Facilitation of adhesion and spreading of endothelial cells on silicone oxide-coated dacron material by microwave-excited low-pressure plasma

Objectives Autologous transplants are still the means of choice for bypass surgery. In addition to good tolerability, there is a reduced thrombogenicity and fewer neointima hyperplasia compared to artificial materials. However, since viable transplants are limited, attempts are being made to improve existing artificial vascular prosthesis material. Next to the reduction of thrombogenicity, a rapid endothelialization of the vascular graft should reduce intimal hyperplasia and thus prevent stenoses. The effect of newly developed silicon oxide coatings on the growth of endothelial cells was therefore the goal of this work in a cell culture study. Methods A woven, uncoated polyethylene terephthalate (PET) vessel prosthesis was used. The coating process was carried out in a low-pressure plasma reactor in a multi-step process. After preparation of the vacuum chamber hexamethyldisiloxane (HDMSO) with oxygen was evaporated using argon plasma. By this an approx. 1 nm thin adhesion promoter layer was separated from plasma and HMDSO. The silicone oxide barrier layer was applied to the PET vessel samples. The carbon content of the layer could be selectively altered by changing the HMDSO oxygen flow ratio, resulting in coatings of 100 nm, 500 nm, and 1,000 nm. In addition, two different oxygen-to-HMDSO ratios were used. To achieve a carbon coating as low as possible, the ratio was set to 200:1. A carbon-rich layer was obtained with the 1:1 setting. The various coatings were then examined for their surface texture by scanning electron microscopy (SEM) as well as by cell culture experiments for cell viability and growth using EA.hy 926 cells. Results SEM showed no changes in the surface morphology; however a layer thickness of 1,000 nm showed peeled off coating areas. Alamar blue assays showed a significantly higher metabolic activity (p=0.026) for the coating 500 nm, ratio 200:1 compared to untreated control samples and a significantly lower metabolic activity (p=0.037) of the coating 500 nm, ratio 1:1 compared to the coating 500 nm, ratio 200:1. This underlines the apparent tendency of the 1:1 coating to inhibit the metabolic activity of the cells, while the 200:1 coating increases the activity. Fluorescence microscopy after calcein acetoxymethyl ester (AM) staining showed no significant difference between the different coatings and the uncoated PET material. However, a tendency of the increased surface growth on the coating 500 nm, ratio 200:1, is shown. The coatings with the ratio 1:1 tend to be less densely covered. Conclusions The results of this work indicate a great potential in the silicon coating of vascular prosthesis material. The plasma coating can be carried out easy and gently. Cell culture experiments demonstrated a tendency towards better growth of the cells on the 200:1 ratio coating and a poorer growth on the carbon-rich coating 1:1 compared to the uncoated material. The coating with silicon oxide with a thickness of 500 nm and an oxygen-HMDSO ratio of 200:1, a particularly low-carbon layer, appears to be a coating, which should therefore be further investigated for its effects on thrombogenicity and intimal hyperplasia.

M6A methylation-mediated elevation of SM22α inhibits the proliferation and migration of vascular smooth muscle cells and ameliorates intimal hyperplasia in type 2 diabetes mellitus

Abnormal proliferation of vascular smooth muscle cells (VSMCs) induced by insulin resistance facilitates intimal hyperplasia of type 2 diabetes mellitus (T2DM) and N6-methyladenosine (m6A) methylation modification mediates the VSMC proliferation. This study aimed to reveal the m6A methylation modification regulatory mechanism. In this study, m6A demethylase FTO was elevated in insulin-treated VSMCs and T2DM mice with intimal injury. Functionally, FTO knockdown elevated m6A methylation level and further restrained VSMC proliferation and migration induced by insulin. Mechanistically, FTO knockdown elevated Smooth muscle 22 alpha (SM22α) expression and m6A-binding protein IGF2BP2 enhanced SM22α mRNA stability by recognizing and binding to m6A methylation modified mRNA. In vivo studies confirmed that the elevated m6A modification level of SM22α mRNA mitigated intimal hyperplasia in T2DM mice. Conclusively, m6A methylation-mediated elevation of SM22α restrained VSMC proliferation and migration and ameliorated intimal hyperplasia in T2DM.

