

17α-ethinylestradiol (EE2), a derivative of 17ß-estradiol (E2), is a potent estrogenic substance that is used as the estrogenic component of oral contraceptives (OCPs). However, women who take OCPs have an increased risk of cardiovascular events. Since few studies have examined EE2 endothelial effects, we explored the effects of EE2 on endothelial function in ovariectomized and isoflavone-free rats. After ovariectomy, 12-week-old female Sprague-Dawley rats were assigned to EE2, E2 or control groups. After 16 weeks, the EE2 and E2 groups were orally administered EE2 (8.3 µg/day) and E2 (12.6 µg/day) for 4 weeks, respectively. At 18 weeks, endothelial denudation of the left common carotid arteries was performed, and they were harvested at 20 weeks. The rats in the EE2 and E2 groups exhibited significantly decreased body weights and significantly increased uterine weights, respectively, but no differences were observed between the EE2 and E2 groups. The EE2 and E2 groups showed significantly enhanced acetylcholine-induced endothelium-dependent relaxation, with apamin plus charybdotoxin inhibiting only the EE2 group. Endothelial nitric oxide (NO) synthase expression was significantly higher in the EE2 group than in the control, but lower than in the E2 group. The intima-to-media ratio of denuded arteries was significantly lower in the E2 group than in the other groups, suggesting that NO decreased in the EE2 group compared to the E2 group. We conclude that EE2 has a weaker ability than E2 to produce NO and, for the first time, we demonstrate the ability of EE2 to enhance the activity of endothelial-derived hyperpolarizing factor.

Comparison of cardiovascular biomarker expression in extracellular vesicles, plasma and carotid plaque for the prediction of MACE in CEA patients.

Extracellular vesicles (EV) are a novel biomarker source for diagnosis and prognosis of cardiovascular disease. A protein comparison of plasma EVs in relation to blood plasma and atherosclerotic plaque has not been performed but would provide insight into the origin and content of biomarker sources and their association with atherosclerotic progression. Using samples of 88 carotid endarterectomy patients in the Athero-Express, 92 proteins (Olink Cardiovascular III panel) were measured in citrate plasma, plasma derived LDL-EVs and atherosclerotic plaque. Proteins were correlated between sources and were related to pre-operative stroke and 3-year major adverse cardiovascular events (MACE). Plasma and EV proteins correlated moderately on average, but with substantial variability. Both showed little correlation with plaque, suggesting that these circulating biomarkers may not originate from the latter. Plaque (n = 17) contained most differentially-expressed proteins in patients with stroke, opposed to EVs (n = 6) and plasma (n = 5). In contrast, EVs contained most differentially-expressed proteins for MACE (n = 21) compared to plasma (n = 9) and plaque (n = 1). EVs appear to provide additional information about severity and progression of systemic atherosclerosis than can be obtained from plasma or atherosclerotic plaque.

Inhibition of Trimethylamine N-Oxide Attenuates Neointimal Formation Through Reduction of Inflammasome and Oxidative Stress in a Mouse Model of Carotid Artery Ligation.

Aims: Trimethylamine-N-oxide (TMAO) is a metabolite generated from dietary choline, betaine, and l-carnitine, after their oxidization in the liver. TMAO has been identified as a novel independent risk factor for atherosclerosis through the induction of vascular inflammation. However, the effect of TMAO on neointimal formation in response to vascular injury remains unclear. Results: This study was conducted using a murine model of acutely disturbed flow-induced atherosclerosis induced by partial carotid artery ligation. 3,3-Dimethyl-1-butanol (DMB) was used to reduce TMAO concentrations. Wild-type mice were divided into four groups [regular diet, high-TMAO diet, high-choline diet, and high-choline diet+DMB] to investigate the effects of TMAO elevation and its inhibition by DMB. Mice fed high-TMAO and high-choline diets had significantly enhanced neointimal hyperplasia and advanced plaques, elevated arterial elastin fragmentation, increased macrophage infiltration and inflammatory cytokine secretion, and enhanced activation of nuclear factor (NF)-κB, the NLRP3 inflammasome, and endoplasmic reticulum (ER) stress relative to the control group. Mice fed high-choline diets with DMB treatment exhibited attenuated flow-induced atherosclerosis, inflammasome expression, ER stress, and reactive oxygen species expression. Human aortic smooth muscle cells (HASMCs) were used to investigate the mechanism of TMAO-induced injury. The HASMCs were treated with TMAO with or without an ER stress inhibitor to determine whether inhibition of ER stress modulates the TMAO-induced inflammatory response. Innovation: This study demonstrates that TMAO regulates vascular remodeling via ER stress. Conclusion: Our findings demonstrate that TMAO elevation promotes disturbed flow-induced atherosclerosis and that DMB administration mitigates vascular remodeling, suggesting a rationale for a TMAO-targeted strategy for the treatment of atherosclerosis. Antioxid. Redox Signal. 38, 215-233.

