Cell Α-smooth Muscle Actin Research Articles

Resolvin D2/GPR18 signaling enhances monocytic myeloid-derived suppressor cell function to mitigate abdominal aortic aneurysm formation.

Abdominal aortic aneurysm (AAA) formation is a chronic vascular pathology characterized by inflammation, leukocyte infiltration, and vascular remodeling. The aim of this study was to delineate the protective role of Resolvin D2 (RvD2), a bioactive isoform of specialized pro-resolving lipid mediators, via G-protein-coupled receptor 18 (GPR18) receptor signaling in attenuating AAAs. Importantly, RvD2 and GPR18 levels were significantly decreased in aortic tissue of AAA patients compared with controls. Furthermore, using an established murine model of AAA in C57BL/6 (WT) mice, we observed that treatment with RvD2 significantly attenuated aortic diameter, pro-inflammatory cytokine production, immune cell infiltration (neutrophils and macrophages), elastic fiber disruption, and increased smooth muscle cell α-actin expression as well as increased TGF-β2 and IL-10 expressions compared to untreated mice. Moreover, the RvD2-mediated protection from vascular remodeling and AAA formation was blocked when mice were previously treated with siRNA for GPR18 signifying the importance of RvD2/GPR18 signaling in vascular inflammation. Mechanistically, RvD2-mediated protection significantly enhanced infiltration and activation of monocytic myeloid-derived suppressor cells (M-MDSCs) by increasing TGF-β2 and IL-10 secretions in a GPR18-dependent manner to attenuate aortic inflammation and vascular remodeling. Collectively, this study demonstrates that RvD2 treatment induces an expansion of myeloid-lineage committed progenitors, such as M-MDSCs, activates GPR18-dependent signaling to enhance TGF-β2 and IL-10 secretion, and mitigates SMC activation that contributes to resolution of aortic inflammation and remodeling during AAA formation.

Specific peptides targeting the myocardiocyte are prognostic markers for heart attack: Function of α-SMA protein

Myocardial infarction (MI) is a serious cardiovascular disease that often results in a significant decline in heart function and associated complications. α-SMA (α-smooth muscle cell actin) is an important biomarker in the process of cardiac remodeling and repair, and its expression level is closely related to myocardial remodeling and prognosis. Therefore, the purpose of this study was to investigate the potential of nanoparticles containing cardiomyocyte targeting peptides in predicting prognosis and α-SMA protein expression after myocardial infarction, with a view to providing new therapeutic strategies and clinical guidelines. In this study, a novel targeting nanoparticle was constructed, using cardiomyocyte specific peptides as targeting ligands, and characterized by loading different drugs. Subsequently, a mouse model of myocardial infarction was used to systematically evaluate the effect of nanoparticles on α-SMA protein expression and prognosis prediction ability after MI. The expression level of α-SMA was analyzed by Western blot and immunohistochemistry, and the prognosis was evaluated by cardiac function assessment. The study found that nanoparticles containing cardiomyocyte targeting peptides significantly increased α-SMA expression levels and improved heart function in animal models of myocardial infarction. Compared with the control group, the application of targeted nanoparticles was closely related to the level of myocardial cell repair and fibrosis, and could effectively predict the prognosis after myocardial infarction. Therefore, nanoparticles containing cardiomyocyte targeting peptides can not only effectively improve the expression of α-SMA, but also serve as an important prognostic tool after myocardial infarction.

Pharmacologic Inhibition of Ferroptosis Attenuates Experimental Abdominal Aortic Aneurysm Formation.

The pathogenesis of abdominal aortic aneurysm (AAA) formation involves vascular inflammation, thrombosis formation and programmed cell death leading to aortic remodeling. Recent studies have suggested that ferroptosis, an excessive iron-mediated cell death, can regulate cardiovascular diseases, including AAAs. However, the role of ferroptosis in immune cells, like macrophages, and ferroptosis-related genes in AAA formation remains to be deciphered. Single cell-RNA sequencing of human aortic tissue from AAA patients demonstrates significant differences in ferroptosis-related genes compared to control aortic tissue. Using two established murine models of AAA and aortic rupture in C57BL/6 (WT) mice, we observed that treatment with liproxstatin-1, a specific ferroptosis inhibitor, significantly attenuated aortic diameter, pro-inflammatory cytokine production, immune cell infiltration (neutrophils and macrophages), increased smooth muscle cell α-actin expression and elastic fiber disruption compared to mice treated with inactivated elastase in both pre-treatment and treatment after a small AAA had already formed. Lipidomic analysis using mass spectrometry shows a significant increase in ceramides and a decrease in intact lipid species levels in murine tissue compared to controls in the chronic AAA model on day 28. Mechanistically, in vitro studies demonstrate that liproxstatin-1 treatment of macrophages mitigated the crosstalk with aortic smooth muscle cells (SMCs) by downregulating MMP2 secretion. Taken together, this study demonstrates that pharmacological inhibition by liproxstatin-1 mitigates macrophage-dependent ferroptosis contributing to inhibition of aortic inflammation and remodeling during AAA formation.