17β-Estradiol Inhibits Proliferation and Oxidative Stress in Vascular Smooth Muscle Cells by Upregulating BHLHE40 Expression

Background: Intimal hyperplasia is a major complication of restenosis after angioplasty. The abnormal proliferation and oxidative stress of vascular smooth muscle cells (VSMCs) are the basic pathological feature of neointimal hyperplasia. 17β-Estradiol can inhibit VSMCs proliferation and inflammation. However, it is still unclear whether and how 17β-Estradiol affects intimal hyperplasia.Methods: The neointima hyperplasia was observed by hematoxylin/eosin staining. The expression of PCNA, cyclin D1, NOX1, NOX4 and p47phox in neointima hyperplasia tissues and VSMCs was determined by qRT-PCR and Western blotting. MTS assay, cell counting and EdU staining were performed to detect cells proliferation. The oxidative stress was assessed by ROS staining.Results: 17β-Estradiol suppressed carotid artery ligation-induced intimal hyperplasia, which is accompanied by an increase of BHLHE40 level. Furthermore, loss- and gain-of-function experiments revealed that BHLHE40 knockdown promotes, whereas BHLHE40 overexpression inhibits TNF-α-induced VSMC proliferation and oxidative stress. 17β-Estradiol inhibited TNF-α-induced VSMC proliferation and oxidative stress by promoting BHLHE40 expression, thereby suppressing MAPK signaling pathways. In addition, enforcing the expression of BHLHE40 leads to amelioration of intimal hyperplasia.Conclusions: Our study demonstrates that 17β-Estradiol inhibits proliferation and oxidative stress in vivo and in vitro by promotion of BHLHE40 expression.

Sophocarpine Alleviates Injury-Induced Intima Hyperplasia of Carotid Arteries by Suppressing Inflammation in a Rat Model

Introduction: Balloon angioplasty is a commonly applied procedure for treating atherosclerotic vascular diseases. However, the maintenance of long-term lumen patency is relatively difficult due to the occurrence of restenosis. Previous research has shown that the occurrence of vascular wall inflammation is associated with higher rates of restenosis. Sophocarpine (SPC) can exert various therapeutic effects such as anti-oxidation, anti-inflammation, anti-tumor, antivirus and immune regulation. This study aimed to investigate whether SPC can alleviate intimal hyperplasia following balloon injury in a rat carotid artery model. Methods: Twenty Sprague–Dawley rats were randomly assigned to four groups: (i) control, (ii) balloon injury, (iii) balloon injury followed by saline injection, and (iv) balloon injury followed by SPC administration. Each group contained five rats. A high-pressure balloon of 3 mm × 20 mm was placed in the carotid artery. The balloon was inflated to a pressure of 8 atmospheres to carry out rat carotid artery balloon injury model. The areas of neointimal and media were determined by Verhoeff_Van Gieson staining, and the intima-to-media (I:M) ratios were subsequently evaluated. After that, the protein levels of IL-6, IL-1β, MCP-1, NF-κB, TNF-α, VCAM-1, ICAM-1 and eNOS were measured. Results: The ratio of I:M was remarkably higher in the balloon injury group than in the control group (p < 0.01). SPC could significantly decrease the ratio of I:M compared with the balloon injury group (p < 0.01). Besides, the protein levels of IL-6, IL-1β, MCP-1, NF-κB, TNF-α, ICAM-1 and VCAM-1 were increased in rat carotid arteries exposed to balloon injury (p < 0.01), and treatment with SPC could attenuate these effects (p < 0.05). Furthermore, balloon injury inhibited the protein expression of eNOS (p < 0.01), and SPC could elevate its level (p < 0.05). Conclusions: SPC could alleviate an intimal hyperplasia in balloon-injured carotid artery, and the mechanisms underlying this protective effect might be due to its inhibitory potency against inflammation signals. Our study also implies the potential applicability of SPC in treating restenosis after balloon angioplasty.