Sustained contractile force regulated by rho-kinase and protein kinase C in sheep carotid arterial smooth muscle.

The time course of smooth muscle contraction can be divided into two phases, the initial phase is associated with force development, whereas the sustained phase is associated with force maintenance. Cumulative evidence suggests that the two phases are regulated by different signaling pathways and that ρ-kinase (ROCK) and protein kinase C (PKC) play an important role in regulating isometric force in sustained contractions. Since the maintenance of sustained force is critical to the function of vascular smooth muscle, unraveling the complex mechanism of force maintenance is crucial for understanding the cell biology of the muscle. The present study examined the effects of ROCK and PKC on the level of phosphorylation of the 20-kD myosin light chain (MLC20) and isometric force during a sustained contraction. We used partial activation and inhibition of ROCK and PKC to reduce the isometric force by 50% of the maximal isometric force in fully activated muscle, Fmax. We then examined the level of MLC20 phosphorylation in each case. We found that in partially activated muscle the level of MLC20 phosphorylation required to maintain 50% Fmax was much lower than that required in muscles where 50% reduction in Fmax was achieved by partial inhibition of ROCK and PKC. The results can be explained by a model containing a contractile apparatus and a cytoskeletal scaffold where force generated by the contractile apparatus is transmitted to the extracellular domain through the cytoskeleton. The results indicate that ROCK and PKC play an important role in force transmission through the cytoskeleton.NEW & NOTEWORTHY The study supports a model that the maintenance of sustained force during a contraction of arterial smooth muscle is dependent on the intracellular transmission of force through the cytoskeleton and that ρ-kinase and protein kinase C plays an important role in the regulation of cytoskeletal integrity and its efficiency in force transmission.

microRNAs Associated with Carotid Plaque Development and Vulnerability: The Clinician's Perspective.

Ischemic stroke (IS) related to atherosclerosis of large arteries is one of the leading causes of mortality and disability in developed countries. Atherosclerotic internal carotid artery stenosis (ICAS) contributes to 20% of all cerebral ischemia cases. Nowadays, atherosclerosis prevention and treatment measures aim at controlling the atherosclerosis risk factors, or at the interventional (surgical or endovascular) management of mature occlusive lesions. There is a definite lack of the established circulating biomarkers which, once modulated, could prevent development of atherosclerosis, and consequently prevent the carotid-artery-related IS. Recent studies emphasize that microRNA (miRNA) are the emerging particles that could potentially play a pivotal role in this approach. There are some research studies on the association between the expression of small non-coding microRNAs with a carotid plaque development and vulnerability. However, the data remain inconsistent. In addition, all major studies on carotid atherosclerotic plaque were conducted on cell culture or animal models; very few were conducted on humans, whereas the accumulating evidence demonstrates that it cannot be automatically extrapolated to processes in humans. Therefore, this paper aims to review the current knowledge on how miRNA participate in the process of carotid plaque formation and rupture, as well as stroke occurrence. We discuss potential target miRNA that could be used as a prognostic or therapeutic tool.

Nicotinamide Mononucleotide Protects against Retinal Dysfunction in a Murine Model of Carotid Artery Occlusion.

Cardiovascular abnormality-mediated retinal ischemia causes severe visual impairment. Retinal ischemia is involved in enormous pathological processes including oxidative stress, reactive gliosis, and retinal functional deficits. Thus, maintaining retinal function by modulating those pathological processes may prevent or protect against vision loss. Over the decades, nicotinamide mononucleotide (NMN), a crucial nicotinamide adenine dinucleotide (NAD+) intermediate, has been nominated as a promising therapeutic target in retinal diseases. Nonetheless, a protective effect of NMN has not been examined in cardiovascular diseases-induced retinal ischemia. In our study, we aimed to investigate its promising effect of NMN in the ischemic retina of a murine model of carotid artery occlusion. After surgical unilateral common carotid artery occlusion (UCCAO) in adult male C57BL/6 mice, NMN (500 mg/kg/day) was intraperitoneally injected to mice every day until the end of experiments. Electroretinography and biomolecular assays were utilized to measure ocular functional and further molecular alterations in the retina. We found that UCCAO-induced retinal dysfunction was suppressed, pathological gliosis was reduced, retinal NAD+ levels were preserved, and the expression of an antioxidant molecule (nuclear factor erythroid-2-related factor 2; Nrf2) was upregulated by consecutive administration of NMN. Our present outcomes first suggest a promising NMN therapy for the suppression of cardiovascular diseases-mediated retinal ischemic dysfunction.

EXPRESSION OF SYNAPTOFYSIN AND VEGF IN THE SENSOMOTOR CORTEX DURING THE CAROTID ARTERY LIGATION, THE BRAIN ANTIGEN SENSITIZATION AND THEIR COMBINATIONS.

