Human Umbilical Vein Smooth Muscle Cells Research Articles

A Dexamethasone-Loaded Polymeric Electrospun Construct as a Tubular Cardiovascular Implant.

Cardiovascular tissue engineering is providing many solutions to cardiovascular diseases. The complex disease demands necessitating tissue-engineered constructs with enhanced functionality. In this study, we are presenting the production of a dexamethasone (DEX)-loaded electrospun tubular polymeric poly(l-lactide) (PLA) or poly(d,l-lactide-co-glycolide) (PLGA) construct which contains iPSC-CMs (induced pluripotent stem cell cardiomyocytes), HUVSMCs (human umbilical vein smooth muscle cells), and HUVECs (human umbilical vein endothelial cells) embedded in fibrin gel. The electrospun tube diameter was calculated, as well as the DEX release for 50 days for 2 different DEX concentrations. Furthermore, we investigated the influence of the polymer composition and concentration on the function of the fibrin gels by imaging and quantification of CD31, alpha-smooth muscle actin (αSMA), collagen I (col I), sarcomeric alpha actinin (SAA), and Connexin 43 (Cx43). We evaluated the cytotoxicity and cell proliferation of HUVECs and HUVSMCs cultivated in PLA and PLGA polymeric sheets. The immunohistochemistry results showed efficient iPSC-CM marker expression, while the HUVEC toxicity was higher than the respective HUVSMC value. In total, our study emphasizes the combination of fibrin gel and electrospinning in a functionalized construct, which includes three cell types and provides useful insights of the DEX release and cytotoxicity in a tissue engineering perspective.

Regulating cell behavior via regional patterned distribution of heparin-like polymers

Molecular patterning on biomaterial surfaces is an effective strategy to regulate biomaterial properties. Among the specific molecules, due to their biological functions, such as regulating cell behavior, heparin-like polymers (HLPs) have attracted much attention. In this study, HLP-distributed regional patterned surfaces (300 μm diameter circular array) were prepared by the combination of visible light-induced graft polymerization, transfer imprinting, and self-assembly to regulate the behavior of human umbilical vein endothelial cells (HUVECs) and human umbilical vein smooth muscle cells (HUVSMCs). The introduction of the regional pattern on HLP-modified surfaces enhanced the promotion effect of sulfonate-containing polymer (pSS) and sulfonate-, and glyco-containing copolymer (pS-co-pM), and slightly weakened the inhibition effect of glyco-containing polymer (pMAG) on the growth of HUVECs and HUVSMCs. Compared with flat surfaces, it was found that the unmodified regional patterned surfaces inhibit the spreading of HUVECs and HUVSMCs, while significantly promoting the spreading of HUVECs and HUVSMCs on all the HLP-distributed regional patterned surfaces. The patterned surface modified with pS-co-pM had the highest average spread area of HUVECs (∼10,554 μm2), which was 193 % higher than that of the unmodified flat surface. This trend was somewhat related to surface VEGF adsorption. The combination of regional divisive patterns and different HLP distributions enriched the potential of further exploring the influences of HLP chemical distributions and complex surface environments on cell-material interactions.

Influence of Diameter and Cyclic Mechanical Stimulation on the Beating Frequency of Myocardial Cell-Laden Fibers.

Atrioventricular block (AVB) is a severe disease for pediatric patients. The repetitive operations needed in the case of the pacemaker implantation to maintain the electrical signal at the atrioventricular node (AVN) affect the patient's life quality. In this study, we present a method of biofabrication of multi-cell-laden cylindrical fibrin-based fibers that can restore the electrical signal at the AVN. We used human umbilical vein smooth muscle cells (HUVSMCs), human umbilical vein endothelial cells (HUVECs) and induced pluripotent stem cell cardiomyocytes (iPSC-CMs) cultivated either statically or dynamically to mimic the native AVN. We investigated the influence of cell composition, construct diameter and cyclic stretch on the function of the fibrin hydrogels in vitro. Immunohistochemistry analyses showed the maturity of the iPSC-CMs in the constructs through the expression of sarcomeric alpha actinin (SAA) and electrical coupling through Connexin 43 (Cx43) signal. Simultaneously, the beating frequency of the fibrin hydrogels was higher and easy to maintain whereas the concentration of iPSC-CMs was higher compared with the other types of cylindrical constructs. In total, our study highlights that the combination of fibrin with the cell mixture and geometry is offering a feasible biofabrication method for tissue engineering approaches for the treatment of AVB.

