Human Umbilical Artery Smooth Muscle 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.

Hydrostatic pressure under hypoxia facilitates fabrication of tissue-engineered vascular grafts derived from human vascular smooth muscle cells in vitro.

Biologically compatible vascular grafts are urgently required. The scaffoldless multi-layered vascular wall is considered to offer theoretical advantages, such as facilitating cells to form cell-cell and cell-matrix junctions and natural extracellular matrix networks. Simple methods are desired for fabricating physiological scaffoldless tissue-engineered vascular grafts. Here, we showed that periodic hydrostatic pressurization under hypoxia (HP/HYP) facilitated the fabrication of multi-layered tunica media entirely from human vascular smooth muscle cells. Compared with normoxic atmospheric pressure, HP/HYP increased expression of N-myc downstream-regulated 1 (NDRG1) and the collagen-cross-linking enzyme lysyl oxidase in human umbilical artery smooth muscle cells. HP/HYP increased N-cadherin-mediated cell-cell adhesion via NDRG1, cell-matrix interaction (i.e., clustering of integrin α5β1 and fibronectin), and collagen fibrils. We then fabricated vascular grafts using HP/HYP during repeated cell seeding and obtained 10-layered smooth muscle grafts with tensile rupture strength of 0.218 to 0.396 MPa within 5 weeks. Implanted grafts into the rat aorta were endothelialized after 1 week and patent after 5 months, at which time most implanted cells had been replaced by recipient-derived cells. These results suggest that HP/HYP enables fabrication of scaffoldless human vascular mimetics that have a spatial arrangement of cells and matrices, providing potential clinical applications for cardiovascular diseases. STATEMENT OF SIGNIFICANCE: : Tissue-engineered vascular grafts (TEVGs) are theoretically more biocompatible than prosthetic materials in terms of mechanical properties and recipient cell-mediated tissue reconstruction. Although some promising results have been shown, TEVG fabrication processes are complex, and the ideal method is still desired. We focused on the environment in which the vessels develop in utero and found that mechanical loading combined with hypoxia facilitated formation of cell-cell and cell-matrix junctions and natural extracellular matrix networks in vitro, which resulted in the fabrication of multi-layered tunica media entirely from human umbilical artery smooth muscle cells. These scaffoldless TEVGs, produced using a simple process, were implantable and have potential clinical applications for cardiovascular diseases.

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.

Functional and Histological Changes in Umbilical Artery and Myometrium Isolated from IUGR Complicated Pregnancies

Objective: To investigate the relaxation responses mediated by L-type Ca2+ channels and big-conductance Ca2+-activated K+ (BKCa) channels and histological changes in the human umbilical artery (HUA) and myometrium smooth muscle isolated from pregnancies complicated with intrauterine growth restriction (IUGR).Methods: The muscle reactivity and the histology of the smooth muscle of the HUA and myometrium retrieved from 14 women with IUGR and 14 controls were investigated by the isolated tissue bath and immunohistochemical method.Results: In HUA, the maximum relaxation responses and pD2 values of nifedipine and NS11021 (BKCa channel opener) were significantly increased and significant histopathological changes are observed in the IUGR group.Conclusions: The pathogenesis of IUGR might be associated with the impairment in the functional responses of L-type Ca2+ channels and BKCa channels in HUA smooth muscle. The increased staining of myometrium and UC with HIF-1α in IUGR may indicate apoptosis, histological damage, and impaired fetal growth.

