Chaojun Tang

Substrate stiffness regulates the proliferation, migration, and differentiation of epidermal cells

The aim of this study was to investigate the role of substrate stiffness on the proliferation, migration, and differentiation of epidermal cells. To investigate the effects of substrate stiffness on wound healing, epidermal cells were chosen and inoculated on silicone substrate with different values of Youngs modulus of elasticity. The cell growth curve, MTT method, and cell cycle detection were used to investigate proliferation, and the scratch test was used to investigate cell migration. Fluorescence flow cytometry was used to study epidermal cell differentiation. The proliferation and migration of epidermal cells favoured stiffer surfaces. A highly stiff surface stimulated epidermal cell proliferation and migration and increased re-epithelialisation, but inhibited differentiation. The candidate pathways mediating epidermal cell proliferation and migration are linked to cell anchoring to substrates by integrin-mediated focal adhesion.

Coronary drug-eluting stents: From design optimization to newer strategies

Compared with early bare-metal stents, drug-eluting stents (DESs) are more effective in treating coronary artery diseases, especially in inhibiting restenosis. However, in-stent restenosis still clinically occurs at a non-negligible rate. More importantly, delayed endothelialization, inflammation, and hypersensitivity trigger subacute or late adverse events, particularly stent thrombosis, and thereby raise more concerns over the long-term safety of DESs. These problems are mostly associated with the permanent polymeric materials, non-optimal therapeutic drugs, and/or metallic stent platforms used in current DES design. It is critically important to further improve and optimize DES design and apply newer strategies for developing next generation DES. These new generation DESs should maintain their clinical efficacy and meanwhile eliminate the long-term safety concerns. In this review article, the current information on the optimization of DES design was critically reviewed based on DESs basic components, namely, stent platform, restenotic drug, and polymer coating. The available strategies for designing next-generation DESs were also summarized, ranging from degradable polymer DESs, to polymer-free DESs, to fully biodegradable DESs.

The Adhesive Properties of Endothelial Cells on Endovascular Stent Coated by Substrates of Poly-L-Lysine and Fibronectin

Optimizing endothelial cell growth and adhesion on the surface of metallic stents implanted in the vascular system is a fundamental issue in understanding and improving their long-term biocompatibility. The ability of the endothelial cell to attach and adhere to the luminal stent surface as well as the capacity to withstand the significant shear stress associated with blood flow are important determinants. The adhesive characteristics of human umbilical vein endothelial cells (HUVEC) on stent surfaces coated with either Poly-L-Lysine (PLL) or fibronectin (FN) were compared with uncoated controls. Increasing concentrations of PLL and FN were measured using a micropipette aspiration system. The adhesive properties of HUVECs under static flow conditions were compared to a dynamic environment on endovascular stents using a parallel-plate-flow chamber. A scanning electron microscope picture was used to measure the number and the adhesive cell ratio as well as the percentage of surface coverage of stent by endothelial cells. The adhesive forces of HUVECs on foreign surfaces coated with PLL and FN were higher compared to uncoated surfaces, and were dependent on increasing concentrations. These coatings resulted in significant increase of the adhesive force of HUVECs. The influence of substrates on the adhesion of the endothelial cell monolayer under static or dynamic flow conditions was highly significant compared with controls (p < 0.01). No significant differences were observed between PLL and FN substrates. Both PLL and FN coated surfaces can significantly increase the adhesion and growth of HUVECs on metallic stent surfaces.

The impact of vascular endothelial growth factor-transfected human endothelial cells on endothelialization and restenosis of stainless steel stents

OBJECTIVES The purpose of this study was to investigate the effects of gene transfection of endothelial cells with vascular endothelial growth factor (VEGF) on re-endothelialization and inhibiting in-stent restenosis. METHODS Stents coated with human umbilical vein endothelial cells (HUVECs) transfected with VEGF(121) were studied both in vitro and in vivo. In vitro studies were performed using a homemade extracorporeal circulation system. In vivo studies were performed using the rabbit abdominal aorta model. RESULTS In vitro studies confirmed that VEGF(121)-transfected cells adhered on the surface of stainless steel stents with over 90% of the surface covered within 24 hours of seeding. In vivo results showed that VEGF(121)-transfected HUVECs-coated stents were covered with seeding cells after implanting, and almost completely covered with cells after stent implantation for 1 week. In contrast, the non-endothelialized areas of bare metal stents and glutin/poly-L-lysine-coated stents were covered at 4 weeks, and the monolayers of cells were not observed, but fragile neointima was found on the surface. After 12 weeks, VEGF(121)-transfected HUVECs-coated stents significantly reduced the neointima area (0.78 ± 0.03 mm(2)) and stenosis (15.69 ± 2.61%) as compared with those for bare metal stents (neointima area = 2.26 ± 0.67 mm(2); the percentage of stenosis = 47.55 ± 7.10%;P < .01) and glutin/poly-L-lysine-coated stents (neointima area = 1.40 ± 0.37 mm(2); the percentage of stenosis = 31.37 ± 8.18%;P < .01). CONCLUSION In this small animal study, VEGF transfected human endothelial cells, when coated on stainless steel stents, reduce neointimal hyperplasia, promote endothelialization, and reduce in-stent restenosis. Additional studies with this technology are necessary to determine its ultimate utility in improving stents performance.