Hypoxia Promotes Human Umbilical Vein Smooth Muscle Cell Phenotypic Switching via the ERK 1/2/c-fos/NF-κB Signaling Pathway

Vein wall hypoxia has long been suggested as a key factor for the development of varicose veins (VVs) and accumulating evidence has revealed the phenotypic transformation of vascular smooth muscle cells (VSMCs) under hypoxic conditions. However, the underlying molecular mechanisms of this process remain poorly understood. Our previous study revealed a positive correlation between c-fos expression and VSMC functional disturbance of VVs. This study aimed to further explore the role of c-fos in the phenotypic switching of VSMCs under hypoxic conditions. Human umbilical vein smooth muscle cells (HUVSMCs) were cultured under hypoxia or normoxia. PD0325901 (10 μmol/L) and pyrrolidine dithiocarbamate (PDTC) (10 μmol/L) were used to inhibit the extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-κ B (NF-κB) signaling pathways, respectively. HUVSMCs stably overexpressing c-fos were constructed to explore the underlying mechanism. The Western blot analysis was performed to detect the protein expression levels of c-fos, phosphorylated p65 (p-p65), interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), osteopontin (OPN), and α-smooth muscle actin (α-SMA). Cell proliferation and migration capacity were detected by a Cell Counting Kit 8 (CCK-8) assay and a wound-healing assay, respectively. The cell apoptotic rate was determined using the Annexin V-FITC Apoptosis Detection Kit. Hypoxic exposure increased the expression levels of indicators of the p-ERK1/2/c-fos and NF-κB signaling pathways, which was accompanied by altered levels of phenotypic biomarkers (α-SMA and OPN). Cells exposed to hypoxia were characterized by a greater proliferative and migratory ability. No significant differences were observed in the rate of cell apoptosis between the normal group and the hypoxic group. In addition, inhibition of the ERK1/2/c-fos signaling pathway by PD0325901 (10 μmol/L) reduced the expression of inflammatory cytokines and attenuated hypoxia-mediated phenotypic transformation. Furthermore, inhibition of the NF-κB signaling pathway by PDTC (10 μmol/L) downregulated the expression level of OPN and reduced the migration of HUVSMCs under hypoxia exposure. However, pretreatment with PDTC did not suppress the expression of c-fos or cell proliferation. Finally, the introduction of exogenous c-fos in HUVSMCs induced increased protein expression levels of p-p65, COX-2, and OPN, accompanied by a remarkable increase in HUVSMC proliferation and migration. Our research demonstrated that hypoxia could promote the phenotypic transformation of HUVSMCs partially through the ERK1/2/c-fos/NF-κB signaling pathway, which provided a novel insight into hypoxia-associated venous wall remodeling to furtheraid the development of a novel therapeutic target for the prevention or treatment of VVs.

Uremic toxin indoxyl sulfate induces dysfunction of vascular smooth muscle cells via integrin-β1/ERK signaling pathway.

Protein-bound uremic toxins (PBUTs) are reported to be one of the major culprits in chronic kidney disease-cardiovascular disease (CKD-CVD) development, yet its mechanism is not fully clear. Our previous study confirmed elevated expression of integrin-β1 (ITGβ1) in vascular smooth muscle cells of uremic patients. Thus, this study aimed to explore the relationship between PBUTs and ITGβ1 in uremic vasculature injury. Human umbilical vein smooth muscle cells (HUVSMCs) and endothelial cells (HUVECs) were treated with two representative PUBTs, indoxyl sulfate (IS) and p-cresyl sulfate (PC). Both cells were measured for the expression of ITGβ1 and downstream signaling pathways and assayed for proliferation, migration, adhesion and apoptosis. The IS treatments were observed with significantly up-regulated ITGβ1 in HUVSMCs but not in HUVECs, while PC did not induce ITGβ1 alteration in either HUVSMCs or HUVECs. Furthermore, overexpression of ITGβ1 revealed activated downstream signal-regulated kinase (ERK) signaling pathway with promoted focal adhesion, migration, proliferation but no apoptosis in HUVSMCs by IS. These functional and pathway alterations could be significantly suppressed by RNA interference of ITGβ1. More importantly, the application of ERK1/2 inhibitor significantly suppressed the focal adhesion, migration and proliferation of HUVSMCs. We first demonstrated that ITGβ1/ERK signaling pathway mediated abnormal focal adhesion, migration and proliferation of vascular smooth muscle cells stimulated by IS. ITGβ1/ERK signaling may serve as a novel therapeutic target for CKD-CVD.