#6062 EFFECT OF KIDNEY TRANSPLANTATION ON ARTERIOLAR MOLECULAR PATHWAYS IN CHILDREN WITH PRECEDING PERITONEAL DIALYSIS

Abstract Background and Aims Patients on peritoneal dialysis suffer from progressive peritoneal membrane transformation and accelerated vascular disease, underlying molecular machineries have recently been described. Molecular effects of kidney transplantation (KTx) on vascular and peritoneal pathophysiology following PD are hardly understood. Method Fat surrounded omental arterioles (i.e. not directly exposed to PD fluids) from age-matched (median 6,5 years) children with chronic kidney disease (CKD5, n = 8), 5 children on PD [neutral pH, low glucose degradation product content, median PD duration 22 (5,50) months] and 6 children, who underwent successful KTx 4-5 weeks after a median 22 (12, 36) months of PD were analyzed by digital histomorphometry. Following microdissection from fat tissue, arterioles underwent transcriptome and proteome analyses, followed by Gene Set Enrichment Analysis (GSEA), weighted gene co-expression network analysis (WGCNA), gene ontology (GO) and protein interaction analysis (string-db.org). Key pathways were validated in independent, matched cohorts by quantitative immunohistochemistry (n = 15/group) and in vitro. Results 5000 most variable arteriolar transcripts (cut off p-value&amp;lt;0.05 and |r| &amp;gt;0.5) were grouped into 23 modules by WGCNA, of which 8 significantly differed between CKD5, PD and KTx. Seven were specifically related to KTx and one module to PD. Four out of seven KTx specific modules resulted in significant GO terms summarized as fatty acid biosynthesis, negative regulation of RNA metabolism, cell cycle arrest, and apoptosis. The PD specific module was associated with muscle cell proliferation, detoxification and complement activation (with thrombospondin as most interconnected gene). Multi-omics analysis of KTx vs. PD demonstrated concordant upregulation of lipid and fatty acid biosynthesis with the hub gene fatty acid synthase (FASN) and downregulation of positive cell cycle regulation. In independent, matched cohorts, arteriolar abundance of thrombospondin and terminal complement complex were higher in children on PD, arterioles from children after KTx had levels comparable to arterioles from children with CKD5. Key drivers of fibrotic process, arteriolar TGF-ß and pSMAD2/3, were also higher in PD arterioles compared to CKD5 and persistently high after KTx. Arteriolar cell cycle arrest marker p16 and apoptosis marker cleaved Casp3 were higher after KTx as compared to PD and CKD5. FASN was abundant in intima and media layers of all arterioles, and three-fold higher after KTx then in children with PD and CKD5, respectively. Neutral lipids (oil red staining) were present in all three groups, more abundant in the media than in the intima, and two-fold increased in the media of children after KTx compared to children on PD (p = 0.018) and with CKD5 (p = 0.002). In vitro, methylprednisolone and tacrolimus, but not mycophenolate mofetil increased FASN abundance and activity in human umbilical arterial endothelial and vascular smooth muscle cells compared to media controls. Conclusion We for the first time comprehensively describe molecular pathways activated after KTx in children following a standard immunosuppressive regime. After KTx, arteriolar fatty acid biosynthesis, apoptosis and cell cycle arrest markers are increased as compared to arterioles from children with PD and CKD5; profibrotic pathways induced by PD are persistently activated. Arteriolar hubgene FASN is three-fold more abundant after KTx and neutral lipid deposition is increased, presumably due to the induction of FASN activity by methylprednisolone and tacrolimus.

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.

Tannic Acid-Assisted Immobilization of Copper(II), Carboxybetaine, and Argatroban on Poly(ethylene terephthalate) Mats for Synergistic Improvement of Blood Compatibility and Endothelialization.

Due to thrombosis and intimal hyperplasia, small-diameter vascular grafts have poor long-term patency. A combination strategy based on nitric oxide (NO) and anticoagulants has the potential to address those issues. In this study, poly(ethylene terephthalate) (PET) mats were prepared by electrospinning and coated with tannic acid (TA)/copper ion complexes. The chelated copper ions endowed the mats with sustained NO generation by catalytic decomposition of endogenous S-nitrosothiol. Subsequently, zwitterionic carboxybetaine acrylate (CBA) and argatroban (AG) were immobilized on the mats. The introduced AG and CBA had synergistic effects on the improvement of blood compatibility, resulting in reduced platelet adhesion and prolonged blood clotting time. The biocomposite mats selectively promoted the proliferation and migration of human umbilical vein endothelial cells while inhibiting the proliferation and migration of human umbilical arterial smooth muscle cells under physiological conditions. In addition, the prepared mats exhibited antibacterial activity against Escherichia coli and Staphylococcus aureus. Collectively, the prepared mats hold great promise as artificial small-diameter vascular grafts.