Anthelmintic nitazoxanide protects against experimental pulmonary fibrosis.

Nitazoxanide is a therapeutic anthelmintic drug. Our previous studies found that nitazoxanide and its metabolite tizoxanide activated adenosine 5'-monophosphate-activated protein kinase (AMPK) and inhibited signal transducer and activator of transcription 3 (STAT3) signals. As AMPK activation and/or STAT3 inhibition are targets for treating pulmonary fibrosis, we hypothesized that nitazoxanide would be effective in experimental pulmonary fibrosis. The mitochondrial oxygen consumption rate of cells was measured by using the high-resolution respirometry system Oxygraph-2K. The mitochondrial membrane potential of cells was evaluated by tetramethyl rhodamine methyl ester (TMRM) staining. The target protein levels were measured by using western blotting. The mice pulmonary fibrosis model was established through intratracheal instillation of bleomycin. The examination of the lung tissues changes were carried out using haematoxylin and eosin (H&E), and Masson staining. Nitazoxanide and tizoxanide activated AMPK and inhibited STAT3 signalling in human lung fibroblast cells (MRC-5 cells). Nitazoxanide and tizoxanide inhibited transforming growth factor-β1 (TGF-β1)-induced proliferation and migration of MRC-5 cells, collagen-I and α-smooth muscle cell actin (α-SMA) expression, and collagen-I secretion from MRC-5 cells. Nitazoxanide and tizoxanide inhibited epithelial-mesenchymal transition (EMT) and inhibited TGF-β1-induced Smad2/3 activation in mouse lung epithelial cells (MLE-12 cells). Oral administration of nitazoxanide reduced the bleomycin-induced mice pulmonary fibrosis and, in the established bleomycin-induced mice, pulmonary fibrosis. Delayed nitazoxanide treatment attenuated the fibrosis progression. Nitazoxanide improves the bleomycin-induced pulmonary fibrosis in mice, suggesting a potential application of nitazoxanide for pulmonary fibrosis treatment in the clinic.

Resolution of inflammation via RvD1/FPR2 signaling mitigates Nox2 activation and ferroptosis of macrophages in experimental abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) formation is characterized by inflammation, leukocyte infiltration, and vascular remodeling. Resolvin D1 (RvD1) is derived from ω-3 polyunsaturated fatty acids and is involved in the resolution phase of chronic inflammatory diseases. The aim of this study was to decipher the protective role of RvD1 via formyl peptide receptor 2 (FPR2) receptor signaling in attenuating abdominal aortic aneurysms (AAA). The elastase-treatment model of AAA in C57BL/6 (WT) mice and human AAA tissue was used to confirm our hypotheses. Elastase-treated FPR2-/- mice had a significant increase in aortic diameter, proinflammatory cytokine production, immune cell infiltration (macrophages and neutrophils), elastic fiber disruption, and decrease in smooth muscle cell α-actin expression compared to elastase-treated WT mice. RvD1 treatment attenuated AAA formation, aortic inflammation, and vascular remodeling in WT mice, but not in FPR2-/- mice. Importantly, human AAA tissue demonstrated significantly decreased FPR2 mRNA expression compared to non-aneurysm human aortas. Mechanistically, RvD1/FPR2 signaling mitigated p47phox phosphorylation and prevented hallmarks of ferroptosis, such as lipid peroxidation and Nrf2 translocation, thereby attenuating HMGB1 secretion. Collectively, this study demonstrates RvD1-mediated immunomodulation of FPR2 signaling on macrophages to mitigate ferroptosis and HMGB1 release, leading to resolution of aortic inflammation and remodeling during AAA pathogenesis.

