Follicle-Stimulating Hormone Provokes Macrophages to Secrete IL-1ß Contributing to Atherosclerosis Progression.

Abnormally high follicle-stimulating hormone (FSH) has been reported to associate with cardiovascular diseases in prostate cancer patients with specific androgen deprivation therapy and in menopausal women. All of the cardiovascular diseases were involved in atherosclerosis. However, the pathogenic mechanism of FSH-associated atherosclerosis remains uncertain. Apolipoprotein E-deficient mice were chosen to develop atherosclerosis, of which the plaques were analyzed with administration of short- and long-term FSH imitating androgen deprivation therapy-induced and menopausal FSH elevation. The study showed that short- and long-term exposure of FSH significantly accelerated atherosclerosis progression in apolipoprotein E-deficient mice, manifested as strikingly increased plaques in the aorta and its roots, increased macrophage content, reduced fibrin, and an enlarged necrotic core, suggesting a decrease in plaque stability. Furthermore, expression profiles from the Gene Expression Omnibus GSE21545 dataset revealed that macrophage inflammation was tightly associated with FSH-induced atherosclerotic progression. The human monocyte cell line THP-1 was induced by PMA and worked as a macrophage model to detect inflammatory factors and cellular functions. FSH remarkably promoted the expression of IL-1ß in macrophages and strikingly increased the chemotactic migratory capacity of macrophages toward MCP-1, but the promigratory capacity of FSH was attenuated in foam cells. Overall, we revealed that FSH significantly promoted the inflammatory response and migration of macrophages, thereby provoking atherosclerosis development.

Follicle-Stimulating Hormone Provokes Macrophages to Secrete IL-1ß Contributing to Atherosclerosis Progression.

Abnormally high follicle-stimulating hormone (FSH) has been reported to associate with cardiovascular diseases in prostate cancer patients with specific androgen deprivation therapy and in menopausal women. All of the cardiovascular diseases were involved in atherosclerosis. However, the pathogenic mechanism of FSH-associated atherosclerosis remains uncertain. Apolipoprotein E-deficient mice were chosen to develop atherosclerosis, of which the plaques were analyzed with administration of short- and long-term FSH imitating androgen deprivation therapy-induced and menopausal FSH elevation. The study showed that short- and long-term exposure of FSH significantly accelerated atherosclerosis progression in apolipoprotein E-deficient mice, manifested as strikingly increased plaques in the aorta and its roots, increased macrophage content, reduced fibrin, and an enlarged necrotic core, suggesting a decrease in plaque stability. Furthermore, expression profiles from the Gene Expression Omnibus GSE21545 dataset revealed that macrophage inflammation was tightly associated with FSH-induced atherosclerotic progression. The human monocyte cell line THP-1 was induced by PMA and worked as a macrophage model to detect inflammatory factors and cellular functions. FSH remarkably promoted the expression of IL-1ß in macrophages and strikingly increased the chemotactic migratory capacity of macrophages toward MCP-1, but the promigratory capacity of FSH was attenuated in foam cells. Overall, we revealed that FSH significantly promoted the inflammatory response and migration of macrophages, thereby provoking atherosclerosis development.

Vitexin inhibits APEX1 to counteract the flow-induced endothelial inflammation.

Vascular endothelial cells are exposed to shear stresses with disturbed vs. laminar flow patterns, which lead to proinflammatory vs. antiinflammatory phenotypes, respectively. Effective treatment against endothelial inflammation and the consequent atherogenesis requires the identification of new therapeutic molecules and the development of drugs targeting these molecules. Using Connectivity Map, we have identified vitexin, a natural flavonoid, as a compound that evokes the gene-expression changes caused by pulsatile shear, which mimics laminar flow with a clear direction, vs. oscillatory shear (OS), which mimics disturbed flow without a clear direction. Treatment with vitexin suppressed the endothelial inflammation induced by OS or tumor necrosis factor-α. Administration of vitexin to mice subjected to carotid partial ligation blocked the disturbed flow-induced endothelial inflammation and neointimal formation. In hyperlipidemic mice, treatment with vitexin ameliorated atherosclerosis. Using SuperPred, we predicted that apurinic/apyrimidinic endonuclease1 (APEX1) may directly interact with vitexin, and we experimentally verified their physical interactions. OS induced APEX1 nuclear translocation, which was inhibited by vitexin. OS promoted the binding of acetyltransferase p300 to APEX1, leading to its acetylation and nuclear translocation. Functionally, knocking down APEX1 with siRNA reversed the OS-induced proinflammatory phenotype, suggesting that APEX1 promotes inflammation by orchestrating the NF-κB pathway. Animal experiments with the partial ligation model indicated that overexpression of APEX1 negated the action of vitexin against endothelial inflammation, and that endothelial-specific deletion of APEX1 ameliorated atherogenesis. We thus propose targeting APEX1 with vitexin as a potential therapeutic strategy to alleviate atherosclerosis.