Function of connexin 43 and RhoA/LIMK2/Cofilin signaling pathway in transient changes of contraction and dilation of human umbilical arterial smooth muscle cells

Post-induction hypotension, a common complication after propofol-based induction regimen, is a life-threatening challenge for anesthesiologists especially when unexpected pre-induction hypertension characterized by angiotensin release and increased vascular tone was presented by the same patient. Gap junctions (GJs) composed of connexin 43 (Cx43) have been considered a key factor in regulating vascular contraction and dilation. We aimed to explore the role of Cx43-GJs during peri-induction blood pressure fluctuation and elucidate the underlying mechanisms. Human umbilical arterial smooth muscle cells (HUASMCs) were pretreated by short-term Angiotensin Ⅱ (Ang Ⅱ) with or without subsequent propofol treatment to simulate transient contraction and dilation of vascular smooth muscle cells during anesthesia induction. F-actin polymerization, a classic indicator of HUASMCs constriction, was determined by F-actin staining assay. Both the function and expression of Cx43-GJs during transient contraction and dilation of HUASMCs, and their potential regulation of downstream Ca2+/RhoA/LIMK2/Cofilin signaling pathway were explored via different targeting inhibitors and siRNAs. Ang Ⅱ pretreatment significantly induced F-actin polymerization that indicate cell contraction, accompanied by enhanced GJs function on HUASMCs. With the inhibition of Cx43 GJs by the specific inhibitor, Gap26, and Cx43-siRNA, Ang Ⅱ-induced F-actin polymerization was reversed accompanied with the decrease of intracellular Ca2+ mobility and the RhoA/LIMK2/Cofilin signaling pathway activity. We also noticed that propofol application could inhibit GJs function, the same as Gap26. Simultaneously, intracellular Ca2+ mobility and RhoA/LIMK2/Cofilin signaling pathway activity on HUASMCs were both downregulated, finally resulting in downstream reduction of F-actin polymerization. The function of Cx43-GJs lies in the center of Ang Ⅱ-induced contraction of HUASMCs, which potentially regulates intracellular Ca2+ mobility as well as RhoA/LIMK2/Cofilin signaling pathway activity. Propofol can reverse this effect induced by Ang Ⅱ through suppressing the function of Cx43-GJs.

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 Protective Effect of Trichosanthes kirilowii Peel Polysaccharide on the Oxidative Damaged HepG2 and HUASMC Cells.

Background Oxidative stress is an important cause of liver disease and atherosclerosis. Natural substances with antioxidant activity are good drugs for treating liver disease and atherosclerosis. Trichosanthes kirilowii Peel Polysaccharide (TKPP) can remove DPPH (2,2-Diphenyl-1-picrylhydrazyl) free radicals and hydroxyl free radicals in vitro, which shows antioxidant activity. Therefore, it is speculated that it can protect human hepatoma cell line (HepG2) and umbilical artery smooth muscle cell (HUASMC) against oxidative damage by hydrogen peroxide (H2O2). Methods Oxidative damage cell models of HepG2 and HUASMC were induced by H2O2. HepG2 and HUASMC were divided into blank group, H2O2 injury group, TKPP treatment group, and glutathione (GSH) positive control group. Cell Counting Kit-8 (CCK-8) was used to detect cell viability. The level of total GSH and the amount of Nitric oxide (NO) secreted by cells were detected by specific kits. The gene and protein expressions of catalase (CAT) and superoxide dismutase (SOD) were detected by fluorescence quantitative PCR and Western Blot. Results In these two kinds of cells, compared with the control group, the survival rate, total GSH level, and NO secretion, CAT and SOD gene and protein expressions were significantly decreased in the H2O2 damaged group. In the TKPP treatment group, the cell survival rate was significantly elevated with the increase of the polysaccharide concentration, and the total GSH level, NO secretion, CAT and SOD gene expression, and protein expression levels were also significantly increased. Conclusion TKPP can improve the activities of HepG2 and HUASMC cells damaged by H2O2 and protect the cellular antioxidant system.