Loss of myeloid lipoprotein lipase exacerbates adipose tissue fibrosis with collagen VI deposition and hyperlipidemia in leptin-deficient obese mice

During obesity, tissue macrophages increase in number and become proinflammatory, thereby contributing to metabolic dysfunction. Lipoprotein lipase (LPL), which hydrolyzes triglyceride in lipoproteins, is secreted by macrophages. However, the role of macrophage-derived LPL in adipose tissue remodeling and lipoprotein metabolism is largely unknown. To clarify these issues, we crossed leptin-deficient Lepob/ob mice with mice lacking the Lpl gene in myeloid cells (Lplm−/m−) to generate Lplm−/m−;Lepob/ob mice. We found the weight of perigonadal white adipose tissue (WAT) was increased in Lplm−/m−;Lepob/ob mice compared with Lepob/ob mice due to substantial accumulation of both adipose tissue macrophages and collagen that surrounded necrotic adipocytes. In the fibrotic epidydimal WAT of Lplm−/m−;Lepob/ob mice, we observed an increase in collagen VI and high mobility group box 1, while α-smooth muscle cell actin, a marker of myofibroblasts, was almost undetectable, suggesting that the adipocytes were the major source of the collagens. Furthermore, the adipose tissue macrophages from Lplm−/m−;Lepob/ob mice showed increased expression of genes related to fibrosis and inflammation. In addition, we determined Lplm−/m−;Lepob/ob mice were more hypertriglyceridemic than Lepob/ob mice. Lplm−/m−;Lepob/ob mice also showed slower weight gain than Lepob/ob mice, which was primarily due to reduced food intake. In conclusion, we discovered that the loss of myeloid Lpl led to extensive fibrosis of perigonadal WAT and hypertriglyceridemia. In addition to illustrating an important role of macrophage LPL in regulation of circulating triglyceride levels, these data show that macrophage LPL protects against fibrosis in obese adipose tissues.

Abstract 11622: Resolution of Inflammation via Rvd1/fpr2 Signaling in Abdominal Aortic Aneurysms

Introduction: Resolvin D1 (RvD1) is derived from a ω-3 polyunsaturated fatty acid and is involved in the resolution phase of inflammation during abdominal aortic aneurysm (AAA) formation. Hypothesis: The aim of this study was to decipher the protective role of RvD1 via formyl peptide receptor 2 (FPR2) receptor signaling in attenuating AAA formation. Methods: C57BL/6 (WT) and FPR2 -/- mice (8-12 week old) underwent treatment with topical elastase or heat-inactivated elastase (control). Separate groups of mice were administered RvD1 (100ng/kg) or saline i.p. daily from day(d)1 to d13 and aortic diameter was measured by video micrometry on d14. mRNA quantification of FPR2 expression in aortic tissue was performed by RT-PCR on d7, 14 and 21. Human aortic tissue from AAA patients and controls (organ transplant donors) were analyzed for quantification of FPR2 expression. Data is presented as mean ± SEM and statistical analysis was performed by using Mann-Whitney test for phenotype or Wilcoxon Signed Rank Test for gene expression data. Results: Elastase-treated WT mice had significantly increased aortic diameter compared to controls (127±8% vs. 1± 0.6%; p<0.0001). Importantly, elastase-treated FPR2 -/- had a significant increase in aortic diameter compared to elastase-treated WT mice (165±16% vs. 127± 8%; p=0.03). RvD1 treatment attenuated AAA formation in WT mice, but not FPR2 -/- mice, on d14 (56± 10% vs.121± 8%; p<0.0001). Immunohistology documented decreased elastin and smooth muscle cell α-actin expression, as well as increased neutrophil and macrophage infiltration in FPR2 -/- mice compared to WT mice, after respective treatments with RvD1. Elastase-treated WT mice had significantly increased FPR2 receptor mRNA expression compared to respective controls on d7, d14, and d21. Human AAA tissue demonstrated significantly decreased FPR2 mRNA expression compared to controls (p=0.02). Conclusions: This data identifies the role of FPR2 in dysregulation of inflammation-resolution during AAA pathogenesis. Immunomodulation of FPR2 signaling by exogenous RvD1 identifies a novel mechanism in resolution of aortic inflammation and vascular remodeling during AAA pathogenesis.

Control of Tissue Fibrosis by 5-Methoxytryptophan, an Innate Anti-Inflammatory Metabolite.