Surface wettability of plasma SiOx:H nanocoating-induced endothelial cells' migration and the associated FAK-Rho GTPases signalling pathways

Vascular endothelial cell (EC) adhesion and migration are essential processes in re-endothelialization of implanted biomaterials. There is no clear relationship and mechanism between EC adhesion and migration behaviour on surfaces with varying wettabilities. As model substrates, plasma SiOx:H nanocoatings with well-controlled surface wettability (with water contact angles in the range of 98.5 ± 2.3° to 26.3 ± 4.0°) were used in this study to investigate the effects of surface wettability on cell adhesion/migration and associated protein expressions in FAK-Rho GTPases signalling pathways. It was found that EC adhesion/migration showed opposite behaviour on the hydrophilic and hydrophobic surfaces (i.e. hydrophobic surfaces promoted EC migration but were anti-adhesions). The number of adherent ECs showed a maximum on hydrophilic surfaces, while cells adhered to hydrophobic surfaces exhibited a tendency for cell migration. The focal adhesion kinase (FAK) inhibitor targeting the Y-397 site of FAK could significantly inhibit cell adhesion/migration, suggesting that EC adhesion and migration on surfaces with different wettabilities involve (p)FAK and its downstream signalling pathways. Western blot results suggested that the FAK-Rho GTPases signalling pathways were correlative to EC migration on hydrophobic plasma SiOx:H surfaces, but uncertain to hydrophilic surfaces. This work demonstrated that surface wettability could induce cellular behaviours that were associated with different cellular signalling events.

Mesenchymal stem cell seeding promotes reendothelialization of the endovascular stent

This study is designed to make a novel cell seeding stent and to evaluate reendothelialization and anti-restenosis after the stent implantation. In comparison with cell seeding stents utilized in previous studies, Mesenchymal stem cells (MSCs) have advantages on promoting of issue repair. Thus it was employed to improve the reendothelialization effects of endovascular stent in present work. MSCs were isolated by density gradient centrifugation and determined as CD29(+) CD44(+) CD34(-) cells by immunofluorescence and immunocytochemistry; gluten and polylysine coated stents were prepared by ultrasonic atomization spray, and MSCs seeded stents were made through rotation culture according to the optimized conditions that were determined in previous studies. The results from animal experiments, in which male New Zealand white rabbits were used, show that the reendothelialization of MSCs coated stents can be completed within one month; in comparison with 316L stainless steel stents (316L SS stents) and gluten and polylysine coated stents, the intimal hyperplasia and in-stent restenosis are significantly inhibited by MSCs coated stents. Endovascular stent seeded with MSCs promotes reendothelialization and inhibits the intimal hyperplasia and in-stent restenosis compared with the 316L SS stents and the gluten and polylysine coated stents.

In vitro and in vivo investigations on the effects of low-density lipoprotein concentration polarization and haemodynamics on atherosclerotic localization in rabbit and zebrafish

Atherosclerosis (AS) commonly occurs in the regions of the arterial tree with haemodynamic peculiarities, including local flow field disturbances, and formation of swirling flow and vortices. The aim of our study was to confirm low-density lipoprotein (LDL) concentration polarization in the vascular system in vitro and in vivo, and investigate the effects of LDL concentration polarization and flow field alterations on atherosclerotic localization. Red fluorescent LDL was injected into optically transparent Flk1: GFP zebrafish embryos, and the LDL distribution in the vascular lumen was investigated in vivo using laser scanning confocal microscopy. LDL concentration at the vascular luminal surface was found to be higher than that in the bulk. The flow field conditions in blood vessel segments were simulated and measured, and obvious flow field disturbances were found in the regions of vascular geometry change. The LDL concentration at the luminal surface of bifurcation was significantly higher than that in the straight segment, possibly owing to the atherogenic effect of disturbed flow. Additionally, a stenosis model of rabbit carotid arteries was generated. Atherosclerotic plaques were found to have occurred in the stenosis group and were more severe in the stenosis group on a high-fat diet. Our findings provide the first ever definite proof that LDL concentration polarization occurs in the vascular system in vivo. Both lipoprotein concentration polarization and flow field changes are involved in the infiltration/accumulation of atherogenic lipids within the location of arterial luminal surface and promote the development of AS.