VAMP3 and SNAP23 mediate the disturbed flow-induced endothelial microRNA secretion and smooth muscle hyperplasia.

Vascular endothelial cells (ECs) at arterial branches and curvatures experience disturbed blood flow and induce a quiescent-to-activated phenotypic transition of the adjacent smooth muscle cells (SMCs) and a subsequent smooth muscle hyperplasia. However, the mechanism underlying the flow pattern-specific initiation of EC-to-SMC signaling remains elusive. Our previous study demonstrated that endothelial microRNA-126-3p (miR-126-3p) acts as a key intercellular molecule to increase turnover of the recipient SMCs, and that its release is reduced by atheroprotective laminar shear (12 dynes/cm2) to ECs. Here we provide evidence that atherogenic oscillatory shear (0.5 ± 4 dynes/cm2), but not atheroprotective pulsatile shear (12 ± 4 dynes/cm2), increases the endothelial secretion of nonmembrane-bound miR-126-3p and other microRNAs (miRNAs) via the activation of SNAREs, vesicle-associated membrane protein 3 (VAMP3) and synaptosomal-associated protein 23 (SNAP23). Knockdown of VAMP3 and SNAP23 reduces endothelial secretion of miR-126-3p and miR-200a-3p, as well as the proliferation, migration, and suppression of contractile markers in SMCs caused by EC-coculture. Pharmacological intervention of mammalian target of rapamycin complex 1 in ECs blocks endothelial secretion and EC-to-SMC transfer of miR-126-3p through transcriptional inhibition of VAMP3 and SNAP23. Systemic inhibition of VAMP3 and SNAP23 by rapamycin or periadventitial application of the endocytosis inhibitor dynasore ameliorates the disturbed flow-induced neointimal formation, whereas intraluminal overexpression of SNAP23 aggravates it. Our findings demonstrate the flow-pattern-specificity of SNARE activation and its contribution to the miRNA-mediated EC-SMC communication.

DNA methyltransferase 1 and Krüppel-like factor 4 axis regulates macrophage inflammation and atherosclerosis.

Macrophage-mediated inflammatory responses occur throughout all stages of atherosclerosis. DNA methylation is one of the critical epigenetic mechanisms and is associated with the development of atherosclerosis. The underlying mechanism of epigenetic regulation of macrophage inflammation (M1 activation) remains unclear. Here we aim to study the role of DNA methyltransferase 1 (DNMT1) in modulating macrophage inflammation and atherosclerosis. DNMT1 expression is up-regulated in THP-1-derived macrophages upon treatment with lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Overexpression of DNMT1 promotes the LPS- and IFN-γ-induced M1 activation whereas inhibition of DNMT1 attenuates it. Consistently, DNMT1 expression is elevated in macrophages in atherosclerotic plaques from human and mouse specimens; compared with the Dnmt1wild-type, myeloid Dnmt1 deficiency in mice in an Apolipoprotein E (ApoE) knockout background or receiving AAV-PSCK9 injection and carotid partial ligation results in ameliorated atheroma formation and suppressed plaque inflammation. The promoter regions of atheroprotective Krüppel-like factor 4 (KLF4) are hypermethylated in M1- activated macrophages. DNMT1 down-regulates the expression of KLF4, probably through catalyzing DNA methylation of the promoter regions of KLF4. Gain- and loss-of function study of KLF4 indicates that the DNMT1-mediated macrophage M1 activation is dependent on KLF4. Our data demonstrate a proatherogenic role for DNMT1 as a defining factor in macrophage inflammation both in vitro and in vivo. DNMT1 promotes macrophage M1 activation by suppressing KLF4 expression. Thus macrophage-specific DNMT1 inhibition may provide an attractive therapeutic potential to prevent or reduce atherosclerosis.

