Hu Xp

Application of decellularized scaffold combined with loaded nanoparticles for heart valve tissue engineering in vitro

The purpose of this study was to fabricate decelluarized valve scaffold modified with polyethylene glycol nanoparticles loaded with transforming growth factor-β1 (TGF-β1), by which to improve the extracellular matrix microenvironment for heart valve tissue engineering in vitro. Polyethylene glycol nanoparticles were obtained by an emulsion-crosslinking method, and their morphology was observed under a scanning electron microscope. Decelluarized valve scaffolds, prepared by using trypsinase and TritonX-100, were modified with nanoparticles by carbodiimide, and then TGF-β1 was loaded into them by adsorption. The TGF-β1 delivery of the fabricated scaffold was measured by asing enzyme-linked immunosorbent assay. Whether unseeded or reseeded with myofibroblast from rats, the morphologic, biochemical and biomechanical characteristics of hybrid scaffolds were tested and compared with decelluarized scaffolds under the same conditions. The enzyme-linked immunosorbent assay revealed a typical delivery of nanoparticles. The morphologic observations and biological data analysis indicated that fabricated scaffolds possessed advantageous biocompatibility and biomechanical property beyond decelluarized scaffolds. Altogether this study proved that it was feasible to fabricate the hybrid scaffold and effective to improve extracellular matrix microenvironment, which is beneficial for an application in heart valve tissue engineering.SummaryThe purpose of this study was to fabricate decelluarized valve scaffold modified with polyethylene glycol nanoparticles loaded with transforming growth factor-β1 (TGF-β1), by which to improve the extracellular matrix microenvironment for heart valve tissue engineering in vitro. Polyethylene glycol nanoparticles were obtained by an emulsion-crosslinking method, and their morphology was observed under a scanning electron microscope. Decelluarized valve scaffolds, prepared by using trypsinase and TritonX-100, were modified with nanoparticles by carbodiimide, and then TGF-β1 was loaded into them by adsorption. The TGF-β1 delivery of the fabricated scaffold was measured by asing enzyme-linked immunosorbent assay. Whether unseeded or reseeded with myofibroblast from rats, the morphologic, biochemical and biomechanical characteristics of hybrid scaffolds were tested and compared with decelluarized scaffolds under the same conditions. The enzyme-linked immunosorbent assay revealed a typical delivery of nanoparticles. The morphologic observations and biological data analysis indicated that fabricated scaffolds possessed advantageous biocompatibility and biomechanical property beyond decelluarized scaffolds. Altogether this study proved that it was feasible to fabricate the hybrid scaffold and effective to improve extracellular matrix microenvironment, which is beneficial for an application in heart valve tissue engineering.

Evaluation of a novel tetra‐functional branched poly(ethylene glycol) crosslinker for manufacture of crosslinked, decellularized, porcine aortic valve leaflets

To address concerns over limitations in the clinical use of glutaraldehyde (GA) fixation in bioprosthetic heart valves, we manufactured novel, branched poly(ethylene glycol) tetraacrylate (PEG-TA) crosslinked valve leaflets and evaluated cytotoxic, thrombogenic, hemolytic, and anticalcification effects, thermal stability, and mechanical properties, in comparison to decellularized valves (control) and GA crosslinked valves. Thermal denaturation temperatures were higher for PEG-TA valve leaflets compared to control and GA crosslinked valves (p < 0.001). Leaflet hydrolyzation rate was lower for the PEG-TA group than for GA and control groups (p < 0.05). Superior cytocompatibility was found for PEG-TA group leaflets (MTT, p < 0.01. apoptosis assay, p > 0.05). No thrombogenesis was found in platelet activation tests (p < 0.0001). Hemolysis assays showed that PEG-TA leaflets would not cause damage to blood cells (p > 0.05). Excellent anticalcification properties were confirmed by von Kossa staining, western blot, and atomic absorption spectroscopy (p < 0.0001) in a rat subcutaneous embedding model. Finally, the novel PEG-TA crosslinked material exhibits improved mechanical properties as compared to GA crosslinked materials (tensile strength, p < 0.001, Youngs modulus, p < 0.001). On the basis of all results presented, it is clear that the performance characteristics of PEG-TA crosslinked valve leaflets make PEG-TA crosslinked leaflets a promising alternative for the next generation of bioprosthetic heart valve.