Vascular cell behavior on glycocalyx–mimetic surfaces: Simultaneous mimicking of the chemical composition and topographical structure of the vascular endothelial glycocalyx

The endothelial glycocalyx is a carbohydrate–rich layer overlying the outermost surface of endothelial cells. It mediates intercellular interactions by specific chemical compositions (e.g., proteoglycans containing glycosaminoglycan (GAG) side chains) and micro/nanotopography. Inspired by the endothelial glycocalyx, we fabricated a series of glycocalyx–mimetic surfaces with tunable chemical compositions (GAG–like polymers with different functional units) and topographical structures (micro/nanopatterns with pillars different in size). The combination of micro/nanopatterns and GAG–like polymers was flexibly and precisely controlled by replica molding using silicon templates (Si templates) and visible light–initiated polymerization. Human umbilical vein endothelial cells (HUVECs) and human umbilical vein smooth muscle cells (HUVSMCs) were suppressed on surfaces modified with polymers of 2–methacrylamido glucopyranose (MAG) but promoted on surfaces modified with polymers of sodium 4–vinyl–benzenesulfonate (SS) and copolymers of SS and MAG. Surface micro/nanopatterns showed highly complicated effects on surfaces grafted with different GAG–like polymers. Moreover, the spread of HUVSMCs was highly promoted on all flat/patterned surfaces containing sulfonate units, and the elongation effect was stronger on surfaces with smaller pillars. On all the flat/patterned surfaces modified with GAG–like polymers, the adsorption of human vascular endothelial growth factor (VEGF) and human basic fibroblast growth factor (bFGF) was improved, and the amount of VEGF and bFGF absorbed on patterned surfaces containing sulfonate units decreased with pattern dimensions. The decreasing trend of VEGF and bFGF adsorption was in accordance with HUVEC density, suggesting that glycocalyx–mimetic surfaces influence the adsorption of VEGF and bFGF and further influence the growth behavior of vascular cells.

Hydroxysafflor Yellow A inhibits the viability and migration of vascular smooth muscle cells induced by serum from rats with chronic renal failure via inactivation of the PI3K/Akt signaling pathway.

It has been reported that the viability and migration of vascular smooth muscle cells contributes to arteriovenous fistula stenosis. Hydroxysafflor Yellow A (HSYA) has been demonstrated to inhibit the viability and migration of VSMCs by regulating Akt signaling. The present study aimed to investigate the role of HSYA on the viability and migration of human umbilical vein smooth muscle cells (HUVSMCs) following stimulation using serum from rats with chronic renal failure (CRF), and to determine the effects of HSYA on PI3K/Akt signaling. Wistar rats were randomly divided into two groups, control and CRF groups. Serum from each group was collected to stimulate the HUVSMCs. Cell Counting Kit-8 and wound healing assays were performed to assess cell viability and migration, respectively. Flow cytometry analysis was performed to assess apoptosis, and western blot analysis was performed to detect protein expression levels of PI3K and Akt. Nitric oxide (NO) production was measured using the Nitrate/Nitrite assay kit. The results demonstrated that serum from CRF rats significantly enhanced cell viability, migration and apoptosis, the effects of which were reversed following treatment with HSYA. In addition, CRF serum decreased NO and endothelial NO synthase expression, whilst increasing the protein expression levels of PI3K and phosphorylated-Akt in HUVSMCs. Notably, treatment with HSYA markedly restored NO production and inactivated the PI3K/Akt signaling pathway. Furthermore, the PI3K/Akt inhibitor, AMG511, exerted similar effects to HSYA. Taken together, the results of the present study suggest that HSYA suppresses cell viability and migration in the presence of CRF serum by inactivating the PI3K/Akt signaling pathway.

Hypoxia and hypoxia-inducible factors promote the development of neointimal hyperplasia in arteriovenous fistula.