Tissue fibrosis causes debilitating human diseases such as liver cirrhosis, heart failure, chronic kidney disease and pulmonary insufficiency. It is a dynamic process orchestrated by specific subsets of monocyte-macrophages, fibroblasts, pericytes and hepatic stellate cells. Fibrosis is linked to tissue inflammation. Pro-inflammatory macrophages promote fibrosis by driving myofibroblast differentiation and macrophage myofibroblast transition. Myofibroblasts express α-smooth muscle cell actin (α-SMA) and secrete extracellular matrix (ECM) proteins notably collagen I and III. Deposition of ECM proteins at injury sites and interstitial tissues distorts normal structure and impairs vital functions. Despite advances in the mechanisms of fibrosis at cellular, molecular and genetic levels, prevention and treatment of fibrotic diseases remain poorly developed. Recent reports suggest that 5-methoxytryptophan (5-MTP) is effective in attenuating injury-induced liver, kidney, cardiac and pulmonary fibrosis. It inhibits macrophage activation and blocks fibroblast differentiation to myofibroblasts. Furthermore, it inhibits hepatic stellate cell differentiation into myofibroblasts. As 5-MTP is an endogenous molecule derived from tryptophan catabolism via tryptophan hydroxylase pathway, it is well-suited as a lead compound for developing new anti-fibrotic drugs. This article provides an overview of 5-MTP synthesis, and a critical review of its anti-fibrotic activities. Its mechanisms of actions and potential therapeutic value will be discussed.

Cardiovascular ACE2 receptor expression in patients undergoing heart transplantation.

AimsMembrane‐bound angiotensin‐converting enzyme (ACE)2 is the main cellular access point for SARS‐CoV‐2, but its expression and the effect of ACE inhibition have not been assessed quantitatively in patients with heart failure. The aim of this study was to characterize membrane‐bound ACE2 expression in the myocardium and myocardial vasculature in patients undergoing heart transplantation and to assess the effect of pharmacological ACE inhibition.Methods and resultsLeft ventricular (LV) tissue was obtained from 36 explanted human hearts from patients undergoing heart transplantation. Immunohistochemical staining with antibodies directed against ACE2 co‐registered with cardiac troponin T (cTnT) and α‐smooth muscle cell actin (SMA) was performed across the entire cohort. ACE2 receptor expression was quantitatively assessed throughout the myocardium and vasculature. ACE2 was consistently expressed throughout the LV myocardium (28.3% ± 22.2% of cardiomyocytes). ACE2 expression was also detected in small calibre blood vessels (range, 2–9 μm), albeit at quantitatively much lower levels (5% ± 9% of blood vessels). There was no significant difference in ACE2 expression between patients receiving ACE inhibitors prior to transplantation and ACE inhibitor‐negative controls (P > 0.05). ACE2 expression did not differ significantly between the different diagnostic groups as the underlying reason for heart transplantation (ANOVA > 0.05). N‐terminal pro‐brain natriuretic peptide (NT‐proBNP) (R 2 = 0.37, P = 0.0006) and pulmonary capillary wedge pressure (PCWP) (R 2 = 0.13, P = 0.043) assessed by right heart catheterization were significantly correlated with greater ACE2 expression in cardiomyocytes.ConclusionsThese data provide a comprehensive characterization of membrane‐bound cardiac ACE2 expression in patients with heart failure with no demonstrable effect exerted by ACE inhibitors.

Pharmacologic inhibition of transient receptor channel vanilloid 4 attenuates abdominal aortic aneurysm formation.

Abdominal aortic aneurysm (AAA) formation is characterized by inflammation, leukocyte infiltration, and vascular remodeling. This study investigates the role of TRPV4 channels, which are transmembrane calcium channels that can regulate vascular tone, in modulating AAA formation. The elastase-treatment model of AAA in C57BL6 (WT) mice and Angiotensin II treatment model in ApoE-/- mice were used to confirm our hypotheses. The administration of a specific TRPV4 antagonist, GSK2193874, in elastase-treated WT mice and in AngII-treated ApoE-/- mice caused a significant attenuation of aortic diameter, decrease in pro-inflammatory cytokines (IL-1β, IL-6, IL-17, MCP-1, MIP-1α, MIP-2, RANTES, and TNF-α), inflammatory cell infiltration (CD3+T cells, macrophages, and neutrophils), elastic fiber disruption, and an increase in smooth muscle cell α-actin expression compared to untreated mice. Similarly, elastase-treated TRPV4-/- mice had a significant decrease in AAA formation, aortic inflammation, and vascular remodeling compared to elastase-treated WT mice on Day 14. In vitro studies demonstrated that the inhibition of TRPV4 channels mitigates aortic smooth muscle cell-dependent inflammatory cytokine production as well as decreases neutrophil transmigration through aortic endothelial cells. Therefore, our results suggest that TRPV4 antagonism can attenuate aortic inflammation and remodeling via decreased smooth muscle cell activation and neutrophil transendothelial migration during AAA formation.