Suppressor of Cytokine Signaling 3 Is a Negative Regulator for Neointimal Hyperplasia of Vein Graft Stenosis

Coronary artery bypass graft (CABG) surgery is one of the most effective treatments for coronary artery disease. However, neointimal hyperplasia and ultimate luminal occlusion that is caused by vascular smooth muscle cell (VSMC) migration, proliferation and inflammatory response impede the long-term prognosis. The SOCS3 protein is involved in modulating various autoimmune and inflammatory diseases. However, the role of SOCS3 in vein graft disease is still unclear. We found that the mRNA and protein expression levels of IL-1β, IL-6, MCP-1, ICAM-1, TNF-α, STAT3, P-STAT3 and SOCS3 were significantly higher in the graft samples compared to normal veins. After transfecting the recombinant adenovirus carrying the rat SOCS3 gene into cultured rat VSMCs or grafting veins in rat, SOCS3 overexpression was found to significantly inhibit VSMC migration and proliferation in vitro and neointimal hyperplasia in vivo, respectively. Furthermore, SOCS3 overexpression inhibited VSMC migration and growth in vitro and alleviated VSMC inflammation in vitro by inhibiting STAT3 activation and phosphorylation. In conclusion, SOCS3 is a crucial physiological negative regulator for vein graft failure and provides a novel target for vein graft stenosis therapy after CABG.

Lazaroid U-74389G inhibits the osteoblastic differentiation of IL-1β-indcued aortic valve interstitial cells through glucocorticoid receptor and inhibition of NF-κB pathway.

BACKGROUND Aortic valve calcification is characterized as the active process of aortic valve interstitial cells (AVICs), and considered as an inflammatory disease. As an antioxidant, the anti-inflammatory activity of Lazaroid has been exhibited in various models. We hypothesized that Lazaroid U-74389G would inhibit the osteoblastic differentiation of AVICs induced by IL-1β. METHODS Normal tricuspid aortic valve leaflets were collected from patients with acute aortic dissection (Type A) undergoing the Bentall procedure. AVICs were isolated and stimulated with IL-1β in presence or absence of U-74389G in culture. Cell lysates were analyzed for osteogenic markers and nuclear factor-κB using real-time PCR and Immunoblotting. Culture media was analyzed for IL-6 and IL-8 with enzyme-linked immunosorbent assay. Alizarin Red Staining was adopted to demonstrate the calcium deposition. RESULTS The expression of alkaline phosphatase and bone morphogenetic protein, accompanied by the production of IL-6 and IL-8, was up-regulated in response to IL-1β and was inhibited by the addition of U-74389G. The NF-κB pathway was activated by IL-1β and involved in the suppression of U-74389G on osteoblastic differentiation in AVICs. The negative effects of U-74389G on ostengenic gene expression and mineralization of AVICs were blocked by glucocorticoid receptor antagonist mifepristone and the NF-κB inhibitor Bay 11-7082. CONCLUSIONS U-74389G inhibits the pro-osteogenic response to IL-1β stimulation in AVICs. The osteoblastic differentiation and mineralization of AVICs were inhabited by U-74389G though the modulation of NF-κB activation, and this pathway could be potential therapeutic targets for medical treatment of calcified aortic valve disease.

