Human Umbilical Artery Smooth Muscle Cells Research Articles

Expression and influence of KATP in umbilical artery smooth muscle cells of patients with hypertensive disorders of pregnancy

The objective of this study is to investigate the expression and influence of adenosine triphosphate-sensitive potassium channel (KATP) in human umbilical arterial smooth muscle cells (HUASMCs) of patients with hypertensive disorders of pregnancy (HDP). Western blotting was used to detect the protein expression levels of KATP inwardly rectifying potassium channel (Kir)6.1 and sulphonylurea receptor (SUR)2B subunits in HUASMCs from patients with normal parturients (NP), gestational hypertension (GH), chronic hypertension (CH), preeclampsia (PE) and chronic hypertension with superimposed preeclampsia (CHSP), respectively. There was no significant difference in the protein expression of Kir6.1 subunit in NP group, GH group, CH group, PE group and CHSP group (P > 0.05). The protein expression of SUR2B subunit was gradually decreased in NP group, GH group, CH group, PE group and CHSP group, with statistically significant difference among the groups (P < 0.05). The altered expression level of KATP SUR2B subunit may be involved in the pathogenesis of HDP. The severity of HDP may be related to the degree of decrease of SUR2B subunit.

Nitric Oxide-Releasing Porous Coating with Antibacterial Activity and Blood Compatibility.

Nitric oxide (NO)-releasing coating is promising to enhance the biocompatibility of medical devices. In this study, polyurethane (PU) and S-nitrosated keratin (KSNO) were dissolved with dimethyl sulfoxide (DMSO) and tetrahydrofuran (THF) to prepare a coating solution. This solution is facile to form a porous coating on various substrates based on solvent-evaporation-induced phase separation (SEIPS). The coating could continuously release NO up to 200 h in the presence of ascorbic acid (Asc). In addition, the coating could accelerate endothelialization by promoting the viability of human umbilical vein endothelial cells (HUVECs) while inhibiting the proliferation of human umbilical artery smooth muscle cells (HUASMCs). Furthermore, the coating had good antibacterial activity and blood compatibility. Taken together, this universal coating provides wider potential applications in the field of cardiovascular implants.

A small-diameter vascular graft promotes rapid and benign remodeling of the neointima through dual release of nitric oxide and hydrogen sulfide

Coronary artery bypass grafting often requires autologous veins as alternatives. The limited source and problem of over-expansion are major obstacles for vein grafts, which could be solved by small-diameter artificial vascular graft. However, their clinical application is limited by thrombosis and restenosis caused by incomplete endothelialization and abnormal smooth muscle cells (SMCs) proliferation. Nitric oxide (NO) and hydrogen sulfide (H2S) are crucial signaling molecules in the cardiovascular system, known to regulate endothelial cells(ECs) and SMCs proliferation, migration, reduce oxidative stress, and inhibit inflammation to prevent intimal hyperplasia. In this study, we developed a new method to prepare a keratin based H2S donor(KAT) complexed with copper ions and electrospun it with PCL to prepare PCL/KAT-Cu small-diameter tissue-engineered vascular graft, which is capable of dual release of NO and H2S. We investigated the effects of NO and H2S release on human umbilical vein endothelial cells (HUVECs) and human umbilical arterial smooth muscle cells(HUASMCs) proliferation and migration, evaluated the graft's selectivity for HUVECs, and assessed its effects on macrophage phenotypic transitions and its protective effect on HUVECs under reactive oxygen species (ROS) conditions. Additionally, we tested the graft's ability to rapidly endothelialize under shear force using an in vitro 3D-perfusion system. After one month of in situ transplantation in rat abdominal aorta, the PCL/KAT-Cu small-diameter tissue-engineered vascular grafts demonstrated satisfactory intimal remodeling, comparable to natural blood vessels. In conclusion, our study presents a promising new strategy for remodeling small-diameter vascular grafts.

Endothelium-Mimicking Bilayer Vascular Grafts with Dual-Releasing of NO/H2S for Anti-Inflammation and Anticalcification.