Biophysical Properties and Motility of Human Mature Dendritic Cells Deteriorated by Vascular Endothelial Growth Factor through Cytoskeleton Remodeling.

Dendritic cells (DCs), the most potent antigen-presenting cells, play a central role in the initiation, regulation, and maintenance of the immune responses. Vascular endothelial growth factor (VEGF) is one of the important cytokines in the tumor microenvironment (TME) and can inhibit the differentiation and functional maturation of DCs. To elucidate the potential mechanisms of DC dysfunction induced by VEGF, the effects of VEGF on the biophysical characteristics and motility of human mature DCs (mDCs) were investigated. The results showed that VEGF had a negative influence on the biophysical properties, including electrophoretic mobility, osmotic fragility, viscoelasticity, and transmigration. Further cytoskeleton structure analysis by confocal microscope and gene expression profile analyses by gene microarray and real-time PCR indicated that the abnormal remodeling of F-actin cytoskeleton may be the main reason for the deterioration of biophysical properties, motility, and stimulatory capability of VEGF-treated mDCs. This is significant for understanding the biological behavior of DCs and the immune escape mechanism of tumors. Simultaneously, the therapeutic efficacies may be improved by blocking the signaling pathway of VEGF in an appropriate manner before the deployment of DC-based vaccinations against tumors.

[The functions and clinical studies of macrophage migration inhibitory factor].

Macrophage migration inhibitory factor (MIF) is a pleiotropic immunoregulator which has a unique structure and a chemokine-like function. Since it was dicovered in 1966, the functions of MIF have been indicated, such as non-specific immunity, the inflammatory cell recruitment, and inflammatory reaction. In addition to its eponymic activity, MIF also has a proinflammatory function and promotes the directed migration and recruitment of leukocytes into infectious and inflammatory sites; it also has functions such as anti-glucocorticoids, anti-apoptosis , and promoting the release of other cytokines. As a result, MIF plays an important role in atherosclerosis, autoimmune diseases, cancer, and metabolic diseases. In this review, we will discuss the molecular pathway, function, related diseases and clinical application of MIF.

[Role of Rho/ROCK in the migration of vascular smooth muscle cells].

The migration of vascular smooth muscle cells (VSMCs) from media to intima is a critical step in the formation of atheroma and vascular stenosis as well as in the restenosis after vascular intervention. As an important downstream effector of RhoA, Rho-associated kinase (ROCK) plays an important role in VSMC migration and vascular remodeling by regulating actin filament cytoskeleton and focal adhesion. There are many bioactive substances such as aldosterone, sphingosine 1 phosphate (S1P), platelet-derived growth factor (PDGF) and angiotensin II (Ang II) that could induce VSMC migration through Rho/ROCK pathway by binding to their specific receptors. Studies on Rho/ROCK pathway could help us to better understand how cardiovascular diseases such as atherosclerosis and hypertension develop.

[Histological character of intimal hyperplasia post jugular vein grafts and the protective effect of lovastatin].

OBJECTIVE: To investigate the characteristics of intimal hyperplasia and lovastatin's effects on canine jugular venous prosthesis bypass grafting. METHODS: In the study, 12 adult mistus dogs were randomly divided into 2 groups: lovastatin group and control group. All the dogs were performed with jugular venous prosthesis bypass grafting (ePTFE, 6 mm in diameter, and 5 cm in length). Four weeks later, all the 12 specimens were harvested. The patency and mural thrombus of grafts were evaluated. The characteristics of intimal hyperplasia were described and measured by HE staining and endothelial nitric oxide synthase (eNOs) immunohistochemical method. The differences between the two groups were compared. RESULTS: Four weeks later, 3 grafts with complete occlusion were found in the two groups separately. Apparent intimal hyperplasia was observed in all the grafts. The neointima of proximal and distal part in lovastatin group were thinner than in control group respectively (proximal P=0.045, distal P=0.040). The endothelial cells were found in the surface of neointima. Newly born vessels could be found in the neointima and the new vessels were more in lovastatin group than in control group (proximal P=0.041, distal P=0.031). CONCLUSION: At the end of 4 weeks, the intimal hyperplasia with neovascularization was obviously near the anastomosis. Lovastatin showed the ability to inhibit the intimal hyperplasia and promote the neovascularization.

