Interferon regulatory factor-1 together with reactive oxygen species promotes the acceleration of cell cycle progression by up-regulating the cyclin E and CDK2 genes during high glucose-induced proliferation of vascular smooth muscle cells.

BACKGROUND: The high glucose-induced proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of diabetic vascular diseases. In a previous study, we confirmed that Interferon regulatory factor-1 (Irf-1) is a positive regulator of the high glucose-induced proliferation of VSMCs. However, the mechanisms remain to be determined. METHODS: The levels of cyclin/CDK expression in two cell models involving Irf-1 knockdown and overexpression were quantified to explore the relationship between Irf-1 and its downstream effectors under normal or high glucose conditions. Subsequently, cells were treated with high glucose/NAC, normal glucose/H2O2, high glucose/U0126 or normal glucose/H2O2/U0126 during an incubation period. Then proliferation, cyclin/CDK expression and cell cycle distribution assays were performed to determine whether ROS/Erk1/2 signaling pathway was involved in the Irf-1-induced regulation of VSMC growth under high glucose conditions. RESULTS: We found that Irf-1 overexpression led to down-regulation of cyclin D1/CDK4 and inhibited cell cycle progression in VSMCs under normal glucose conditions. In high glucose conditions, Irf-1 overexpression led to an up-regulation of cyclin E/CDK2 and an acceleration of cell cycle progression, whereas silencing of Irf-1 suppressed the expression of both proteins and inhibited the cell cycle during the high glucose-induced proliferation of VSMCs. Treatment of VSMCs with antioxidants prevented the Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression in high glucose conditions. In contrast, under normal glucose conditions, H2O2 stimulation and Irf-1 overexpression induced cell proliferation, up-regulated cyclin E/CDK2 expression and promoted cell cycle acceleration. In addition, overexpression of Irf-1 promoted the activation of Erk1/2 and when VSMCs overexpressing Irf-1 were treated with U0126, the specific Erk1/2 inhibitor abolished the proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression under high glucose or normal glucose/H2O2 conditions. CONCLUSIONS: These results demonstrate that the downstream effectors of Irf-1 are cyclin E/CDK2 during the high glucose-induced proliferation of VSMCs, whereas they are cyclin D1/CDK4 in normal glucose conditions. The Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression are associated with ROS/Erk1/2 signaling pathway under high glucose conditions.

Biocompatibility evaluation of tissue-engineered valved venous conduit by reseeding autologous bone marrow-derived endothelial progenitor cells and multipotent adult progenitor cells into heterogeneous decellularized venous matrix.

Clinical treatment of chronic deep venous insufficiency remains difficult despite the availability of various therapies. Previous experimental efforts have demonstrated that the tissue-engineered valvedvenous conduit (TEVV) is a promising option to replace the damaged venous valve. The aim of the present study was to evaluate the TEVV by reseeding bone marrow-derived endothelial progenitor cells and multipotent adult progenitor cells into acellular matrix according to International Standard ISO10993, and to clarify their interactions with blood, the local effect after implantation both in vitro and vivo, and immunogenicity. The results showed that the 2-cm long TEVV did not cause haemolysis in vitro and remained patent without thrombosis formation in vivo. However, the luminal surface of TEVV was partially covered by multilayer cells. Compared with the native ovine femoral vein segment, the TEVV beneath the mouse skin produced significant mononuclear cell infiltration, with serum interleukin-6 and tumour necrosis factor-α similar to normal. The TEVV maintained its structural integrity, while the native ovine femoral vein segments fell apart at postoperative week nine. The TEVV implantation did not change serum immunoglobulin G. In addition, the seeds and extracts of the scaffold did not affect the proliferation of mouse lymphocytes. These findings suggest that the histocompatibility, haemocompatibility and immunogenicity of this TEVV are acceptable owing to complete removal of the cellular components of autologous seeds and residues of chemical regents, thus providing an experimental basis for further clinical translation. Copyright © 2014 John Wiley & Sons, Ltd.

Functional analysis in vivo of engineered valved venous conduit with decellularized matrix and two bone marrow-derived progenitors in sheep.

Tissue engineering has been considered a promising approach for creating grafts to replace autologous venous valves. Here, ovine bone marrow-derived endothelial progenitor cells (EPCs) and multipotent adult progenitor cells (MAPCs) were harvested and then loaded into decellularized venous matrix to create tissue-engineered (TE) valved vein. Subsequently, the ovine femoral veins containing the valve were removed and replaced by TE grafts or acellular matrix only. The morphology and function were analysed for up to 1 year by ultrasonography, angiography, H&E staining and scanning electron microscopy (SEM). The differentiation of seeded cells was traced immunofluorochemically. The results showed that decellularized venous matrix could initially and feebly attract endogenous cells, but failed afterwards and were insufficient to restore valve function. On the contrary, the seeded cells differentiated into endothelial cells (ECs) in vivo and formed a monolayer endothelium, and smooth muscle cells within the scaffold therefore produced TE grafts comparable to the native vein valve. This TE graft remained patent and sufficient after implantation into the venous circuit of the ovine lower extremity for at least 6 months. Unfortunately, cells seeded on the luminal surface and both sides of the leaflets lost their biological functions at 12 months, resulting in thrombosis formation and leading to complete occlusion of the TE grafts and impotent venous valves. These findings suggest that this TE valved venous conduit can function physiologically in vivo in the medium term. Before translating this TE venous valve into clinical practice, the durability should be improved and thrombogenicity should be suppressed. Copyright © 2016 John Wiley & Sons, Ltd.