Downregulation of lysyl oxidase in venous malformations: Association with vascular destabilization and sclerotherapy.

Venous malformations (VM) are localized defects in vascular morphogenesis manifested by dilated venous channels with reduced perivascular cell coverage. As a vital enzyme for extracellular matrix (ECM) deposition, lysyl oxidase (LOX) plays important roles in vascular development and diseases. However, the expression and significance of LOX are unknown in VM. Herein, 22 VM specimens and eight samples of normal skin tissues were evaluated immunohistochemically for the expression of LOX, α-smooth muscle cell actin (α-SMA) and transforming growth factor-β (TGF-β). In vitro studies on human umbilical vein endothelial cells (HUVEC) were employed for determining potential mechanisms. Our results showed that LOX expression was significantly reduced in VM compared with normal skin tissues, in parallel with attenuated perivascular α-SMA+ cell coverage and TGF-β downregulation in VM. Further correlation analysis indicated that LOX expression was positively correlated with perivascular α-SMA+ cell coverage and TGF-β expression in VM. Moreover, marked elevation of LOX, TGF-β and α-SMA was observed in bleomycin-treated VM samples. Furthermore, our in vitro data demonstrated that both recombinant TGF-β and bleomycin induced obvious increase of LOX expression and activity and a concomitant increase in ECM components in HUVEC, which could be reversed by LOX inhibition. To our best knowledge, this study revealed for the first time the downregulation of LOX in VM and its correlation with vascular destabilization and TGF-β-induced endothelial ECM deposition. Moreover, our results highlighted that LOX may be implicated in the sclerotherapy of VM and holds promise as a therapeutic target.

Homologous and heterologous assessment of a novel biomaterial for venous patch

ObjectiveThis study evaluated swine and bovine pulmonary visceral pleura (PVP) as a vascular patch. Venous patches are frequently used in surgery for repair or reconstruction of veins. Autologous patches are often limited by the number and dimension of donor tissue and can result in donor complications. Bovine pericardium is the most common heterologous patch used by vascular surgeons. Researchers, however, are continually seeking to improve heterologous and synthetic patches for improved outcome. MethodsThe PVP was peeled from swine and bovine lungs and cross-linked with glutaraldehyde. After sterilization and rinsing, the PVP patches were implanted in the jugular vein (10 × 35 mm) of pigs and dogs. Patency was evaluated by ultrasound, and animals were euthanized at 2 and 4 months. Neoendothelium and neomedia were evaluated by histologic analysis. ResultsThe jugular vein patched by PVP in pigs and dogs remained patent at 2 and 4 months with no adhesions, inflammation, or aneurysm in the patches. The biomarkers of endothelial cells—factor VIII, platelet/endothelial cell adhesion molecule 1, and endothelial nitric oxide synthase—were detected in the neoendothelial cells. The expression of vascular smooth muscle cell (VSMC) α-actin was robust in the neomedia at 2 and 4 months. Neomedia composed of VSMCs developed to nearly double the thickness of adjacent jugular vein. The circumferential orientation of VSMCs in neomedia further increased in the 4-month group. ConclusionsThe cross-linked swine and bovine PVP patch has a nonthrombogenic surface that maintains patency. The PVP patch may overcome the pitfall of compliance mismatch of synthetic patches. The proliferation of vascular cells assembled in the neoendothelium and neomedia in the patches may support long-term patency.

The Role of MicroRNA-21 in Venous Neointimal Hyperplasia: Implications for Targeting miR-21 for VNH Treatment.

The molecular mechanism of hemodialysis access arteriovenous fistula (AVF) failure due to venous neointimal hyperplasia (VNH) is not known. The role of microRNA-21 (miR-21) in VNH associated with AVF failure was investigated by performing in vivo and in vitro experiments. In situ hybridization results revealed that miR-21 expression increased and was associated with fibroblasts in failed AVFs from patients. In a murine AVF model, qRT-PCR gene expression results showed a significant increase in miR-21 and a decrease in miR-21 target genes in graft veins (GVs) compared to contralateral veins in mouse AVF. miR-21 knockdown in GVs was performed using a lentivirus-mediated small hairpin RNA (shRNA), and this improved AVF patency with a decrease in neointima compared to control GVs. Moreover, loss of miR-21 in GVs significantly decreased the Tgfβ1, Col-Ia, and Col-Iva genes. Immunohistochemistry demonstrated a significant decrease in myofibroblasts and proliferation with an increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in miR-21-knockdown vessels, along with a decrease in hypoxia-inducible factor-1 alpha (HIF-1α) and phospho-SMAD2 (pSMAD-2) and phospho-SMAD3 (pSMAD-3) and an increase in phosphatase and tensin homolog (PTEN) staining. Hypoxic fibroblast knockdown for miR-21 showed a significant decrease in Tgfβ-1 expression and pSMAD-2 and -3 levels and a decrease in myofibroblasts. These results indicate that miR-21 upregulation causes VNH formation by fibroblast-to-myofibroblast differentiation.