[The capping protein on the slow-growing end of actin filament: erythrocyte tropomodulin].

Erythrocyte tropomodulin (E-Tmod) is first isolated from human erythrocyte membrane as a TM-binding protein. Its N-terminus contains two TM-binding sites and one TM-dependent actin capping domain and C-terminus contains 5 leucine-rich repeats and a TM-independent actin capping domain. As the unique capping protein at the slow-growing end of F-actin, E-Tmod binds to N-terminus of TM and actin and decreases the TM-coated F-actin depolymerization. E-Tmod encoding gene is highly conserved and E-Tmod is distributed widely in erythrocytes and cardiomyocytes, etc. E-Tmod plays a pivotal role in organizing F-actin and cytoskeleton and maintaining the mechanical properties of the cells.

VAMP3 and SNAP23 as Potential Targets for Preventing the Disturbed Flow-Accelerated Thrombus Formation.

Disturbed blood flow has been recognized to promote platelet aggregation and thrombosis via increasing accumulation of von Willebrand factor (VWF) at the arterial post-stenotic sites. The mechanism underlying the disturbed-flow regulated endothelial VWF production remains elusive. Here we described a mouse model, in which the left external carotid artery (LECA) is ligated to generate disturbed flow in the common carotid artery. Ligation of LECA increased VWF accumulation in the plasma. Carotid arterial thrombosis was induced by ferric chloride (FeCl3) application and the time to occlusion in the ligated vessels was reduced in comparison with the unligated vessels. In vitro, endothelial cells were subjected to oscillatory shear (OS, 0.5 ± 4 dynes/cm2) or pulsatile shear (PS, 12 ± 4 dynes/cm2). OS promoted VWF secretion as well as the cell conditioned media-induced platelet aggregation by regulating the intracellular localization of vesicle-associated membrane protein 3 (VAMP3) and synaptosomal-associated protein 23 (SNAP23). Disruption of vimentin intermediate filaments abolished the OS-induced translocation of SNAP23 to the cell membrane. Knockdown of VAMP3 and SNAP23 reduced the endothelial secretion of VWF. Systemic inhibition of VAMP3 and SNAP23 by treatment of mice with rapamycin significantly ameliorated the FeCl3-induced thrombogenesis, whereas intraluminal overexpression of VAMP3 and SNAP23 aggravated it. Our findings demonstrate VAMP3 and SNAP23 as potential targets for preventing the disturbed flow-accelerated thrombus formation.

Hypermethylation of mitochondrial DNA in vascular smooth muscle cells impairs cell contractility.

Vascular smooth muscle cell (SMC) from arterial stenotic-occlusive diseases is featured with deficiency in mitochondrial respiration and loss of cell contractility. However, the regulatory mechanism of mitochondrial genes and mitochondrial energy metabolism in SMC remains elusive. Here, we described that DNA methyltransferase 1 (DNMT1) translocated to the mitochondria and catalyzed D-loop methylation of mitochondrial DNA in vascular SMCs in response to platelet-derived growth factor-BB (PDGF-BB). Mitochondrial-specific expression of DNMT1 repressed mitochondrial gene expression, caused functional damage, and reduced SMC contractility. Hypermethylation of mitochondrial D-loop regions were detected in the intima-media layer of mouse carotid arteries subjected to either cessation of blood flow or mechanical endothelial injury, and also in vessel specimens from patients with carotid occlusive diseases. Likewise, the ligated mouse arteries exhibited an enhanced mitochondrial binding of DNMT1, repressed mitochondrial gene expression, defects in mitochondrial respiration, and impaired contractility. The impaired contractility of a ligated vessel could be restored by ex vivo transplantation of DNMT1-deleted mitochondria. In summary, we discovered the function of DNMT1-mediated mitochondrial D-loop methylation in the regulation of mitochondrial gene transcription. Methylation of mitochondrial D-loop in vascular SMCs contributes to impaired mitochondrial function and loss of contractile phenotype in vascular occlusive disease.