Vascular complications caused by diabetes impair the activities of endothelial nitric oxide synthase (eNOS) and cystathionine γ-lyase (CSE), resulting in decreased physiological levels of nitric oxide (NO) and hydrogen sulfide (H2S). The low bioavailability of NO and H2S hinders the endothelialization of vascular grafts. In this study, endothelium-mimicking bilayer vascular grafts were designed with spatiotemporally controlled dual releases of NO and H2S for in situ endothelialization and angiogenesis. Keratin-based H2S donor was synthesized and electrospun with poly(l-lactide-co-ε-caprolactone) (PLCL) as the outer layer of the graft to release H2S. Hyaluronic acid, one of the major glycosaminoglycans in endothelial glycocalyx, was complexed with Cu ions as the inner layer to mimic glutathione peroxidase (GPx) and maintain long-term physiological NO flux. The synergistic effects of NO and H2S of bilayer grafts selectively promoted the regeneration and migration of human umbilical vascular endothelial cells (HUVECs), while inhibiting the overproliferation of human umbilical artery smooth muscle cells (HUASMCs). Bilayer grafts could effectively prevent vascular calcification, reduce inflammation, and alleviate endothelial dysfunction. The in vivo study in a rat abdominal aorta replacement model for 1 month showed that the graft had a good patency rate and had potential for vascular remodeling in situ.

Bilayer vascular grafts with on-demand NO and H2S release capabilities

Nitric oxide (NO) and hydrogen sulfide (H2S) gasotransmitters exhibit potential therapeutic effects in the cardiovascular system. Herein, biomimicking multilayer structures of biological blood vessels, bilayer small-diameter vascular grafts (SDVGs) with on-demand NO and H2S release capabilities, were designed and fabricated. The keratin-based H2S donor (KTC) with good biocompatibility and high stability was first synthesized and then electrospun with poly (l-lactide-co-caprolactone) (PLCL) to be used as the outer layer of grafts. The electrospun poly (ε-caprolactone) (PCL) mats were aminolyzed and further chelated with copper (II) ions to construct glutathione peroxidase (GPx)-like structural surfaces for the catalytic generation of NO, which acted as the inner layer of grafts. The on-demand release of NO and H2S selectively and synergistically promoted the proliferation and migration of human umbilical vein endothelial cells (HUVECs) while inhibiting the proliferation and migration of human umbilical artery smooth muscle cells (HUASMCs). Dual releases of NO and H2S gasotransmitters could enhance their respective production, resulting in enhanced promotion of HUVECs and inhibition of HUASMCs owing to their combined actions. In addition, the bilayer grafts were conducive to forming endothelial cell layers under flow shear stress. In rat abdominal aorta replacement models, the grafts remained patency for 6 months. These grafts were capable of facilitating rapid endothelialization and alleviating neointimal hyperplasia without obvious injury, inflammation, or thrombosis. More importantly, the grafts were expected to avoid calcification with the degradation of the grafts. Taken together, these bilayer grafts will be greatly promising candidates for SDVGs with rapid endothelialization and anti-calcification properties.

Bioinspired PLCL/Elastin Nanofibrous Vascular Tissue Engineering Scaffold Enhances Endothelial Cells and Inhibits Smooth Muscle Cells.

In vascular tissue engineering, a scaffold that can enhance the proliferation of endothelial cells (ECs) while inhibiting the synthetic differentiation of smooth muscle cells (SMCs) is crucial to prevent thrombus and restenosis after graft implantation. However, it is always challenging to incorporate both properties simultaneously in a vascular tissue engineering scaffold. In this study, a novel composite material was developed by combining a synthetic biopolymer of poly(l-lactide-co-caprolactone) (PLCL) and a natural biopolymer of elastin through electrospinning. Cross-linking of the PLCL/elastin composite fibers using EDC/NHS was performed to stabilize the elastin component. The incorporation of elastin into PLCL was found to enhance the hydrophilicity and biocompatibility of the resulting PLCL/elastin composite fibers, as well as the mechanical properties. Additionally, as a natural component of the extracellular matrix, elastin displayed antithrombotic properties reducing platelet adhesion and improving blood compatibility. Results of cell culture experiments with human umbilical vein ECs (HUVECs) and human umbilical artery SMCs (HUASMCs) showed that the composite fiber membrane had high cell viability, promoting the proliferation and adhesion of HUVECs and inducing a contractile phenotype in HUASMCs. These results indicate that the PLCL/elastin composite material has great potential for use in vascular graft applications due to its favorable properties and rapid endothelialization and contractile phenotypes of cells.