Cardiomyocyte differentiation induced in cardiac progenitor cells by cardiac fibroblast-conditioned medium.

Our previous study showed that after being treated with 5-azacytidine, Nkx2.5(+) human cardiac progenitor cells (CPCs) derived from embryonic heart tubes could differentiate into cardiomyocytes. Although 5-azacytidine is a classical agent that induces myogenic differentiation in various types of cells, the drug is toxic and unspecific for myogenic differentiation. To investigate the possibility of inducing CPCs to differentiate into cardiomyocytes by a specific and non-toxic method, CPCs of passage 15 and mesenchymal stem cells (MSCs) were treated with cardiac ventricular fibroblast-conditioned medium (CVF-conditioned medium). Following this treatment, the Nkx2.5(+) CPCs underwent cardiomyogenic differentiation. Phase-contrast microscopy showed that the morphology of the treated CPCs gradually changed. Ultrastructural observation confirmed that the cells contained typical sarcomeres. The expression of cardiomyocyte-associated genes, such as alpha-cardiac actin, cardiac troponin T, and beta-myosin heavy chain (MHC), was increased in the CPCs that had undergone cardiomyogenic differentiation compared with untreated cells. In contrast, the MSCs did not exhibit changes in morphology or molecular expression after being treated with CVF-conditioned medium. The results indicated that Nkx2.5(+) CPCs treated with CVF-conditioned medium were capable of differentiating into a cardiac phenotype, whereas treated MSCs did not appear to undergo cardiomyogenic differentiation. Subsequently, following the addition of Dkk1 and the blocking of Wnt signaling pathway, CVF-conditioned medium-induced morphological changes and expression of cardiomyocyte-associated genes of Nkx2.5(+) CPCs were inhibited, which indicates that CVF-conditioned medium-induced cardiomyogenic differentiation of Nkx2.5(+) CPCs is associated with Wnt signaling pathway. In addition, we also found that the activation of Wnt signaling pathway was accompanied by higher expression of GATA-4 and the blocking of the pathway inhibited the expression of GATA-4 in CVF-conditioned medium-incubated Nkx2.5(+) CPCs. This finding suggests that Wnt signaling pathway may alter GATA-4 expression and activate the cardiogenic program in the regulation of differentiation. In conclusion, Nkx2.5(+) CPCs have enormous potential for cardiomyogenic differentiation and the CVF-conditioned medium specifically induces CPCs to differentiate into a cardiac phenotype. Wnt signaling pathway is involved in CVF-conditioned medium-induced cardiomyogenic differentiation of Nkx2.5(+) CPCs.

Applied Anatomy of the Femoral Veins in Macaca fascicularis / Anatomía Aplicada de las Venas Femorales en Macaca fascicularis

The aim was to understand the anatomical features of the venous valve in Macaca fascicularis and to compare it with that of humans. The bilateral lower limbs (24 limbs from 12 animals) of Macaca fascicularis cadavers were dissected, and the femoral veins (FVs) were equally divided into distal, intermediate, and proximal sections. The external diameter of the FV in each section was measured. The venous valves were observed microscopically and stained with hematoxylin and eosin as well as trichrome. Data describing the human venous valve were collected from the current literature. No great saphenous veins were found among the 24 lower limbs from the Macaca fascicularis cadavers. The external diameters of the FVs in the distal, intermediate, and proximal sections were 3.53 ± 0.37 mm, 3.42 ± 0.55 mm, and 3.37 ± 0.54 mm, respectively. In most cases, there was one venous bivalve located in the FV approximately 0-2.71 mm below the junction of the FV and the deep femoral vein. Endothelium covered the luminal and sinusal surfaces of the leaflets. Abundant collagen fibers were found under the endothelial cells beneath the luminal surface of the leaflets. An elastin fiber network was located under the sinus endothelial surface. Smooth muscle cells in the FV extend to the edge of the valve. The venous valve of Macaca fascicularis is similar to that of humans, both morphologically and histologically. However, there is only one venous bivalve and no great saphenous vein in Macaca fascicularis.

El objetivo fue comprender las características anatómicas de la válvula venosa en Macaca fascicularis y compararla con la de los humanos. Fueron disecados bilateralmente los miembros pélvicos (24 miembros de 12 animales) de cadáveres de Macaca fascicularis; las venas femorales (VF) fueron divididas en secciones distal, media y proximal. Se midió el diámetro externo de las VFs en cada sección. Las válvulas venosas se observaron microscópicamente y se tiñeron con H-E y tricrómico. Los datos para describir la válvula venosa humana se obtuvieron desde la literatura. No se encontraron venas safenas magnas entre los 24 miembros inferiores. Los diámetros externos de las VFs en las secciones distal, media y proximal fueron 3,53±0,37 mm, 3,42 mm±0,55, y 3,37±0,54 mm, respectivamente. En la mayoría de los casos, hubo vena bivalva situada aproximadamente 0-2,71 mm debajo de la unión de la VF y la vena femoral profunda. El endotelio cubrió las superficies luminal y sinusal. Se observaron abundantes fibras de colágeno en las células endoteliales bajo la superficie luminal de las válvulas. Una red de fibras de elastina se encontró bajo la superficie del seno endotelial. Las células musculares lisas en las VFs se extiendían hasta el margen de la válvula. La válvula venosa del Macaca fascicularis es similar a la de los seres humanos, morfológica e histológicamente. Sin embargo, sólo hubo una vena bivalvular, y no se observaron venas safenas en Macaca fascicularis.