Id1 induces tubulogenesis by regulating endothelial cell adhesion and cytoskeletal organization through β1-integrin and Rho-kinase signalling

The inhibitor of differentiation 1 (Id1) protein is required for tubulogenesis, but the molecular signalling pathways remain unclear. Overexpression (Id1-t) or down-regulation (si-Id1) of Id-1 in cell lines, were used to study the function of Id1. The expression of Id1 and β1-integrin was assessed by Western blotting. Up-regulation of Id1 in human umbilical vascular endothelial cells (HUVECs) activated the expression of β1-integrin and promoted cell adhesion and spreading. Conversely, down-regulation of Id1 suppressed β1-integrin expression and inhibited tubulogenesis. By using a β1-integrin antibody to inhibit β1-integrin function, we demonstrated that Id1-induced cell adhesion and tubulogenesis were mediated by β1-integrin. In addition, HUVECs overexpressing Id1 were able to promote capillary tube formation through cytoskeleton reorganization and cell contraction. Finally, the Rho-kinase inhibitor Y27632 inhibited tubulo-genesis induced by Id1. Our findings provide evidence that Id1 regulates tubulogenesis in vitro through β1-integrin and Rho-kinase signalling.

OxLDL stimulates Id1 nucleocytoplasmic shuttling in endothelial cell angiogenesis via PI3K pathway.

Angiogenesis plays remarkable roles in the development of atherosclerotic rupture plaques. However, its essential mechanism remains unclear. The purpose of the study was to investigate whether inhibitor of DNA binding-1 or inhibitor of differentiation 1 (Id1) promoted angiogenesis when exposed to oxidised low-density lipoprotein (oxLDL), and to determine the molecular mechanism involved. Using aortic ring assay and tube formation assay as a model system, a low concentration of oxLDL was found to induce angiogenic sprouting and capillary lumen formation of endothelial cell. But the Id1 expression was significantly upregulated by oxLDL at low and high concentrations. The Id1 was localised in the nuclei of the human umbilical vein endothelial cells in the control group and in the high-concentration oxLDL group. Id1 was translocated to the cytoplasm at low oxLDL concentrations. The nucleocytoplasmic shuttling at low oxLDL concentration was inhibited by treatment with the nuclear export inhibitor leptomycin B. Protein kinase A (PKA) inhibitor H89 promoted nuclear export of Id1, and phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 reduced the nuclear export of Id1. PI3K inhibition blocked oxLDL-induced angiogenesis. Low concentrations of oxLDL promoted angiogenic sprouting and capillary formation. And this process depends on nuclear export of Id1, which in turn is controlled by the PI3K pathway. This report presents a new link between oxLDL and Id1 localisation, and may provide a new insight into the interactions of ox-LDL and Id1 in the context of atherosclerosis.

Distinctive effects of CD34- and CD133-specific antibody-coated stents on re-endothelialization and in-stent restenosis at the early phase of vascular injury

It is not clear what effects of CD34- and CD133-specific antibody-coated stents have on re-endothelialization and in-stent restenosis (ISR) at the early phase of vascular injury. This study aims at determining the capabilities of different coatings on stents (e.g. gelatin, anti-CD133 and anti-CD34 antibodies) to promote adhesion and proliferation of endothelial progenitor cells (EPCs). The in vitro study revealed that the adhesion force enabled the EPCs coated on glass slides to withstand flow-induced shear stress, so that allowing for the growth of the cells on the slides for 48 h. The in vivo experiment using a rabbit model in which the coated stents with different substrates were implanted showed that anti-CD34 and anti-CD133 antibody-coated stents markedly reduced the intima area and restenosis than bare mental stents (BMS) and gelatin-coated stents. Compared with the anti-CD34 antibody-coated stents, the time of cells adhesion was longer and earlier present in the anti-CD133 antibody-coated stents and anti-CD133 antibody-coated stents have superiority in re-endothelialization and inhibition of ISR. In conclusion, this study demonstrated that anti-CD133 antibody as a stent coating for capturing EPCs is better than anti-CD34 antibody in promoting endothelialization and reducing ISR.