Preparation of biomolecules-mimic poly (ether sulfone)-based hydrogels and their anti-thrombotic, endothelialization and anti-microbial properties

The development of novel hydrogels with high biocompatibility is a vital and active topic of study. Here, we used a facile polycondensation approach to develop multifunctional poly (ether sulfone) (PES)-based hydrogels with diverse functional groups (−SO3Na, −COONa, −OH, and − NH2), as indicated by flourier transformed infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). The chemical structure of the PES-based hydrogels could be modulated by monomer selection and post-treatment, resulting in a wide range of biomolecule-mimic structures and matching biological functions. In the current investigation, heparin-mimic PES-based hydrogels demonstrated good hemocompatibility, as evident by reduced platelet adhesion and activation, delayed clotting times, suppressed blood-related complement activations, and CD11b expression on leukocytes in the blood. Moreover, hyper-sulfonated PES-based hydrogels showed exceptional cytocompatibility with human umbilical vein endothelial cells (HUVECs) and considerable antiproliferative activity against human umbilical artery smooth muscle cells (HUASMCs), as revealed by cell proliferation and cytotoxicity assay findings. Finally, chitosan-mimic PES-based hydrogels had marked anti-microbial properties against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), as shown by the number of colonies. The aforementioned bio-functions were attributed to the formation of various chemical structures on the surfaces of PES-based hydrogels. The distinctive PES-based hydrogels synthesized in this work would be of high utility not only in the synthesis of biomedical hydrogels but also in the rational design of various types of biocompatible materials.

Alpha-2-macroglobulin is involved in the occurrence of early-onset pre-eclampsia via its negative impact on uterine spiral artery remodeling and placental angiogenesis

BackgroundPre-eclampsia (PE) is one of the leading causes of maternal and fetal morbidity/mortality during pregnancy, and alpha-2-macroglobulin (A2M) is associated with inflammatory signaling; however, the pathophysiological mechanism by which A2M is involved in PE development is not yet understood.MethodsHuman placenta samples, serum, and corresponding clinical data of the participants were collected to study the pathophysiologic mechanism underlying PE. Pregnant Sprague–Dawley rats were intravenously injected with an adenovirus vector carrying A2M via the tail vein on gestational day (GD) 8.5. Human umbilical artery smooth muscle cells (HUASMCs), human umbilical vein endothelial cells (HUVECs), and HTR-8/SVneo cells were transfected with A2M-expressing adenovirus vectors.ResultsIn this study, we demonstrated that A2M levels were significantly increased in PE patient serum, uterine spiral arteries, and feto-placental vasculature. The A2M-overexpression rat model closely mimicked the characteristics of PE (i.e., hypertension in mid-to-late gestation, histological and ultrastructural signs of renal damage, proteinuria, and fetal growth restriction). Compared to the normal group, A2M overexpression significantly enhanced uterine artery vascular resistance and impaired uterine spiral artery remodeling in both pregnant women with early-onset PE and in pregnant rats. We found that A2M overexpression was positively associated with HUASMC proliferation and negatively correlated with cell apoptosis. In addition, the results demonstrated that transforming growth factor beta 1 (TGFβ1) signaling regulated the effects of A2M on vascular muscle cell proliferation described above. Meanwhile, A2M overexpression regressed rat placental vascularization and reduced the expression of angiogenesis-related genes. In addition, A2M overexpression reduced HUVEC migration, filopodia number/length, and tube formation. Furthermore, HIF-1α expression was positively related to A2M, and the secretion of sFLT-1 and PIGF of placental origin was closely related to PE during pregnancy or A2M overexpression in rats.ConclusionsOur data showed that gestational A2M overexpression can be considered a contributing factor leading to PE, causing detective uterine spiral artery remodeling and aberrant placental vascularization.

PDGF-BB signaling via PDGFR-β regulates the maturation of blood vessels generated upon vasculogenic differentiation of dental pulp stem cells.

