Nianguo Dong

Decreased adiponectin and increased inflammation expression in epicardial adipose tissue in coronary artery disease

BackgroundDisorders of endocrine substances in epicardial adipose tissue are known causes of coronary artery disease (CAD). Adiponectin is associated with cardiovascular disease. However, expression of adiponectin in epicardial adipose tissue and its function in CAD pathogenesis is unclear. This study investigates adiponectin expression in epicardial adipose tissue in CAD patients.MethodsVessels or adipose tissue samples collected from CAD patients and non-CAD controls were examined after immunochemical staining. Adiponectin, cytokines of interleukin-6 (IL-6) and necrosis factor-α (TNF-α) and toll-like receptor 4 (TLR4) expression level in adipose tissue were measured using real-time quantitative RT-PCR. Adiponectin concentrations in peripheral and coronary sinus vein plasma were measured with enzyme-linked immunosorbent assay. Peripheral vein plasma biochemistries were performed with routine laboratory techniques. Monocytes were collected from blood using lymphocyte separation medium. Expression level of cytokines and transcription factor NF-κB were measured to learn the effect of adiponectin on stearic acid-stimulated monocytes. Percentage of TLR4 positive monocytes was analyzed using flow cytometry.ResultsHistological examination revealed increased macrophage infiltration into epicardial adipose tissue of CAD patients. Decreased adiponectin displayed by real-time quantitative RT-PCR was associated with enhanced cytokines of IL-6 and TNF-α or TLR4 expression level in epicardial adipose tissue, suggesting decreased circulating adiponectin may be useful as a more sensitive predictor for coronary atherosclerosis than routine laboratory examinations. Adiponectin suppressed secretion of IL-6 and TNF-α in stimulated monocytes and TLR4 was expressed on cell surfaces.ConclusionsEndocrine disorders in epicardial adipose tissue are strongly linked to CAD, and adiponectin has a protective effect by inhibiting macrophage-mediated inflammation.

Fabrication of a Novel Hybrid Heart Valve Leaflet for Tissue Engineering: An In Vitro Study

The objective of this study was to fabricate biomatrix/polymer hybrid heart valve leaflet scaffolds using an electrospinning technique and seeded by mesenchymal stem cells. Mesenchymal stem cells were obtained from rats. Porcine aortic heart valve leaflets were decellularized, coated with basic fibroblast growth factor/chitosan/poly-4-hydroxybutyrate using an electrospinning technique, reseeded, and cultured over a time period of 14 days. Controls were reseeded and cultured over an equivalent time period. Specimens were examined biochemically, histologically, and mechanically. Recellularization of the hybrid heart valve leaflet scaffolds was significantly improved compared to controls. Biochemical and mechanical analysis revealed a significant increase of cell mass, 4-hydroxyproline, collagen, and strength in the hybrid heart valve leaflets compared to controls. This is the first attempt in tissue-engineered heart valves to fabricate hybrid heart valve leaflets using mesenchymal stem cells combined with a slow release technique and an electrospinning technique.

Endoplasmic Reticulum Stress Participates in Aortic Valve Calcification in Hypercholesterolemic Animals

Objectives—Aortic valve (AV) calcification occurs via a pathophysiological process that includes lipoprotein deposition, inflammation, and osteoblastic differentiation of valvular interstitial cells. Here, we investigated the association between endoplasmic reticulum (ER) stress and AV calcification. Approach and Results—We identified ER stress activation in AV of patients with calcified AV stenosis. We generated an AV calcification model in hypercholesterolemic rabbits and mice, respectively, and found marked AV ER stress induction. Classical ER stress inhibitor, tauroursodeoxycholic acid, administration markedly prevented AV calcification, and attenuated AV osteoblastic differentiation and inflammation in both rabbit and mouse models of AV calcification via inhibition of ER stress. In cultured valvular interstitial cells (VICs), we found that oxidized low density lipoprotein (oxLDL) caused ER stress in a cytosolic [Ca]2+i-dependent manner. OxLDL promoted osteoblastic differentiation via ER stress–mediated protein kinase-like ER kinase/activating transcription factor 4/osteocalcin and inositol-requiring transmembrane kinase and endonuclease-1&agr; (IRE1&agr;)/spliced X-box–binding protein 1/Runx2 pathway, and induced inflammatory responses through IRE1&agr;/c-Jun N-terminal kinase and IRE1&agr;/nuclear factor kappa-light-chain-enhancer of activated B cells signaling in VICs. Inhibition of ER stress by either tauroursodeoxycholic acid or 4-phenyl butyric acid could both suppress oxLDL–induced osteoblastic differentiation and inflammatory responses in VICs. Conclusions—These data provide novel evidence that ER stress participates in AV calcification development, and suggest that ER stress may be a novel target for AV calcification prevention and treatment.