Effects of low-dose oxygen ions and protons on cardiac function and structure in male C57BL/6J mice

PurposeAstronauts traveling beyond low-Earth orbit will be exposed to high linear-energy transfer charged particles. Because there is concern about the adverse effects of space radiation on the cardiovascular system, this study assessed cardiac function and structure and immune cell infiltration in a mouse model of charged-particle irradiation. Materials and methodsMale C57BL/6 J mice were exposed to oxygen ions (16O, 600 MeV/n at 0.25−0.26 Gy/min to a total dose of 0, 0.05, 0.1, 0.25, or 1 Gy), protons (150 MeV, 0.35−0.55 Gy/min to 0, 0.5, or 1 Gy), or protons (150 MeV, 0.5 Gy) followed by 16O (600 MeV/n, 0.1 Gy). Separate groups of mice received 137Cs γ-rays (1 Gy/min to 0, 0.5, 1, or 3 Gy) as a reference. Cardiac function and blood velocity were measured with ultrasonography at 3, 5, 7, and 9 months after irradiation. At 2 weeks, 3 months, and 9 months, cardiac tissue was collected to assess apoptosis, tissue remodeling, and markers of immune cells. ResultsEjection fraction and fractional shortening decreased at 3 and 7 months after 16O. These parameters did not change in mice exposed to γ-rays, protons, or protons followed by 16O. Each of the radiation exposures caused only small increases in cleaved caspase-3 and numbers of apoptotic nuclei. Changes in the levels of α-smooth muscle cell actin and a 75-kDa peptide of collagen type III in the left ventricle suggested tissue remodeling, but there was no significant change in total collagen deposition at 2 weeks, 3 months, and 9 months. Increases in protein amounts of cluster of differentiation (CD)2, CD68, and CD45 as measured with immunoblots at 2 weeks, 3 months, and 9 months after exposure to protons or 16O alone suggested immune cell infiltration. For type III collagen, CD2 and CD68, the efficacy in inducing protein abundance of CD2, CD68, and CD45 was 16O > protons > γ-rays > protons followed by 16O. ConclusionsLow-dose, high-energy charged-particle irradiation caused mild changes in cardiac function and tissue remodeling in the mouse.

Characterization of Atherosclerosis Formation in a Murine Model of Type IIa Human Familial Hypercholesterolemia.

A murine genetic model of LDL-cholesterol- (LDL-C-) driven atherosclerosis, based on complete deficiencies of both the LDL-receptor (Ldlr−/−) and key catalytic component of an apolipoprotein B-edisome complex (Apobec1−/−), which converts apoB-100 to apoB-48, has been extensively characterized. These gene deficiencies allow high levels of apoB-100 to be present and inefficiently cleared, thus leading to very high levels of LDL-C in mice on a normal diet. Many key features of atherosclerotic plaques observed in human familial hypercholesterolemia are found in these mice as they are allowed to age through 72 weeks. The general characteristics include the presence of high levels of LDL-C in plasma and macrophage-related fatty streak formation in the aortic tree, which progressively worsens with age. More specifically, plaque found in the aortic sinuses contains a lipid core with relatively high numbers of macrophages and a smooth muscle cell α-actin- and collagen-containing cap, which thins with age. These critical features of plaque progression suggest that the Ldlr−/−/Apobec1−/− mouse line presents a superior model of LDL-C-driven atherosclerosis.

Human mesenchymal stromal cell-derived extracellular vesicles attenuate aortic aneurysm formation and macrophage activation via microRNA-147.