Inhibitory effects of suppressor of cytokine signaling 3 on inflammatory cytokine expression and migration and proliferation of IL-6/IFN-γ-induced vascular smooth muscle cells

The main pathogenesis of saphenous vein graft neointimal hyperplasia after coronary artery bypass grafting (CABG) is inflammation-caused migration and proliferation of vascular smooth muscle cells (VSMCs). Janus kinase 2/signal transducer and activators of transcription 3 (JAK2/STAT3) pathway is an important signaling pathway through which VSMCs phenotype conversion occurs. Suppressor of cytokine signaling 3 (SOCS3) is the classic negative feedback inhibitor of JAK2/STAT3 pathway. Growing studies show that SOCS3 plays an important anti-inflammatory role in numerous autoimmune diseases, inflammatory diseases and inflammation-related tumors. However, the effect and mechanism of SOCS3 on vein graft disease is unclear. The purpose of this study was to investigate the effects of SOCS3 on the inflammation, migration and proliferation of VSMCs in vitro and the mechanism. The small interference RNA plasmid targeting rat SOCS3 (SiRNA-rSOCS3) and the recombinant adenovirus vector carrying rat SOCS3 gene (pYrAd-rSOCS3) were constructed, and the empty plamid (SiRNA-control) and vector (pYrAd-GFP) only carrying GFP reported gene were constructed as control. The rat VSMCs were cultured. There were two large groups of A (SOCS3 up-regulated): control group, IL-6/IFN-γ group, IL-6/IFN-γ+pYrAd-rSOCS3 group, IL-6/IFN-γ+pYrAd-GFP group; and B (SOCS3 down-regulated): control group, IL-6/IFN-γ group, IL-6/IFN-γ+SiRNA-rSOCS3 group and IL-6/ IFN -γ+SiRNA-control group. The pYrAd-rSOCS3 and SiRNA-rSOCS3 were transfected into VSMCs induced by IL-6/IFN-γ. After 24 h, real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expression of SOCS3, STAT3 (only by Western blotting), P-STAT3 (only by Western blotting), IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1. The MTT, Transwell assay and flow cytometry were used to examine VSMCs proliferation, migration and cell cycle progression, respectively. As compared with control group, the mRNA and protein expression of SOCS3, STAT3, P-STAT3, IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1 was significantly up-regulated in VSMCs stimulated by IL-6/IFN-γ. However, in VSMCs transfected with pYrAd-rSOCS3 before stimulation with IL-6/IFN-γ, the expression of SOCS3 mRNA and protein was further up-regulated, and that of STAT3, P-STAT3, IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1 was significantly down-regulated as compared with IL-6/IFN-γ group and IL-6/IFN-γ+pYrAd-GFP group. The expression of those related-cytokines in IL-6/IFN-γ+SiRNA-rSOCS3 group was markedly increased as compared with IL-6/IFN-γ group and IL-6/IFN-γ+SiRNA-control group. The absorbance (A) values, the number of cells migrating to the lower chamber, and percentage of cells in the G2/M+S phase were increased in VSMCs stimulated by IL-6/IFN-γ. In VSMCs incubated with pYrAd-rSOCS3 or SiRNA-rSOCS3 before IL-6/IFN-γ stimulation, the A values, the number of cells migrating to the lower chamber, and the percentage of cells in the G2/M+S phase were significantly decreased, and increased respectively. These results imply that IL-6/IFN-γ, strong inflammatory stimulators, can promote transformation of VSMCs phenotype form a quiescent contractile state to a synthetic state by activating JAK2/STAT3 pathway. Over-expresssed SOCS3 might inhibit pro-inflammatory effect, migration and growth of VSMCs by blocking STAT3 activation and phosphorylation. These data in vitro confirm that SOCS3 may play a negatively regulatory role in development and progression of vein graft failure. These conclusions can provide a novel strategy for clinical