A functional vascular network requires that blood vessels are invested by mural cells. We have shown that dental pulp stem cells (DPSC) can undergo vasculogenic differentiation, and that the resulting vessels anastomize with the host vasculature and become functional (blood carrying) vessels. However, the mechanisms underlying the maturation of DPSC-derived blood vessels remains unclear. Here, we performed a series of studies to understand the process of mural cell investment of blood vessels generated upon vasculogenic differentiation of dental pulp stem cells. Primary human DPSC were co-cultured with primary human umbilical artery smooth muscle cells (HUASMC) in 3D gels in presence of vasculogenic differentiation medium. We observed DPSC capillary sprout formation and SMC recruitment, alignment and remodeling that resulted in complex vascular networks. While HUASMC enhanced the number of capillary sprouts and stabilized the capillary network when co-cultured with DPSC, HUASMC by themselves were unable to form capillary sprouts. In vivo, GFP transduced human DPSC seeded in biodegradable scaffolds and transplanted into immunodeficient mice generated functional human blood vessels invested with murine smooth muscle actin (SMA)-positive, GFP-negative cells. Inhibition of PDGFR-β signaling prevented the SMC investment of DPSC-derived capillary sprouts in vitro and of DPSC-derived blood vessels in vivo. In contrast, inhibition of Tie-2 signaling did not have a significant effect on the SMC recruitment in DPSC-derived vascular structures. Collectively, these results demonstrate that PDGF-BB signaling via PDGFR-β regulates the process of maturation (mural investment) of blood vessels generated upon vasculogenic differentiation of human dental pulp stem cells.

The Effect of High and Variable Glucose on the Viability of Endothelial Cells Co-Cultured with Smooth Muscle Cells.

Diabetes mellitus causes endothelial dysfunction. The aim of this study was to investigate the effect of normal (5 mmol/L), high (20 mmol/L), and fluctuating (5 and 20 mmol/L changed every day) glucose concentration in the culture medium on the viability of human umbilical vein endothelial cells (HUVECs) co-cultured with human umbilical artery smooth muscle cells (HUASMCs). The cultures were conducted on semi-permeable flat polysulfone (PSU) fibronectin-coated membranes immobilized in self-made inserts. The insert contained either HUVECs on a single membrane or HUASMCs and HUVECs on two membranes close to each other. Cultures were conducted for 7 or 14 days. Apoptosis, mitochondrial potential, and the production of reactive oxygen species and lactate by HUVECs were investigated. The results indicate that fluctuations in glucose concentration have a stronger negative effect on HUVECs viability than constant high glucose concentration. High and fluctuating glucose concentrations slow down cell proliferation compared to the culture carried out in the medium with normal glucose concentration. In conclusion, HUASMCs affect the viability of HUVECs when both types of cells are co-cultured in medium with normal or variable glucose concentration.

Circular RNA circLIFR regulates the proliferation, migration, invasion and apoptosis of human vascular smooth muscle cells via the miR-1299/KDR axis.

Dysfunction of vascular smooth muscle cells (VSMCs) plays a critical role in the development of intracranial aneurysm (IA). Here, we explored the detailed role and mechanism of circular RNA (circRNA) LIF receptor subunit alpha (circLIFR, circ_0072309) in human umbilical artery smooth muscle cells (HUASMCs). CircLIFR, microRNA (miR)-1299 and kinase insert domain receptor (KDR) expression levels were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot assays. Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) and 5-Ethynyl-2'-Deoxyuridine (EdU) assays. Cell migration was gauged by wound-healing and transwell assays. Cell invasion and apoptosis were detected by transwell assay and flow cytometry, respectively. Direct relationship between miR-1299 and circLIFR or KDR was verified by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. CircLIFR and KDR were down-regulated and miR-1299 was up-regulated in the artery wall tissues and ASMCs of IA patients. Enforced expression of circLIFR enhanced HUASMC proliferation, migration, invasion, and impeded apoptosis. Mechanistically, circLIFR directly targeted miR-1299, and miR-1299 was a downstream mediator of circLIFR in regulating the proliferation, migration, invasion and apoptosis of HUASMCs. KDR was identified as a direct and functional target of miR-1299 in HUASMCs. Furthermore, circLIFR was a post-transcriptional regulator of KDR expression through miR-1299. Our findings suggest that circLIFR, an underexpressed circRNA in IA, can regulate the proliferation, migration, invasion and apoptosis of HUASMCs depending on the miR-1299/KDR axis.