The formation of an abdominal aortic aneurysm (AAA) is characterized by inflammation, macrophage infiltration, and vascular remodeling. In this study, we tested the hypothesis that mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) immunomodulate aortic inflammation, to mitigate AAA formation via modulation of microRNA-147. An elastase-treatment model of AAA was used in male C57BL/6 wild-type (WT) mice. Administration of EVs in elastase-treated WT mice caused a significant attenuation of aortic diameter and mitigated proinflammatory cytokines, inflammatory cell infiltration, an increase in smooth muscle cell α-actin expression, and a decrease in elastic fiber disruption, compared with untreated mice. A 10-fold up-regulation of microRNA (miR)-147, a key mediator of macrophage inflammatory responses, was observed in murine aortic tissue in elastase-treated mice compared with controls on d 14. EVs derived from MSCs transfected with miR-147 mimic, but not with miR-147 inhibitor, attenuated aortic diameter, inflammation, and leukocyte infiltration in elastase-treated mice. In vitro studies of human aortic tissue explants and murine-derived CD11b+ macrophages induced proinflammatory cytokines after elastase treatment, and the expression was attenuated by cocultures with EVs transfected with miR-147 mimic, but not with miR-147 inhibitor. Thus, our findings define a critical role of MSC-derived EVs in attenuation of aortic inflammation and macrophage activation via miR-147 during AAA formation.-Spinosa, M., Lu, G., Su, G., Bontha, S. V., Gehrau, R., Salmon, M. D., Smith, J. R., Weiss, M. L., Mas, V. R., Upchurch, G. R., Sharma, A. K. Human mesenchymal stromal cell-derived extracellular vesicles attenuate aortic aneurysm formation and macrophage activation via microRNA-147.

Effects of low-dose rate γ-irradiation combined with simulated microgravity on markers of oxidative stress, DNA methylation potential, and remodeling in the mouse heart.

PurposeSpace travel is associated with an exposure to low-dose rate ionizing radiation and the microgravity environment, both of which may lead to impairments in cardiac function. We used a mouse model to determine short- and long-term cardiac effects to simulated microgravity (hindlimb unloading; HU), continuous low-dose rate γ-irradiation, or a combination of HU and low-dose rate γ-irradiation.MethodsCardiac tissue was obtained from female, C57BL/6J mice 7 days, 1 month, 4 months, and 9 months following the completion of a 21 day exposure to HU or a 21 day exposure to low-dose rate γ-irradiation (average dose rate of 0.01 cGy/h to a total of 0.04 Gy), or a 21 day simultaneous exposure to HU and low-dose rate γ-irradiation. Immunoblot analysis, rt-PCR, high-performance liquid chromatography, and histology were used to assess inflammatory cell infiltration, cardiac remodeling, oxidative stress, and the methylation potential of cardiac tissue in 3 to 6 animals per group.ResultsThe combination of HU and γ-irradiation demonstrated the strongest increase in reduced to oxidized glutathione ratios 7 days and 1 month after treatment, but a difference was no longer apparent after 9 months. On the other hand, no significant changes in 4-hydroxynonenal adducts was seen in any of the groups, at the measured endpoints. While manganese superoxide dismutase protein levels decreased 9 months after low-dose γ-radiation, no changes were observed in expression of catalase or Nrf2, a transcription factor that determines the expression of several antioxidant enzymes, at the measured endpoints. Inflammatory marker, CD-2 protein content was significantly decreased in all groups 4 months after treatment. No significant differences were observed in α-smooth muscle cell actin protein content, collagen type III protein content or % total collagen.ConclusionsThis study has provided the first and relatively broad analysis of small molecule and protein markers of oxidative stress, T-lymphocyte infiltration, and cardiac remodeling in response to HU with simultaneous exposure to low-dose rate γ-radiation. Results from the late observation time points suggest that the hearts had mostly recovered from these two experimental conditions. However, further research is needed with larger numbers of animals for a more robust statistical power to fully characterize the early and late effects of simulated microgravity combined with exposure to low-dose rate ionizing radiation on the heart.

Chemokine binding protein 'M3' limits atherosclerosis in apolipoprotein E-/- mice.