treatment of vein graft diseases and a new theoretic clue for related drug development.SummaryThe main pathogenesis of saphenous vein graft neointimal hyperplasia after coronary artery bypass grafting (CABG) is inflammation-caused migration and proliferation of vascular smooth muscle cells (VSMCs). Janus kinase 2/signal transducer and activators of transcription 3 (JAK2/STAT3) pathway is an important signaling pathway through which VSMCs phenotype conversion occurs. Suppressor of cytokine signaling 3 (SOCS3) is the classic negative feedback inhibitor of JAK2/STAT3 pathway. Growing studies show that SOCS3 plays an important anti-inflammatory role in numerous autoimmune diseases, inflammatory diseases and inflammation-related tumors. However, the effect and mechanism of SOCS3 on vein graft disease is unclear. The purpose of this study was to investigate the effects of SOCS3 on the inflammation, migration and proliferation of VSMCs in vitro and the mechanism. The small interference RNA plasmid targeting rat SOCS3 (SiRNA-rSOCS3) and the recombinant adenovirus vector carrying rat SOCS3 gene (pYrAd-rSOCS3) were constructed, and the empty plamid (SiRNA-control) and vector (pYrAd-GFP) only carrying GFP reported gene were constructed as control. The rat VSMCs were cultured. There were two large groups of A (SOCS3 up-regulated): control group, IL-6/IFN-γ group, IL-6/IFN-γ+pYrAd-rSOCS3 group, IL-6/IFN-γ+pYrAd-GFP group; and B (SOCS3 down-regulated): control group, IL-6/IFN-γ group, IL-6/IFN-γ+SiRNA-rSOCS3 group and IL-6/ IFN -γ+SiRNA-control group. The pYrAd-rSOCS3 and SiRNA-rSOCS3 were transfected into VSMCs induced by IL-6/IFN-γ. After 24 h, real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expression of SOCS3, STAT3 (only by Western blotting), P-STAT3 (only by Western blotting), IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1. The MTT, Transwell assay and flow cytometry were used to examine VSMCs proliferation, migration and cell cycle progression, respectively. As compared with control group, the mRNA and protein expression of SOCS3, STAT3, P-STAT3, IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1 was significantly up-regulated in VSMCs stimulated by IL-6/IFN-γ. However, in VSMCs transfected with pYrAd-rSOCS3 before stimulation with IL-6/IFN-γ, the expression of SOCS3 mRNA and protein was further up-regulated, and that of STAT3, P-STAT3, IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1 was significantly down-regulated as compared with IL-6/IFN-γ group and IL-6/IFN-γ+pYrAd-GFP group. The expression of those related-cytokines in IL-6/IFN-γ+SiRNA-rSOCS3 group was markedly increased as compared with IL-6/IFN-γ group and IL-6/IFN-γ+SiRNA-control group. The absorbance (A) values, the number of cells migrating to the lower chamber, and percentage of cells in the G2/M+S phase were increased in VSMCs stimulated by IL-6/IFN-γ. In VSMCs incubated with pYrAd-rSOCS3 or SiRNA-rSOCS3 before IL-6/IFN-γ stimulation, the A values, the number of cells migrating to the lower chamber, and the percentage of cells in the G2/M+S phase were significantly decreased, and increased respectively. These results imply that IL-6/IFN-γ, strong inflammatory stimulators, can promote transformation of VSMCs phenotype form a quiescent contractile state to a synthetic state by activating JAK2/STAT3 pathway. Over-expresssed SOCS3 might inhibit pro-inflammatory effect, migration and growth of VSMCs by blocking STAT3 activation and phosphorylation. These data in vitro confirm that SOCS3 may play a negatively regulatory role in development and progression of vein graft failure. These conclusions can provide a novel strategy for clinical treatment of vein graft diseases and a new theoretic clue for related drug development.