Effects of Porphyromonas gingivalis lipopolysaccharide on the proliferation and migration of human umbilical artery smooth muscle cells under co-culture conditions

Objective: To investigate the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) of type Ⅰand Ⅳ fimA on the proliferation and migration of human umbilical artery smooth muscle cells (HUASMC) under co-culture conditions and to explore the biological basis and possible mechanisms of the relationship between periodontitis and atherosclerosis (As). Methods: Type Ⅰ and Ⅳ fimA Pg were anaerobically cultured and the Pg-LPS was extracted, purified and identified. Human umbilical vein endothelial cells (HUVEC) and HUASMC were cultured in vitro and the HUVEC-HUASMC co-cultured cell model was established using rat tail type Ⅰ collagen. The experiment was divided into three groups: group T1 (co-cultured cells were stimulated with type Ⅰ fimA Pg-LPS at the mass concentrations of 0.5, 1.0, 2.0, 5.0, 10.0 mg/L), group T2 (co-cultured cells were stimulated with type Ⅳ fimA Pg-LPS at the mass concentrations of 0.5, 1.0, 2.0, 5.0, 10.0 mg/L), and negative control group (without LPS). Cell counting kit-8 (CCK-8) was used to detect the proliferation ability of HUASMC and the migration ability of HUASMC was observed by using the Transwell migration chamber. Comparasons of the changes in the proliferation and migration ability of HUASMC after 2, 8, 24 and 48 h of Pg-LPS stimulated co-cultured cells with various mass concentrations of Pg-LPS were conducted. Results: For the co-cultured cells under the action of type Ⅰ and Ⅳ fimA Pg-LPS, at 24 and 48 h, in each mass concentration group of the two types of Pg-LPS (0.5, 1.0, 2.0, 5.0, 10.0 mg/L), HUASMC's A values were significantly up-regulated compared to the negative control group (P<0.05) and for the co-cultured cells after the stimulation of type Ⅳ fimA Pg-LPS at concentrations of 5 and 10 mg/L at 48 h, the A values (1.386±0.044, 1.455±0.058) of HUASMC were significantly higher than that of HUASMC (1.168±0.064, 1.204±0.088) in the same concentrations of type Ⅰ fimA Pg-LPS (P<0.05). In addition, the A values of HUASMC in stimulated co-cultured cells under concentrations of 5.0 and 10.0 mg/L of type Ⅳ fimA Pg-LPS at 48 h were significantly higher than that in stimulated co-culture cells under the concentrations of 0.5 and 1.0 mg/L of type Ⅳ fimA Pg-LPS (1.170±0.082, 1.239±0.089) (P<0.05). The migration results of HUASMC showed that at 8, 24, and 48 h, the numbers of migration of HUASMC in each mass concentration group of type Ⅰ and Ⅳ fimA Pgas-LPS were significantly higher than that of HUASMC under the same Pg-LPS mass concentration at 2 h in the same group (P<0.05). The migration quantities of HUASMC in the other Pg-LPS mass concentration groups at 48 h were significantly higher than that of HUASMC under the same Pg-LPS mass concentration at 24 h in the same group (P<0.05), except for 10.0 mg/L type Ⅳ fimA Pg-LPS. The numbers of HUASMC migration in 2.0 mg/L type Ⅳ fimA Pg-LPS stimulated co-cultured cells at 48 h (204.00±20.98) were significantly higher than that in type Ⅰ fimA Pg-LPS stimulated co-culture cells at the same concentration (141.89±18.28) (P<0.05). Further more, at the same observation time point, the higher the Pg-LPS concentration, the greater the number of HUASMC migration (P<0.05). Conclusions: Both Ⅰ and Ⅳ fimA Pg-LPS could enhance the proliferation and migration ability of HUASMC in the co-culture system, and the virulence of Pg-LPS might be related to the fimA genotype. Ⅳ fimA Pg-LPS showed a more significant stimulating effect and more likely to cause HUASMC dysfunction than Ⅰ fimA Pg-LPS, which provided part of the basis for the progression of As in severe periodontitis.