MED1 Deficiency in Macrophages Accelerates Intimal Hyperplasia via ROS Generation and Inflammation

Mediator complex subunit 1 (MED1) is a component of the mediator complex and functions as a coactivator involved in the regulated transcription of nearly all RNA polymerase II-dependent genes. Previously, we showed that MED1 in macrophages has a protective effect on atherosclerosis; however, the effect of MED1 on intimal hyperplasia and mechanisms regulating proinflammatory cytokine production after macrophage MED1 deletion are still unknown. In this study, we report that MED1 macrophage-specific knockout (MED1ΔMac) mice showed aggravated neointimal hyperplasia, vascular smooth muscle cells (VSMCs), and macrophage accumulation in injured arteries. Moreover, MED1ΔMac mice showed increased proinflammatory cytokine production after an injury to the artery. After lipopolysaccharide (LPS) treatment, MED1ΔMac macrophages showed increased generation of reactive oxygen species (ROS) and reduced expression of peroxisome proliferative activated receptor gamma coactivator-1α (PGC1α) and antioxidant enzymes, including catalase and glutathione reductase. The overexpression of PGC1α attenuated the effects of MED1 deficiency in macrophages. In vitro, conditioned media from MED1ΔMac macrophages induced more proliferation and migration of VSMCs. To explore the potential mechanisms by which MED1 affects inflammation, macrophages were treated with BAY11-7082 before LPS treatment, and the results showed that MED1ΔMac macrophages exhibited increased expression of phosphorylated-p65 and phosphorylated signal transducer and activator of transcription 1 (p-STAT1) compared with the control macrophages, suggesting the enhanced activation of NF-κB and STAT1. In summary, these data showed that MED1 deficiency enhanced inflammation and the proliferation and migration of VSMCs in injured vascular tissue, which may result from the activation of NF-κB and STAT1 due to the accumulation of ROS.

Macrophage membrane camouflaged reactive oxygen species responsive nanomedicine for efficiently inhibiting the vascular intimal hyperplasia

Background Intimal hyperplasia caused by vascular injury is an important pathological process of many vascular diseases, especially occlusive vascular disease. In recent years, Nano-drug delivery system has attracted a wide attention as a novel treatment strategy, but there are still some challenges such as high clearance rate and insufficient targeting. Results In this study, we report a biomimetic ROS-responsive MM@PCM/RAP nanoparticle coated with macrophage membrane. The macrophage membrane with the innate “homing” capacity can superiorly regulate the recruitment of MM@PCM/RAP to inflammatory lesion to enhance target efficacy, and can also disguise MM@PCM/RAP nanoparticle as the autologous cell to avoid clearance by the immune system. In addition, MM@PCM/RAP can effectively improve the solubility of rapamycin and respond to the high concentration level of ROS accumulated in pathological lesion for controlling local cargo release, thereby increasing drug availability and reducing toxic side effects. Conclusions Our findings validate that the rational design, biomimetic nanoparticles MM@PCM/RAP, can effectively inhibit the pathological process of intimal injury with excellent biocompatibility. Graphical Abstract

Hydraulic expansion facilitates remodeling of arteriovenous fistulas without increasing venous intimal hyperplasia in rabbits

Background An arteriovenous fistula (AVF) is considered essential for chronic hemodialysis. Objective To determine the effects of hydraulic expansion on the intimal hyperplasia of an AVF. Methods We divided 12 healthy male New Zealand white rabbits into a control group (vein without special handling and direct anastomosis with an artery, n = 6) and a hydraulic expansion group (vein dilated by hydraulic pressure before anastomosis, n = 6). Histopathomorphology was examined with hematoxylin and eosin staining and immunohistochemistry. Analysis of covariance (ANCOVA) was used to compare the data between the groups. Results Immediately and 1 day after surgery, the diameter of the fistula vein in rabbits in the hydraulic expansion group was significantly larger than it was in the control group (P = 0.02 and 0.03 respectively), but not on subsequent days. After hydraulic expansion and before construction of the fistula, the wall of vein was noticeably thinner on macroscopic observation, and the anterior and posterior walls were indistinguishable. At 3 weeks after surgery in the hydraulic expansion group, cells in the vein wall were disordered, there were fewer elastic fibers, tissues from the endothelium to tunica externa were less dense, and there was less extracellular matrix than in the control group. Expression of connective tissue growth factor in the hydraulic expansion group was significantly less than that in the control group (P = 0.01). No differences were found in intimal thickness or immunohistochemistry scores for transforming growth factor-β1 between the groups. Conclusion Hydraulic expansion did not increase intimal hyperplasia of an AVF, but facilitates remodeling of AVFs in rabbits.