Patients with end-stage renal failure need arteriovenous fistulas (AVF) to undergo dialysis. However, AVFs present a high rate of failure as a result of excessive venous thickness. Excessive venous thickness may be a consequence of surgical dissection and change in oxygen concentration within the venous wall. We show that venous cells adapt their metabolism and growth depending on oxygen concentration, and drugs targeting the hypoxic response pathway modulate this response in vitro. We used the same drugs on a mouse model of AVF and show that direct or indirect inhibition of the hypoxia-inducible factors (HIFs) help decrease excessive venous thickness. Hypoxia and HIFs can be targets of therapeutic drugs to prevent excessive venous thickness in patients undergoing AVF surgical creation. Because the oxygen concentration changes in the venous wall, surrounding tissue and the blood during surgical creation of arteriovenous fistula (AVF), we hypothesized that hypoxia could contribute to AVF failure as a result of neointimal hyperplasia. We postulated that modulation of the hypoxia-inducible factors (HIF) with pharmacological compounds could promote AVF maturation. Fibroblasts [normal human fibroblasts (NHF)], smooth muscle cells [human umbilical vein smooth muscle cells (HUVSMC)] and endothelial cells [human umbilical vein endothelial cells (HUVEC)], representing the three layers of the venous wall, were tested in vitro for proliferation, cell death, metabolism, reactive oxygen species production and migration after silencing of HIF1/2-α or after treatment with deferioxamine (DFO), everolimus (Eve), metformin (Met), N-acetyl-l-cysteine (NAC) and topoisomerase I (TOPO), which modulate HIF-α stability or activity. Compounds that were considered to most probably modify intimal hyperplasia were applied locally to the vessels in a mouse model of aortocaval fistula. We showed, in vitro, that NHF and HUVSMC can adapt their metabolism and thus their growth depending on oxygen concentration, whereas HUVEC appears to be less flexible. siHIF1/2α, DFO, Eve, Met, NAC and TOPO can modulate metabolism and proliferation depending on the cell type and the oxygen concentration. In vivo, siHIF1/2α, Eve and TOPO decreased neointimal hyperplasia by 32%-50%, 7 days after treatment. Within the vascular wall, hypoxia and HIF-1/2 mediate early failure of AVF. Local delivery of drugs targeting HIF-1/2 could inhibit neointimal hyperplasia in a mouse model of AVF. Such compounds may be delivered during the surgical procedure for AVF creation to prevent early AVF failure.

The role of carboxylic groups in heparin-mimicking polymer-functionalized surfaces for blood compatibility: Enhanced vascular cell selectivity

Blood compatibility is an eternal topic of biomedical materials. The effect of heparin-mimicking polymers (HMPs) on blood compatibility has been well studied, especially the synergistic effect of sugar unit and sulfonate/sulfate unit. However, carboxylic groups also play an important role in HMPs. In this work, copolymers of sodium 4-vinyl-benzenesulfonate (SS) and 2-methacrylamido glucopyranose (MAG) (poly(SS-co-MAG)) and poly(acrylate acid) (PAA) were self-assembled on Au surfaces with different feed ratios. When self-assembly of poly(SS-co-MAG) alone, the optimized feed ratio of SS and MAG for vascular cell selectivity was 1:1 (PS1M1); at this ratio the Au-PS1M1 surface showed the highest human umbilical vein endothelial cells (HUVECs) density and the lowest human umbilical vein smooth muscle cells (HUVSMCs) density. When self-assembly of PAA alone (surface designated as Au-PAA), the proliferation of both HUVECs and HUVSMCs was inhibited. Compared with either PS1M1 or PAA alone, the surfaces modified with both PAA and PS1M1 at the feed ratio of 1:1 (material designated as Au-PSM/PAA-2) showed enhanced promoting effect on HUVECs as well as enhanced inhibiting effect on HUVSMCs, indicating stronger vascular cell selectivity of carboxylic groups in the presence of sugar and sulfonate units.

All-Trans Retinoic Acid Prevented Vein Grafts Stenosis by Inhibiting Rb-E2F Mediated Cell Cycle Progression and KLF5-RARα Interaction in Human Vein Smooth Muscle Cells.