Effect of Porphyromonas gingivalis lipopolysaccharide on calcification of human umbilical artery smooth muscle cells co-cultured with human periodontal ligament cells.

Periodontitis is an independent risk factor for coronary heart disease. Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) was considered to be one of the main virulence factors. In addition, vascular smooth muscle cells transform into osteoblast-like cells in an arterial calcification process under chronic inflammatory conditions. The present study aimed to determine the calcification induced by Pg-LPS in human umbilical artery smooth muscle cells (HUASMCs) co-cultured with human periodontal ligament cells (HPDLCs). An in vitro co-culture system was established using Transwell inserts. HUASMC proliferation and alkaline phosphatase (ALP) activity were measured with a Cell Counting Kit-8 and an ALP kit, respectively. Calcium nodule formation was detected using alizarin red S staining. The effects of Pg-LPS on the mRNA expression of the calcification genes of ALP, core-binding factor α1 (Runx2) and bone sialoprotein (BSP) were assessed using reverse transcription-quantitative PCR. The results indicated that Pg-LPS increased HUASMC proliferation and ALP activity. Furthermore, among all of the groups, calcium nodule formation was most extensive in co-cultured cells in the mineralization-inducing medium containing Pg-LPS. In addition, the expression of specific osteogenic genes (Runx2, ALP and BSP) significantly increased in the presence of Pg-LPS and mineralization-inducing medium, which was further enhanced in co-culture with HPDLCs. In conclusion, co-culture with HPDLCs increased the effect of Pg-LPS to stimulate the calcification of HUASMCs. It was suggested that besides the inflammation, periodontitis may promote the occurrence of vascular calcification. The study indicated that periodontal treatment of subgingival scaling to reduce and/or control Porphyromonas gingivalis may decrease the occurrence or severity of vascular calcification.

Circular RNA circ-ARFIP2 regulates proliferation, migration and invasion in human vascular smooth muscle cells via miR-338-3p-dependent modulation of KDR

Dysfunction of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of intracranial aneurysm (IA). Circular RNAs (circRNAs) have been implicated in the pathogenesis of IA by reducing microRNA (miRNA) activity. In this paper, we investigated the precise roles of circRNA ADP ribosylation factor interacting protein 2 (circ-ARFIP2, circ_0021001) in VSMC dysfunction. The levels of circ-ARFIP2, miR-338-3p and kinase insert domain receptor (KDR) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Ribonuclease (RNase) R and subcellular fractionation assays were used to assess the stability and localization of circ-ARFIP2, respectively. Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay, and cell invasion was measured by transwell assay. Cell proliferation was gauged by 5-Ethynyl-2'-Deoxyuridine (EdU) assay. Cell migration was evaluated by transwell and wound-healing assays. Targeted correlations among circ-ARFIP2, miR-338-3p and KDR were validated by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Circ-ARFIP2 and KDR were underexpressed and miR-338-3p was overexpressed in the arterial wall tissues of IA patients. Overexpression of circ-ARFIP2 in human umbilical artery smooth muscle cells (HUASMCs) showed a significant promotion in cell proliferation, migration and invasion. Mechanistically, circ-ARFIP2 targeted miR-338-3p, and circ-ARFIP2 regulated cell behaviors by miR-338-3p. KDR was a direct and functional target of miR-338-3p. Moreover, KDR was a downstream effector of circ-ARFIP2 function. Circ-ARFIP2 regulated KDR expression by targeting miR-338-3p. Our present findings demonstrated that the increased level of circ-ARFIP2 enhanced HUASMC proliferation, migration and invasion at least in part by the miR-338-3p/KDR axis.