Early surgery versus conventional treatment for asymptomatic severe aortic regurgitation with normal ejection fraction and left ventricular dilatation

OBJECTIVES The management of patients with asymptomatic severe aortic regurgitation (AR) remains controversial. The aim of the present study was to assess and compare the clinical results between early surgery and conventional treatment for asymptomatic severe AR in patients with normal left ventricular (LV) systolic function and LV dilatation. METHODS This retrospective cohort study included 230 consecutive patients with severe AR with left ventricular ejection function (LVEF ≥50%) and left ventricular end-diastolic dimension (LVEDD >70 mm) from 2003 to 2014. A total of 154 patients received early aortic valve replacement (AVR), whereas 76 patients received conventional treatment at the discretion of the surgeon. Overall and cardiovascular survival rates were evaluated between 2 groups with propensity score analysis. RESULTS The patients had a mean age of 54.3 ± 14.1 years; 60.4% were men. Mean LVEF and LVEDD were 58.3 ± 7.7% and 75.3 ± 5.2 mm, respectively. Kaplan-Meier analysis revealed that early AVR was associated with better 10-year cardiovascular ( P  = 0.037) survivals in the overall population. In the 62 propensity score-matched pairs, early AVR was still associated with a significantly better 10-year overall survival ( P  = 0.018). Furthermore, patients who underwent early AVR had significantly improved 3-, 5- and 10-year cardiovascular survival rates of 98.3, 96.2 and 93.6%, respectively, compared with 93.5, 88.3 and 80.0% for those in the conventional treatment group ( P  = 0.008). CONCLUSIONS Compared with conservative management, early AVR is associated with an improved long-term outcome in patients with asymptomatic severe AR with LVEF ≥50% and LVEDD >70 mm. These data provide new evidence to strongly support current guidelines, which recommend a strategy of early operative intervention.

Synthesis and Applications of Tetra-functional Branched Poly（ethylene glycol） Derivative for the Decellularized Valve Leaflets Cross-linking

To investigate the effects of polyethylene glycol cross-linking on the mechanical properties, 80 porcine aortic valves were harvested, decellularized, and introduced with sulfhydryl. Then the valves were randomly assigned into 5 experimental groups and 1 control group (n=16). For the valves in those experimental groups, branched polyethylene glycol diacrylate (PEG) of 5 different molecular weights (3.4, 8, 12, 20, 40 kDa) were synthesized and cross-linked with them respectively. The efficiency of the cross-linking was determined by measuring the amount of residual thiol group and the mechanical properties of the cross-linked valve leaflets were assessed by uni-axial planar tensile testing. The efficiency of the PEG 20 kDa group was 70.72±2.33%, obviously superior to that of the other groups (p<0.05). Tensile test proved that branched PEG cross-linking can significantly enhance the mechanical behaviors of the decellularized valve leaflet and the Young’s modulus of each group was positively correlated with the molecular weight of PEG. It was concluded that branched PEG with the molecular weight of 20 kDa can effectively cross-link the decellularized porcine aortic valves and improve their mechanical properties, which makes it a promising cross-linker that can be used in the modification of decellularized tissue engineering valves.

Status on Heart Transplantation in China

IntroductIon “End‐stage heart disease” commonly refers to an irreversible stage of cardiac decompensation caused by a variety of pathologies that cannot be treated using conventional drugs or traditional surgical treatments. The life expectancy of patients with end‐stage heart disease ranges from <6 months to 1 year. Therapeutic strategies for end‐stage heart disease patients are primarily based on three approaches: Internal medicine therapy, surgical therapy (heart transplantation), and multiple organ protection therapy via the core method of mechanical circulation assistance. Among these approaches, heart transplantation has become recognized as the most effective treatment.