Vein graft failure (VGF) is an important limitation for coronary artery bypass graft (CABG) surgery. Inhibition of the excessive proliferation and migration of venous smooth muscle cells (SMCs) is an effective strategy to alleviate VGF during the CABG perioperative period. In the present study, we aimed to explore the role and potential mechanism of all-trans retinoic acid (ATRA) on preventing vein grafts stenosis. The autogenous vein grafts model was established in the right jugular artery of rabbits. Immunohistochemistry staining and western blot assays were used to detected the protein expression, while real-time PCR assay was applied for mRNAs expression detection. The interaction between proteins was identified by co-immunoprecipitation assay. The Cell Counting Kit-8 and wound-healing assays were used to investigate the role of ATRA on human umbilical vein smooth muscle cells (HUVSMCs) function. Cell cycle progression was identified by flow cytometry assay. Vein graft stenosis and SMCs hyperproliferation were confirmed in vein grafts by histological and Ki-67 immunohistochemistry assays. Treatment of ATRA (10mg/kg/day) significantly mitigated the stenosis extent of vein grafts, demonstrated by the decreased thickness of intima-media, and decreased Ki-67 expression. ATRA could repress the PDGF-bb-induced excessive proliferation and migration of HUVSMCs, which was mediated by Rb-E2F dependent cell cycle inhibition. Meanwhile, ATRA could reduce the interaction between KLF5 and RARα, thereby inhibiting the function of cis-elements of KLF5. KLF5-induced inducible nitric oxide synthase (iNOS) expression activation could be significantly inhibited by ATRA. These results suggested that ATRA treatment may represent an effective prevention and therapy avenue for VGF.

Ilimaquinone inhibits neovascular age-related macular degeneration through modulation of Wnt/β-catenin and p53 pathways

Neovascular age-related macular degeneration (nAMD) is a common cause of irreversible vision loss in the elderly. Anti-vascular endothelial growth factor has been effective in treating pathological ocular neovascularization, but it has limitations including the need for repeated intraocular injections for the maintenance of therapeutic effects in most patients and poor or non-response to this agent in some patients. in vitro cellular studies were conducted using retinal pigment epithelial cell lines (ARPE-19 and hTERT-RPE1), human umbilical vein endothelial cells (HUVECs), and human umbilical vein smooth muscle cells (HUVSMCs). in vivo efficacy of ilimaquinone (IQ) was tested in laser-induced choroidal neovascularization mouse and rabbit models. Tissue distribution study was performed in male C57BL6/J mice. IQ, 4,9-friedodrimane-type sesquiterpenoid isolated from the marine sponge, repressed the expression of angiogenic/inflammatory factors and restored the expression of E-cadherin in retinal pigment epithelial cells by inhibiting the Wnt/β-catenin pathway. In addition, it selectively inhibited proliferation and tube formation of HUVECs by activating the p53 pathway. Topical and intraperitoneal administration of IQ significantly reduced choroidal neovascularization in rabbits and mice with laser-induced choroidal neovascularization. Notably, IQ by the oral route of exposure was highly permeable to the eyes and suppressed abnormal vascular leakage by downregulation of β-catenin and stabilization of p53 in vivo. Our findings demonstrate that IQ functions through regulation of p53 and Wnt/β-catenin pathways with conceivable advantages over existing cytokine-targeted anti-angiogenic therapies.

3D Printing of Cell-Container-Like Scaffolds for Multicell Tissue Engineering

The development of an engineered non-contact multicellular coculture model that can mimic the in vivo cell microenvironment of human tissues remains challenging. In this study, we successfully fabricated a cell-container-like scaffold composed of β-tricalcium phosphate/hydroxyapatite (β-TCP/HA) bioceramic that contains four different pore structures, including triangles, squares, parallelograms, and rectangles, by means of three-dimensional (3D) printing technology. These scaffolds can be used to simultaneously culture four types of cells in a non-contact way. An engineered 3D coculture model composed of human bone-marrow-derived mesenchymal stem cells (HBMSCs), human umbilical vein endothelial cells (HUVECs), human umbilical vein smooth muscle cells (HUVSMCs), and human dermal fibroblasts (HDFs) with a spatially controlled distribution was constructed to investigate the individual or synergistic effects of these cells in osteogenesis and angiogenesis. The results showed that three or four kinds of cells cocultured in 3D cell containers exhibited a higher cell proliferation rate in comparison with that of a single cell type. Detailed studies into the cell–cell interactions between HBMSCs and HUVECs revealed that the 3D cell containers with four separate spatial structures enhanced the angiogenesis and osteogenesis of cells by amplifying the paracrine effect of the cocultured cells. Furthermore, the establishment of multicellular non-contact systems including three types of cells and four types of cells, respectively, cocultured in 3D cell containers demonstrated obvious advantages in enhancing osteogenic and angiogenic differentiation in comparison with monoculture modes and two-cell coculture modes. This study offers a new direction for developing a scaffold-based multicellular non-contact coculture system for tissue regeneration.