MicroRNA-29b targeting of cell division cycle 7-related protein kinase (CDC7) regulated vascular smooth muscle cell (VSMC) proliferation and migration

BackgroundProliferation and migration of vascular smooth muscle cells (VSMCs) are vital processes in vascular remodeling and pathology. This study aimed to explore the expression of miR-29b and cell division cycle 7-related protein kinase (CDC7) in patients with cerebral aneurysm (CA) and their effects on the proliferation and mobility of human umbilical artery smooth muscle cells (HUASMCs).MethodsRNA levels of miR-29b and CDC7 were evaluated in the CA tissues and adjacent normal cerebral arteries from 18 patients undergoing surgery for CA rupture. The targeting of CDC7 by miR-29b was verified with luciferase reporter assay. Both CDC7 and miR-29b overexpression and silencing vectors were introduced to validate their effects on the proliferation and mobility of HUASMCs.ResultsThe mRNA level of miR-29b was down-regulated (P<0.05), while the mRNA level of CDC7 was markedly elevated in CA patients (P<0.05). A Luciferase reporter assay showed CDC7 is a target gene of miR-29b, and miR-29b mimic down-regulated the mRNA and protein levels of CDC7 (P<0.05). Furthermore, miR-29b mimic inhibited, while miR-29b inhibitor or CDC7 over-expression promoted the proliferation and mobility of HUASMCs (P<0.05).ConclusionsmiR-29-3p inhibits cell proliferation and mobility via directly targeting CDC7, which could be a potential therapeutic target for vascular dysfunction related diseases, including atherosclerosis and CA.

Early modulation of macrophage ROS-PPAR\u03b3-NF-\u03baB signalling by sonodynamic therapy attenuates neointimal hyperplasia in rabbits

Disruption of re-endothelialization and haemodynamic balance remains a critical side effect of drug-eluting stents (DES) for preventing intimal hyperplasia. Previously, we found that 5-aminolevulinic acid-mediated sonodynamic therapy (ALA-SDT) suppressed macrophage-mediated inflammation in atherosclerotic plaques. However, the effects on intimal hyperplasia and re-endothelialization remain unknown. In this study, 56 rabbits were randomly assigned to control, ultrasound, ALA and ALA-SDT groups, and each group was divided into two subgroups (n = 7) on day 3 after right femoral artery balloon denudation combined with a hypercholesterolemic diet. Histopathological analysis revealed that ALA-SDT enhanced macrophage apoptosis and ameliorated inflammation from day 1. ALA-SDT inhibited neointima formation without affecting re-endothelialization, increased blood perfusion, decreased the content of macrophages, proliferating smooth muscle cells (SMCs) and collagen but increased elastin by day 28. In vitro, ALA-SDT induced macrophage apoptosis and reduced TNF-α, IL-6 and IL-1β via the ROS-PPARγ-NF-κB signalling pathway, which indirectly inhibited human umbilical artery smooth muscle cell (HUASMC) proliferation, migration and IL-6 production. ALA-SDT effectively inhibits intimal hyperplasia without affecting re-endothelialization. Hence, its clinical application combined with bare-metal stent (BMS) implantation presents a potential strategy to decrease bleeding risk caused by prolonged dual-antiplatelet regimen after DES deployment.

Preparation of Ascidian-Inspired Hydrogel Thin Films to Selectively Induce Vascular Endothelial Cell and Smooth Muscle Cell Growth.

Hydrogel thin films (HTFs), which have multiple functions including hemocompatibility and ability of enhancing growth of endothelia cells (ECs) while suppressing proliferation of smooth muscle cells (SMCs), have garnered intense scientific interest due to their potential applications in the field of blood-contact materials. In the present study, we developed stable, multifunctional HTFs containing multiple functional groups (-SO3Na, -COONa, -OH, and -NH2) by layer-by-layer (LbL) self-assembly of a modified sulfonated sodium alginate (SSA) and chitosan (CS), followed by ascidian-inspired post-cross-linking. The prepared HTFs with a 3D porous structure, as revealed by scanning electron microscopy (SEM), maintained outstanding long-term stability. The results of protein adsorption, platelet adhesion and activation, and clotting time confirmed that the hemocompatibility of the HTFs was significantly enhanced compared with the matrix. From the cytocompatibility test, the multiple functional groups at the HTF surface led to remarkable enhancement in human umbilical vein endothelial cell (HUVEC) growth and significant inhibition effects on human umbilical artery smooth muscle cell (HUASMC) proliferation. The ratio of HUVEC to HUASMC increased from 0.25 on the Ti surface to 2.87 on the surface of the HTF with the highest degree of sulfonation (DS). The improved blood compatibility was ascribed to the introduction of the multiple functional groups, and the strongly selective effects on vascular cells were attributed to the synergistic effect of the high degree of sulfonation and the existence of phenolic hydroxyl groups. The unique HTFs prepared in this study, demonstrating excellent hemocompatibility and high selectivity toward vascular cells, may help guide the design of cardiovascular biomaterials for endothelialization.