Mid- to Long-term Clinical Outcomes of Hancock II Bioprosthesis in Chinese Population

Background: Compared to the Western countries, Chinese patients present a special primary disease spectrum, diverse valvular pathogenesis, and different postoperational anticoagulation strategy. This research aimed to evaluate the mid- to long-term clinical performance of Hancock II bioprosthesis in the Chinese population. Methods: This study retrospectively reviewed all patients who received surgical treatments with at least one Hancock II bioprosthesis implantation from January 2004 to December 2013 at a single center in China. Totally 647 patients were included in the clinical evaluation, and 629 patients were successfully discharge, among whom 605 patients were completely followed-up. The follow-up rate was 96.2%. The mean and median follow-up time was 62.0 ± 59.0 and 56.0 months, respectively. Postoperative outcomes of survival rates, reoperations and valve related morbidities were assessed. Continuous and categorical variables were compared using the t -test and Chi-square test, respectively. Survival and freedom from adverse events were calculated by using a Kaplan–Meier method. Results: The overall in-hospital mortality was 2.8% (18/647) while there were 34 deaths (5.6%, 34/605) in the follow-up stage after discharge. The overall survival rate was 94.6% and 82.7% at 5 years and 10 years, respectively. The cumulative survival rate of 10 years was 82.8% in AVR group, 84.4% in MVR group, and 78.4% in DVR group. The overall rate of freedom from reoperations was 95.5% at 5 years and 86.8% at 10 years. The freedom from reoperation at 10 years was 87.0%, 88.1%, and 84.0% in AVR, MVR, and DVR group, respectively. The freedom from morbidities at 10 years was: 90.3% for thromboembolism, 95.2% for hemorrhage, 97.5% for prosthesis endocarditis, 95.9% for paravalvular leak, and 94.6% for structural valve deterioration, respectively. Conclusions: Hancock II bioprosthesis exhibited a satisfactory mid- to long-term durability and promising clinical performance in the Chinese population. The occurrence rates of death and other adverse events in this single-center study were overall coincident and quite acceptable when compared with existing data.

Role of TGF-β1 Signaling in Heart Valve Calcification Induced by Abnormal Mechanical Stimulation in a Tissue Engineering Model

A tissue engineering model of heart valve calcification induced in a bioreactor was established to evaluate the calcification induced by abnormal mechanical stimulation and explore the underlying molecular mechanisms. Polyethylene glycol (PEG)-modified decellularized porcine aortic leaflets seeded with human valve interstitial cells (huVICs) were mounted on a Ti-Ni alloy frame to fabricate two-leaflet and threeleaflet tissue engineered valves. The two-leaflet model valves were exposed to abnormal pulsatile flow stimulation with null (group A), low (1000 mL/min, group B), medium (2000 mL/min, group C), and high velocity (3000 mL/min, group D) for 14 days. Morphology and calcification were assessed by von Kossa staining, alkaline phosphatase (ALP) content, and Runx2 immunostaining. Leaflet calcification and mRNA and protein expression of transforming growth factor (TGF)-β1, bone morphogenetic protein 2 (BMP2), Smad1, and MSX2 were measured at different time points. ALP content was examined in two-leaflet valves seeded with BMP2 shRNA plasmid-infected huVICs and exposed to the same stimulation conditions. The results showed that during 14 days of flow stimulation, huVICs on the leaflet surface proliferated to generate normal monolayer coverage in groups A, B, and C. Under mechanical stimulation, huVICs showed a parallel growth pattern in the direction of the fluid flow, but huVICs exhibited disordered growth in the high-velocity flow environment. von Kossa staining, ALP measurement, and immunohistochemical staining for Runx2 confirmed the lack of obvious calcification in group A and significant calcification in group D. Expression levels of TGF-β1, BMP2, and MSX2 mRNA and protein were increased under fluid stimulation. ALP production by BMP2 shRNA plasmid-infected huVICs on model leaflets was significantly reduced. In conclusion, abnormal mechanical stimulation in a bioreactor induced calcification in the tissue engineering valve model. The extent of calcification correlated positively with the flow velocity, as did the mRNA and protein levels of TGF-β1, BMP2, and MSX2. These findings indicate that TGF-β1/BMP2 signaling is involved in valve calcification induced by abnormal mechanical stimulation.