BMP9 attenuates occurrence of venous malformation by maintaining endothelial quiescence and strengthening vessel walls via SMAD1/5/ID1/α-SMA pathway

Venous malformation (VM) is a type of vascular morphogenic defect in humans with an incidence of 1%. Although gene mutation is considered as the most common cause of VM, the pathogenesis of those without gene mutation remains to be elucidated. Here, we aimed to explore the relation of bone morphogenetic protein 9 (BMP9) and development of VM. At first, we found serum and tissue BMP9 expression in VM patients was significantly lower than that in healthy subjects, detected via enzyme-linked immunosorbent assay. Next, with wound healing assay, transwell assay and tube formation assay, we discovered BMP9 could inhibit migration and enhance tube formation activity of human umbilical vein endothelial cells (HUVECs) via receptor activin receptor-like kinase 1 (ALK1). Besides, BMP9 improved the expression of structural proteins alpha-smooth muscle actin (α-SMA) and Desmin in human umbilical vein smooth muscle cells (HUVSMCs) via activation of the SMAD1/5-ID1 pathway, determined by RNA-based next-generation sequencing, qPCR, immunofluorescence and western blotting. Intriguingly, this effect could be blocked by receptor ALK1 inhibitor, SMAD1/5 inhibitor and siRNAs targeting ID1, verifying the BMP9/ALK1/SMAD1/5/ID1/α-SMA pathway. Meanwhile, knocking out BMP9 in C57BL/6 mice embryo led to α-SMA scarcity in walls of lung and mesenteric vessels, as well as walls of small trachea. BMP9−/− zebrafish also exhibited abnormal vascular maturity, indicating a critical role of BMP9 in vascular maturity and remodeling. Finally, a VM mice model revealed that BMP9 might have therapeutic effect in VM progression. Our study discovered that BMP9 might inhibit the occurrence of VM by strengthening the vessel wall and maintaining endothelium quiescence. These findings provide promising evidences of new therapeutic targets that might be used for the management of VM.

Estrogen and estrogen receptor affects MMP2 and MMP9 expression through classical ER pathway and promotes migration of lower venous vascular smooth muscle cells.

To explore the role of estrogen and estrogen receptors in the migration of vascular smooth muscle cells in varicose lower-extremity veins. Tissue samples of normal lower extremity vein (56 cases) and varicose lower extremity vein (47 cases) were collected. Western blot and real-time fluorescent qPCR were performed to measure the expression of Estrogen receptor α (ERα) in tissues. Two cell co-culture systems were established for human umbilical vein endothelial cells (HUVECs) and human umbilical vein smooth muscle cells (HUVSMCs). One system was incubated under normal oxygen conditions (normal oxygen group), and the other was under oxygen-poor conditions (hypoxia group). The two systems were treated with 10-7 mM Estrogen E2, 10-7 mM BSA-conjugated Estrogen E2-BSA, 10-7 mM Estrogen E2+10-3 mM Tamoxifen (TAM), respectively for 24 h. The treated cells were subjected to cell scratch assay, transwell assay, and Western blot analysis of MMP2 and MMP9 protein expression. The expression of ERα in varicose lower extremity vein was significantly up-regulated compared with that in normal lower extremity vein. The cell migration rate and the number of migrating cells in untreated hypoxia group and E2-treated normal oxygen group were comparable (p>0.05) to those in untreated normal oxygen group. The cell migration rate and the number of migrating cells were significantly increased (p<0.05) in E2-treated hypoxia group, compared with E2-treated normal oxygen group and untreated hypoxia group. The cell migration rate, the number of migrating cells, and expression levels of MMP2 and MMP9 were significantly decreased in E2/TAM-treated hypoxia group, compared with those in E2-treated hypoxia group. In summary, E2 can promote the migration of vascular smooth muscle cells and induce varicose veins of the lower extremities, which may be related to the promotion of MMP2 and MMP9 expression through the classical pathway of ER.

3D Coaxial Bioprinting of Vasculature.