Poly(ε‐caprolactone)/keratin/heparin/VEGF biocomposite mats for vascular tissue engineering

Vascular endothelial growth factor (VEGF) is an effective growth and angiogenic cytokine, which stimulates proliferation and survival of endothelial cells, and promotes angiogenesis and vascular permeability. Binding VEGF with heparin could protect it from rapid degradation, subsequently allowing it to be controlled release. Primarily, poly(ε-caprolactone) (PCL) and keratin were coelectrospun, followed by conjugating with heparin and subsequently binding VEGF. The loaded heparin and VEGF on these mats were quantified, respectively. The surface characteristics of mats were investigated by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The VEGF delivery results indicated these mats could sustainably release VEGF for 2 weeks. Cell viability assays suggested these mats were valid to accelerate human umbilical vein endothelial cells (HUVECs) proliferation, while inhibit human umbilical arterial smooth muscle cells (HUASMCs) growth under the combined actions of VEGF and heparin. The results tested by blood clotting times (APTT, PT, and TT), hemolysis, and platelet adhesion indicated the mats were blood compatible. To sum up, these biocomposite mats are ideal scaffolds for vascular tissue engineering.

Inhibitory effect of rapamycin on proliferation of human umbilical arterial smooth muscle cells

Objective: Rapamycin has a protective cardiovascular effect and inhibits proliferation and migration of vascular smooth muscle cells. We investigated the effects of rapamycin on proliferation of cultured human umbilical arterial smooth muscle cells (HUASMCs) by determining interleukin-6 (IL-6) levels.Materials and methods: Adherent third-generation primary-cultured HUASMCs were used in the study, and MTT assay was used to measure the effects of different rapamycin concentrations on cell proliferation at various time points (3–96 h). RT-PCR was used to measure IL-6 mRNA expression and ELISA was used to measure IL-6 protein expression.Results: After three passages, HUASMCs displayed >90% confluence. Inhibition of cell proliferation by rapamycin was both time and dose dependent. When the action concentration of rapamycin was 100 ng·mL–1, the inhibitory effect was strongest after 48 h (30.25 ± 2.40)%, and the follow-up study was conducted after 48 h. When the action time of rapamycin was 48 h, the inhibitory effect of 150 ng·mL–1 at the action concentration was the strongest, and the inhibitory rate was (42.88 ± 3.84)%. There was no significant difference between the inhibitory effect and the action concentration of 100 ng·mL–1 (p>.05). Moreover, low (2 ng·mL–1), moderate (10 ng·mL–1), and high (100 ng·mL–1) rapamycin concentrations down-regulated both IL-6 mRNA and expression factor in a dose-dependent manner.Discussion and conclusions: Rapamycin inhibits proliferation of HUASMCs in vitro and through down-regulation of IL-6 expression.

Dengue Virus Infection of Primary Human Smooth Muscle Cells.

Dengue virus (DENV) infection of humans is presently the most important arthropod-borne viral global threat, for which no suitable or reliable animal model exists. Reports addressing the effect of DENV on vascular components other than endothelial cells are lacking. Dengue virus infection of vascular smooth muscle cells, which play a physiological compensatory response to hypotension in arteries and arterioles, has not been characterized, thus precluding our understanding of the role of these vascular components in dengue pathogenesis. Therefore, we studied the permissiveness of primary human umbilical artery smooth muscle cells (HUASMC) to DENV 1-4 infection and compared with the infection in the previously reported primary human umbilical vein endothelial cells (HUVEC) and the classically used, non-transformed, and highly permissive Lilly Laboratories Cell-Monkey Kidney 2 cells. Our results show that HUASMC are susceptible and productive to infection with the four DENV serotypes, although to a lesser extent when compared with the other cell lines. This is the first report of DENV permissiveness in human smooth muscle cells, which might represent an unexplored pathophysiological contributor to the vascular collapse observed in severe human dengue infection.