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.

Downregulation of large-conductance Ca2+-activated K+ channels in human umbilical arterial smooth muscle cells in gestational diabetes mellitus

AimsWe investigated the changes in large-conductance Ca2+-activated K+ (BKCa) channels from human umbilical arterial smooth muscle cells experiencing gestational diabetes mellitus (GDM). Main methodsWhole-cell patch-clamp technique, arterial tone measurement, RT-PCR, Quantitative real-time PCR, western blot were performed in human umbilical arterial smooth muscle cells. Key findingsWhole-cell BKCa current density was decreased in the GDM group compared with the normal group. The vasorelaxant effects of the synthetic BKCa channel activator NS-1619 (10 μM) were impaired in the GDM group compared with the normal group. Reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and western blot analyses suggested that the mRNA, total RNA, and protein expression levels of the BKCa channel were decreased in the GDM group relative to the normal group. In addition, the expression levels of protein kinase A and protein kinase G, which regulate BKCa channel activity, remained unchanged between the groups. Applying the BKCa channel inhibitor paxilline (10 μM) induced vasoconstriction and membrane depolarization of isolated umbilical arteries in the normal group but showed less of an effect on umbilical arteries in the GDM group. SignificanceOur results demonstrate for the first time impaired BKCa current and BKCa channel-induced vasorelaxation activities that were not caused by impaired BKCa channel-regulated protein kinases, but by decreased expression of the BKCa channels, in the umbilical arteries of GDM patients.

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.

Synthesis, Characterization, and Electrospinning of a Functionalizable, Polycaprolactone-Based Polyurethane for Soft Tissue Engineering.

We synthesized a biodegradable, elastomeric, and functionalizable polyurethane (PU) that can be electrospun for use as a scaffold in soft tissue engineering. The PU was synthesized from polycaprolactone diol, hexamethylene diisocyanate, and dimethylolpropionic acid (DMPA) chain extender using two-step polymerization and designated as PU-DMPA. A control PU using 1,4-butanediol (1,4-BDO) as a chain extender was synthesized similarly and designated as PU-BDO. The chemical structure of the two PUs was verified by FT-IR and 1H-NMR. The PU-DMPA had a lower molecular weight than the PU-BDO (~16,700 Da vs. ~78,600 Da). The melting enthalpy of the PU-DMPA was greater than that of the PU-BDO. Both the PUs exhibited elastomeric behaviors with a comparable elongation at break ( 13.2). The PU-DMPA had a higher initial modulus (19.8 MPa vs. 8.7 MPa) and a lower linear modulus (0.7 MPa vs. 1.2 MPa) and ultimate strength (9.5 MPa vs. 13.8 MPa) than the PU-BDO. The PU-DMPA had better hydrophilicity than the PU-BDO. Both the PUs displayed no cytotoxicity, although the adhesion of human umbilical artery smooth muscle cells on the PU-DMPA surface was better. Bead free electrospun PU-DMPA membranes with a narrow fiber diameter distribution were successfully fabricated. As a demonstration of its functionalizability, gelatin was conjugated to the electrospun PU-DMPA membrane using carbodiimide chemistry. Moreover, hyaluronic acid was immobilized on the amino-functionalized PU-DMPA. In conclusion, the PU-DMPA has the potential to be used as a scaffold material for soft tissue engineering.

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.