Development of a suitable vascular network for an efficient mass exchange is crucial to generate three-dimensional (3D) viable and functional thick construct in tissue engineering. Different technologies have been reported for the fabrication of vasculature conduits, such as decellularized tissues and biomaterial-based blood vessels. Recently, bioprinting has also been considered as a promising method in vascular tissue engineering. In this work, human umbilical vein smooth muscle cells (HUVSMCs) were encapsulated in sodium alginate and printed in the form of vasculature conduits using a coaxial nozzle deposition system. Protocols for cell encapsulation and 3D bioprinting are presented. Investigations including dehydration, swelling, degradation characteristics, and patency, permeability, and mechanical properties were also performed and presented to the reader. In addition, in vitro studies such as cell viability and evaluation of extra cellular matrix deposition were performed.

Bioprinting of coaxial multicellular structures for a 3D co-culture model

With three dimensional (3D) bioprinting technology, it is feasible to fabricate various types of tissue models, in which multiple kinds of cells can be co-cultured. In this study, a novel 3D coaxial co-culture model in vitro was established by direct bioprinting. Using a four-layer coaxial nozzle, novel coaxial multicellular structures were fabricated, which contained two layers of alginate hydrogel as shell and two layers of hollow channels for cell perfusion. Sodium alginate (Na-Alg) was crosslinked by calcium cation with the contact of coaxial flows, forming a tubular coaxial structure of hydrogel. Human umbilical vein endothelial cells (HUVECs) and human umbilical vein smooth muscle cells (HUVSMCs) were used for a typical vascular co-culture model. Collagen solutions loaded with HUVECs and HUVSMCs were perfused into the layers of hollow channels and gelled at 37 ℃. The forming effect was favorable with good integrity. Pertinent process parameters of the bioprinting were investigated. Mechanical properties of four-layer coaxial structures were evaluated, indicating the higher mechanical strength than monolayer hydrogel structure. Cell viability assay after cell perfusion showed that the process of cell perfusion was feasible. In 7-day culture, cells maintained a steady proliferation. The immunohistochemical expression of HUVECs and HUVSMCs was detected in the structure. Cell work showed good biological functions of this 3D coaxial co-culture model.

Homocysteine induces vascular inflammatory response via SMAD7 hypermethylation in human umbilical vein smooth muscle cells

Homocysteine (Hcy) can induce atherosclerosis through the inflammatory response and DNA methylation disorder. Our recent study has reported a novel epigenetic modified gene related to atherosclerosis –SMAD7. To further understand the pathogenesis of atherosclerosis, the current study was designed to investigate an inflammatory role of Hcy in human umbilical vein smooth muscle cells (HUVSMCs) through interfering with SMAD7 methylation. Using MALDI-TOF MS, we found that Hcy increased DNA methylation levels of SMAD7 promoter in a dose and time-dependent manner in HUVSMCs. Meanwhile, both SMAD7 mRNA and protein levels were decreased along with the increase of Hcy concentrations and treating time. Decreased SMAD7 levels led to up regulation of pro-inflammatory cytokines (TNF-α and IL-1β) expression in HUVSMCs. Furthermore, we found that activation of NF-κB pathway was the mechanism by which reduced Smad7 levels enhanced vascular inflammation. Thus, Hcy is able to activate NF-κB-mediated vascular inflammatory response via inducing hypermethylation of SMAD7 promoter in HUVSMCs. The in vitro findings supplement our recent clinical study that SMAD7 methylation as a novel marker in atherosclerosis and further elucidate the role of Hcy in atherogenesis.

Effects of Different Simple Triglycerides on Cell Fatty Acid Compositions, Proliferation-Related Protein, and Gene Expressions Induced by Oxidized-LDL in HUVSMCs.

The proliferating effects of 3 different simple triglycerides (tristearin, triolein, and trilinolein) on the human umbilical vein smooth muscle cells (HUVSMCs) induced by oxidized-LDL (ox-LDL) were investigated in this study. The protein and mRNA gene expressions of proliferating cell nuclear antigen (PCNA), smooth muscle-α-actin (SM-α-actin), and monocyte chemoattractant protein-1 (MCP-1) in HUVSMCs were measured by Western blotting and real-time quantitative polymerase chain reaction (PCR). It was shown that in tristearin (SSS) treated HUVSMCs, the saturated fatty acid content was increased, and the compositions of polyunsaturated fatty acid (PUFA) and monounsaturated fatty acid were decreased significantly. On the other hand, triolein (OOO) and trilinolein (LLL) significantly increased the levels of some typical PUFA such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. Moreover, LLL and OOO could upregulate the protein and mRNA expressions of PCNA, MCP-1 as well as downregulate the expression of SM-α-actin. The results also showed that, SSS had significant promotion effects on the proliferation of HUVSMCs induced by ox-LDL. Although both LLL and OOO could inhibit the proliferation of HUVSMCs induced by ox-LDL, and might have certain inhibition of the atherosclerotic process.