Effects of myakuryu on hemorheological characteristics and mesenteric microcirculation of rats fed with a high-fat diet.

There is evidence that hyperlipidemia can induce hemorheological and microcirculatory disturbances. Myakuryu, a Chinese traditional medicine is efficacious in promoting lipid metabolism and protecting oxidative stress, but whether this drug can ameliorate rheologic disturbances caused by hyperlipidemia is still unknown. The present study was conducted to investigate the effects of myakuryu on hemorheological and microcirculatory disturbances induced by hyperlipidemia. Wistar rats were divided into a group on control diet (n=8) and a group on high-fat diet (HFD, n=44). Eight weeks later, plasma triglyceride (TG) and total cholesterol (TC) were determined. Sixteen animals with the highest levels of hyperlipidemia from the HFD group were randomly divided into two sub-groups: the untreated hyperlipidemia group (n=8) and the group treated with myakuryu (n=8). At the end of the sixteenth week, rheological and microcirculatory parameters were measured. Chemical analysis showed that myakuryu treatment caused significant reductions of plasma TG and TC levels (P<0.01), and the cholesterol/phospholipid ratio in the erythrocyte membrane (P<0.05). Rheological and microcirculatory measurements showed that myakuryu treatment led to a significant decrease in the erythrocyte aggregation index, plasma viscosity and blood viscosity at shear rates of 50, 100 and 150 s(-1) and in adherent leukocytes in mesenteric venules. There was a significant increase in erythrocyte deformation, electrophoretic mobility, membrane fluidity and F-actin content in the erythrocyte membrane as well as in red cell velocity in mesenteric venules. Our findings suggest that myakuryu treatment can improve blood flow and reduce adherent leukocytes in the venules of rats fed with HFD by ameliorating blood viscosity, erythrocyte deformability and aggregation, and other hemorheological characteristics.

Matrix stiffness determines the phenotype of vascular smooth muscle cell in vitro and in vivo: Role of DNA methyltransferase 1.

Cells perceive the physical cues such as perturbations of extracellular matrix (ECM) stiffness, and translate these stimuli into biochemical signals controlling various aspects of cell behavior, which contribute to the physiological and pathological processes of multiple organs. In this study, we tested the hypothesis that during arterial stiffening, vascular smooth muscle cells (SMCs) sense the increase of ECM stiffness, which modulates the cellular phenotype through the regulation in DNA methyltransferases 1 (DNMT1) expression. Moreover, we hypothesized that the mechanisms involve intrinsic stiffening and deficiency in contractility of vascular SMCs. Substrate stiffening was mimicked in vitro with polyacrylamide gels. A contractile-to-synthetic phenotypic transition was induced by substrate stiffening in vascular SMCs through the down-regulation of DNMT1 expression. DNMT1 repression was also observed in the tunica media of mice aortas in an acute aortic injury model and a chronic kidney failure model, as well as in the tunica intima of human carotid arteries with calcified atherosclerotic lesions. DNMT1 inhibition facilitates arterial stiffening in vivo and promotes osteogenic transdifferentiation, calcification and cellular stiffening of vascular SMCs in vitro. These effects may be attributable, at least in part, to the role of DNMT1 in regulating the promoter activities of Transgelin (SM22α) and α-smooth muscle actin (SMA) and the functional contractility of SMCs. We conclude that DNMT1 is a critical regulator that negatively regulates arterial stiffening via maintaining the contractile phenotype of vascular SMCs. This research may facilitate elucidation of the complex crosstalk between vascular SMCs and their surrounding matrix in healthy and in pathological conditions and provide new insights into the implications for potential targeting of the phenotypic regulatory mechanisms in material-related therapeutic applications.

The measurement of shear modulus and membrane surface viscosity of RBC membrane with Ektacytometry: a new technique.