Chemokines are important in macrophage recruitment and the progression of atherosclerosis. The ‘M3’ chemokine binding protein inactivates key chemokines involved in atherosclerosis (e.g. CCL2, CCL5 and CX3CL1). We aimed to determine the effect of M3 on plaque development and composition. In vitro chemotaxis studies confirmed that M3 protein inhibited the activity of chemokines CCL2, CCL5 and CX3CL1 as primary human monocyte migration as well as CCR2-, CCR5- and CX3CR1-directed migration was attenuated by M3. In vivo, adenoviruses encoding M3 (AdM3) or green fluorescence protein (AdGFP; control) were infused systemically into apolipoprotein (apo)-E-/- mice. Two models of atherosclerosis development were used in which the rate of plaque progression was varied by diet including: (1) a ‘rapid promotion’ model (6-week high-fat-fed) and (2) a ‘slow progression’ model (12-week chow-fed). Plasma chemokine activity was suppressed in AdM3-infused mice as indicated by significantly less monocyte migration towards AdM3 mouse plasma ex vivo (29.56%, p = 0.014). In the ‘slow progression’ model AdM3 mice had reduced lesion area (45.3%, p = 0.035) and increased aortic smooth muscle cell α-actin expression (60.3%, p = 0.014). The reduction in lesion size could not be explained by changes in circulating inflammatory monocytes as they were higher in the AdM3 group. In the ‘rapid promotion’ model AdM3 mice had no changes in plaque size but reduced plaque macrophage content (46.8%, p = 0.006) and suppressed lipid deposition in thoracic aortas (66.9%, p<0.05). There was also a reduction in phosphorylated p65, the active subunit of NF-κb, in the aortas of AdM3 mice (37.3%, p<0.0001). M3 inhibited liver CCL2 concentrations in both models with no change in CCL5 or systemic chemokine levels. These findings show M3 causes varying effects on atherosclerosis progression and plaque composition depending on the rate of lesion progression. Overall, our studies support a promising role for chemokine inhibition with M3 for the treatment of atherosclerosis.

Primary culture and functional identification of distal pulmonary artery smooth muscle cells in mice

Objective: To establish a method of isolation and primary culture of mice distal pulmonary artery smooth muscle cells (PASMCs) and identify the functional properties. Methods: PASMCs were harvested from the distal pulmonary artery (PA) tissue of mice by enzymatic digestion of collagenaseⅠand papain; and the growth characteristics were observed under inverted microscope and identified by Immunofluorescence technique. Effects on the intracellular calcium ion concentration of distal PASMCs were detected by Fura-2-AM fluorescent probe tracer under a fluorescence microscope in Krebs solution containing clopiazonic acid (CPA) and nifedipin (Nif). Results: PASMCs density reached approximately to 80% in a typical valley-peak-like shape after 6 days. Cell α-smooth muscle actin (α-SMA) immunofluorescence identified that 95% of the cultured cells were PASMCs. More than 95% PASMCs responded well to calcium-potassium Krebs solution (potassium ion concentration of 60 mmol/L) and showed a rapid increase in basal [Ca(2+) ](i) after 1 minute's perfusion (Δ[Ca(2+) ](i)>50), which demonstrated that the voltage-dependent calcium channels (VDCC) of distal PASMCs were in good function; after the perfusion of calcium Krebs, calcium-free/calcium-Krebs containing CPA and Nif, distal PASMCs showed two typical peaks, indicated the full function of store-operated calcium channel (SOCC) in distal PASMCs. Conclusion: This experiment successfully established a stable and reliable mice distal PASMCs model and the study of pulmonary vascular diseases could benefit from its higher purity and better functional condition.

The Role of Repeat Administration of Adventitial Delivery of Lentivirus-shRNA-Vegf-A in Arteriovenous Fistula to Prevent Venous Stenosis Formation

PurposeTo determine if a second dose of a lentivirus mediated small hairpin RNA that inhibits Vegf-A gene expression (LV-shRNA-Vegf-A) can improve lumen vessel area (LVA) of the outflow vein of an arteriovenous fistula (AVF) and decrease venous neointimal hyperplasia. Materials and MethodsChronic kidney disease was created in C57BL/6 mice; 28 days later, an AVF was created by connecting the right carotid artery to the ipsilateral jugular vein. Immediately after AVF creation, 5 × 106 plaque-forming units of LV-shRNA-Vegf-A or control shRNA was administered to the adventitia of the outflow vein, and a second dose of the same treatment was administered 14 days later. Animals were sacrificed at 21 days, 28 days, and 42 days after AVF creation for reverse transcription polymerase chain reaction and histomorphometric analyses. ResultsBy day 21, there was a 125% increase in the average LVA (day 21, P = .11), with a decrease in cell proliferation (day 21, P = .0079; day 28, P = .28; day 42, P = .5), decrease in α-smooth muscle cell actin staining (day 21, P < .0001; day 28, P < .05; day 42, P = .59), and decrease in hypoxic stress (day 21, P < .001; day 28, P = .28; day 42, P = .46) in LV versus control shRNA vessels. ConclusionsA second dose of LV-shRNA-Vegf-A administration results in a moderate improvement in LVA at day 21.