Role of UBIAD1 in Intracellular Cholesterol Metabolism and Vascular Cell Calcification.

Vascular calcification is an important risk factor associated with mortality among patients with chronic kidney disease. Intracellular cholesterol metabolism is involved in the process of vascular cell calcification. In this study, we investigated the role of UbiA prenyltransferase domain containing 1 (UBIAD1) in intracellular cholesterol metabolism and vascular cell calcification, and identified its subcellular location. Primary human umbilical vein smooth muscle cells (HUVSMCs) were incubated with either growth medium (1.4 mmol/L Pi) or calcification medium (CM) (3.0 mmol/L Pi). Under treatment with CM, HUVSMCs were further incubated with exogenous cholesterol, or menaquinone-4, a product of UBIAD1. The plasmid and small interfering RNA were transfected in HUVSMCs to alter the expression of UBIAD1. Matrix calcium quantitation, alkaline phosphatase activity, intracellular cholesterol level and menaquinone-4 level were measured. The expression of several genes involved in cholesterol metabolism were analyzed. Using an anti-UBIAD1 antibody, an endoplasmic reticulum marker and a Golgi marker, the subcellular location of UBIAD1 in HUVSMCs was analyzed. CM increased matrix calcium, alkaline phosphatase activity and intracellular cholesterol level, and reduced UBIAD1 expression and menaquinone-4 level. Addition of cholesterol contributed to increased matrix calcification and alkaline phosphatase activity in a dose-dependent manner. Elevated expression of UBIAD1 or menaquinone-4 in HUVSMCs treated with CM significantly reduced intracellular cholesterol level, matrix calcification and alkaline phosphatase activity, but increased menaquinone-4 level. Elevated expression of UBIAD1 or menaquinone-4 reduced the gene expression of sterol regulatory element-binding protein-2, and increased gene expression of ATP binding cassette transporters A1, which are in charge of cholesterol synthesis and efflux. UBIAD1 co-localized with the endoplasmic reticulum marker and the Golgi marker in HUVSMCs. In conclusion, high intracellular cholesterol content contributes to phosphate-induced vascular cell differentiation and calcification. UBIAD1 or menaquinone-4 could decrease vascular cell differentiation and calcification, probably via its potent role of inversely modulating cellular cholesterol.

Novel hydroxybutyl chitosan nanoparticles for siRNA delivery targeting tissue factor inhibits proliferation and induces apoptosis in human vascular smooth muscle cells.

Chitosan, a polysaccharide isolated from shrimp and other crustacean shells, has been widely investigated for DNA and siRNA delivery. Despite substantial effort having been made to improve chitosan as a non-viral gene delivery vector, the application is severely limited by its poor solubility under physiological conditions. Hydroxybutyl chitosan (HBC), a modified chitosan, is soluble under neutral conditions. Tissue factor (TF) is involved in the pathogenesis of cardiovascular diseases by promoting thrombus formation and inducing the migration and proliferation of vascular smooth muscle cells. Targeting TF is an attractive therapeutic strategy for cardiovascular diseases. In the present study, the use of HBC for the transfer of TF-siRNAs into human umbilical vein smooth muscle cells (HUVSMCs) was investigated, and the effects of TF knockdown on cell proliferation and apoptosis were examined. HBC/siRNA nanoparticles were produced by mixing HBC and siRNA solutions with the assistance of tripolyphosphate buffer. The transfection efficiency with these nanoparticles was 74±2.5%, which was determined using a fluorescence-labeled siRNA under fluorescence microscopy. The delivery of HBC/TF-siRNA resulted in reductions in the production of cellular and soluble TF protein in HUVMSCs, which were measured using western blotting and enzyme-linked immunosorbent assay, respectively. TF knockdown led to inhibited cell proliferation, as assessed using a Cell Counting Kit-8 assay, and increased cell apoptosis, determined using Annexin V-fluorescein isothiocyanate staining. These findings suggested that HBC may be a promising vector for siRNA delivery, and that in vivo HBC/siRNA nanoparticle delivery targeting TF may be a potential option for the treatment of cardiovascular diseases, which warrants further investigation.