A new technique is proposed to estimate the shear modulus (mu) and membrane surface viscosity (eta(m)) of red blood cell (RBC). Theoretical formulae for finding these two parameters are first derived based on the force balance on a RBC in a flow field of low viscosity. Different types of Ektacytometry are then used to measure relevant quantities. The obtained values (mu=6.1 x 10(-6)N/m, eta(m)=8.8 x10 (-7)Ns/m for normal RBC) are consistent with those previously found by micropipette technique and in AC electric field. The present technique is, however, much easier to operate and more advantageous in reflecting the average properties of a large quantity of RBCs, and it is much cheaper to be applied in clinical practice than any other method of measuring the two parameters. The sensitivity of the technique is demonstrated by testing RBCs treated with glutaraldehyde of different concentrations. This technique was demonstrated by the flow chamber.

The Mammalian Target of Rapamycin and DNA methyltransferase 1 axis mediates vascular endothelial dysfunction in response to disturbed flow.

The earliest atherosclerotic lesions preferentially develop in arterial regions experienced disturbed blood flow, which induces endothelial expression of pro-atherogenic genes and the subsequent endothelial dysfunction. Our previous study has demonstrated an up-regulation of DNA methyltransferase 1 (DNMT1) and a global hypermethylation in vascular endothelium subjected to disturbed flow. Here, we determined that DNMT1-specific inhibition in arterial wall ameliorates the disturbed flow-induced atherosclerosis through, at least in part, targeting cell cycle regulator cyclin A and connective tissue growth factor (CTGF). We identified the signaling pathways mediating the flow-induction of DNMT1. Inhibition of the mammalian target of rapamycin (mTOR) suppressed the DNMT1 up-regulation both in vitro and in vivo. Together, our results demonstrate that disturbed flow influences endothelial function and induces atherosclerosis in an mTOR/DNMT1-dependent manner. The conclusions obtained from this study might facilitate further evaluation of the epigenetic regulation of endothelial function during the pathological development of atherosclerosis and offer novel prevention and therapeutic targets of this disease.

Salvianolic acids attenuate rat hippocampal injury after acute CO poisoning by improving blood flow properties.

Carbon monoxide (CO) poisoning causes the major injury and death due to poisoning worldwide. The most severe damage via CO poisoning is brain injury and mortality. Delayed encephalopathy after acute CO poisoning (DEACMP) occurs in forty percent of the survivors of acute CO exposure. But the pathological cause for DEACMP is not well understood. And the corresponding therapy is not well developed. In order to investigate the effects of salvianolic acid (SA) on brain injury caused by CO exposure from the view point of hemorheology, we employed a rat model and studied the dynamic of blood changes in the hemorheological and coagulative properties over acute CO exposure. Compared with the groups of CO and 20% mannitol + CO treatments, the severe hippocampal injury caused by acute CO exposure was prevented by SA treatment. These protective effects were associated with the retaining level of hematocrit (Hct), plasma viscosity, fibrinogen, whole blood viscosities and malondialdehyde (MDA) levels in red blood cells (RBCs). These results indicated that SA treatment could significantly improve the deformation of erythrocytes and prevent the damage caused by CO poisoning. Meanwhile, hemorheological indexes are good indicators for monitoring the pathological dynamic after acute CO poisoning.

Influence of expressed TRAIL on biophysical properties of the human leukemic cell line Jurkat.

The cDNA fragment of human TRAIL (TNF-related apoptosis inducing ligand) was cloned into RevTet-On, a Tet-regulated and high-level gene expression system. The gene expression system was constructed in a human leukemic cell line: Jurkat. By using RevTet-On TRAIL gene expression system in Jurkat as a cell model, we studied the influence of TRAIL gene on the changes of cellular apoptosis before and after the TRAIL gene expression, which was induced by adding tetracycline derivative doxycycline (Dox). The results indicated that the cellular apoptosis ratio was largely dependent on the trail gene expression level. Moreover, it was found that the apoptosis-inducing TRAIL could cause significant changes in the biophysical properties of Jurkat cells. The cell surface charge density decreased, the membrane fluidity declined, the elastic coefficients K1 increased, and the proportion of a-helix in membrane protein secondary structure decreased. Thus, the apoptosis-inducing TRAIL gene caused significant changes on the biomechanic properties of Jurkat cells.