OBJECTIVE: The aim: To study changes of the expression of synaptophysin (Syn) and vascular endothelial growth factor (VEGF) in neurons of the sensorimotor cortex (SMC) to reveal after unilateral ligation of the carotid artery, sensitization with brain antigen and their combination. PATIENTS AND METHODS: Materials and methods: Experimental animals - Wistar rats (260-290 g). Experimental models: mobilization of the left common carotid artery, ligation of the indicated artery, sensitization with cerebral antigen, combination of sensitization with cerebral antigen and ligation of the carotid artery. Methods: immunohistochemistry, quantitative densitometric assessment. RESULTS: Results: Dyscirculatory disorders of cerebral blood supply during unilateral mobilization or ligation of the common carotid artery, sensitization with cerebral antigen lead in rats to a transient decrease in synaptophysin expression and phase changes in VEGF expression in the SMC from the lesion side. These changes occur in the absence of morphological changes in the cerebral cortex. CONCLUSION: Conclusions: The absence of morphological changes in the SMC in the short term (10-30 days) after minor trauma to the common carotid artery (separation from the bed and n.vagus) or its ligation is accompanied by a transient decrease in Syn expression and some increase in VEGF, which may reflect a violation of synaptic function and the general metabolic activity of neurons. Sensitization with a brain antigen, leading to an increase in the level of anti-brain antibodies and immune complexes in the blood of rats, can act as an independent damaging factor for the brain, and also potentiates and prolongs changes caused by impaired blood circulation.

DN200434 Inhibits Vascular Smooth Muscle Cell Proliferation and Prevents Neointima Formation in Mice after Carotid Artery Ligation.

BACKGRUOUND: Excessive proliferation and migration of vascular smooth muscle cells (VSMCs), which contributes to the development of occlusive vascular diseases, requires elevated mitochondrial oxidative phosphorylation to meet the increased requirements for energy and anabolic precursors. Therefore, therapeutic strategies based on blockade of mitochondrial oxidative phosphorylation are considered promising for treatment of occlusive vascular diseases. Here, we investigated whether DN200434, an orally available estrogen receptor-related gamma inverse agonist, inhibits proliferation and migration of VSMCs and neointima formation by suppressing mitochondrial oxidative phosphorylation. METHODS: VSMCs were isolated from the thoracic aortas of 4-week-old Sprague-Dawley rats. Oxidative phosphorylation and the cell cycle were analyzed in fetal bovine serum (FBS)- or platelet-derived growth factor (PDGF)-stimulated VSMCs using a Seahorse XF-24 analyzer and flow cytometry, respectively. A model of neointimal hyperplasia was generated by ligating the left common carotid artery in male C57BL/6J mice. RESULTS: DN200434 inhibited mitochondrial respiration and mammalian target of rapamycin complex 1 activity and consequently suppressed FBS- or PDGF-stimulated proliferation and migration of VSMCs and cell cycle progression. Furthermore, DN200434 reduced carotid artery ligation-induced neointima formation in mice. CONCLUSION: Our data suggest that DN200434 is a therapeutic option to prevent the progression of atherosclerosis.

Plasma CD16+ Extracellular Vesicles Associate with Carotid Artery Intima-Media Thickness in HIV+ Adults on Combination Antiretroviral Therapy.

HIV-infected individuals have increased risk for cardiovascular disease (CVD) despite suppressive antiretroviral therapy (ART). This is likely a result of persistent immune activation and systemic inflammation. Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and may drive inflammation contributing to CVD. EVs were characterized in plasma from 74 HIV-infected individuals on combination antiretroviral therapy (cART) and 64 HIV-uninfected controls with paired carotid intima-media thickness (cIMT) assessment. EVs were profiled with markers reflecting lymphoid, myeloid, and endothelial origin. Seventeen plasma inflammatory biomarkers were also assessed. Human umbilical vein endothelial cell (HUVEC) apoptosis was quantified after EV exposure. A significant correlation was observed in HIV-infected participants between cIMT and EVs expressing CD16, and the monocyte-related markers CD4, CD14, and CX3CR1 showed a similar but nonsignificant association with cIMT. No significant correlation between cIMT measurements from HIV-uninfected individuals and EVs was observed. Levels of serum amyloid A, C-reactive protein, and myeloperoxidase significantly correlated with CD14+, CD16+, and CX3CR1+ EVs. No correlation was noted between cIMT and soluble inflammatory markers. HUVECs showed increased necrosis after exposure to the EV-containing fraction of plasma derived from HIV-infected individuals compared to uninfected controls. Our study reveals that EVs expressing monocyte markers correlated with cIMT in HIV-infected individuals on cART. Moreover, EV fractions derived from HIV-infected individuals lead to greater endothelial cell death via necrotic pathways. Collectively, EVs have potential as biomarkers of and therapeutic targets in the pathogenesis of CVD in the setting of treated HIV disease. IMPORTANCE HIV-infected individuals have a 2-fold-increased risk of cardiovascular disease compared with the general population, yet the mechanisms underlying this comorbidity are unclear. Extracellular vesicles have emerged as important mediators in cell-cell communication and, given what we know of their biology, may drive inflammation contributing to cardiovascular disease in this vulnerable population.