LncRNA MALAT1 sponges miR-204 to promote osteoblast differentiation of human aortic valve interstitial cells through up-regulating Smad4

BACKGROUND Emerging evidences have indicated that long non-coding RNAs (lncRNAs) play vital roles in cardiovascular physiology and pathology. The lncRNA MALAT1, a highly abundant and conserved imprinted gene, has been implicated in many cardiovascular diseases. However, the function of MALAT1 in calcific aortic valve disease (CAVD) remains unknown. This study sought to document the function and underlying mechanism of MALAT1 in regulating CAVD. METHODS Protein level was determined by immunoblotting and immunofluorescence staining. MALAT1, miR-204 and mRNA expressions were detected by qRT-PCR. Mineralized bone matrix formation was assessed by Alizarin Red staining. The interaction between MALAT1 and miR-204 was studied using luciferase reporter assay, RNA pull-down assay and RNA-binding protein immunoprecipitation assay. RESULTS Ectopic expression of MALAT1 was observed in calcific valves and after osteogenic induction in human aortic valve interstitial cells (VICs). In vitro experiments revealed that MALAT1 acted as a positive regulator of osteogenic differentiation by repressing miR-204 expression and activity and thereby promoting expression of osteoblast-specific markers, including alkaline phosphatase, mineralized bone matrix formation and osteocalcin. Mechanistically, we identified Smad4 as a direct target of miR-204. Importantly, MALAT1 could directly interact with miR-204 and overexpression of miR-204 efficiently reversed the upregulation of Smad4 induced by MALAT1. Thus, MALAT1 positively regulated the expression of Smad4 through sponging miR-204, and promoted osteogenic differentiation of VICs. CONCLUSIONS Our study provides novel mechanistic insights into a critical role for lncRNA MALAT1 as a miRNA sponge in CAVD and sheds new light on lncRNA-directed diagnostics and therapeutics in CAVD.

Matricellular protein CCN3 mitigates abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a major cause of morbidity and mortality; however, the mechanisms that are involved in disease initiation and progression are incompletely understood. Extracellular matrix proteins play an integral role in modulating vascular homeostasis in health and disease. Here, we determined that the expression of the matricellular protein CCN3 is strongly reduced in rodent AAA models, including angiotensin II-induced AAA and elastase perfusion-stimulated AAA. CCN3 levels were also reduced in human AAA biopsies compared with those in controls. In murine models of induced AAA, germline deletion of Ccn3 resulted in severe phenotypes characterized by elastin fragmentation, vessel dilation, vascular inflammation, dissection, heightened ROS generation, and smooth muscle cell loss. Conversely, overexpression of CCN3 mitigated both elastase- and angiotensin II-induced AAA formation in mice. BM transplantation experiments suggested that the AAA phenotype of CCN3-deficient mice is intrinsic to the vasculature, as AAA was not exacerbated in WT animals that received CCN3-deficient BM and WT BM did not reduce AAA severity in CCN3-deficient mice. Genetic and pharmacological approaches implicated the ERK1/2 pathway as a critical regulator of CCN3-dependent AAA development. Together, these results demonstrate that CCN3 is a nodal regulator in AAA biology and identify CCN3 as a potential therapeutic target for vascular disease.

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.

Retinoic acid attenuates acute heart rejection by increasing regulatory T cell and repressing differentiation of Th17 cell in the presence of TGF‐β

Retinoic acid (RA), in a transforming growth factor beta (TGF‐β)‐dependent manner, promotes differentiation of regulatory T cells (Tregs) but inhibits the differentiation of Th17 cells in vitro from naive CD4+T cells. In addition, transfer of induced Tregs (iTregs) reduces rejection. We therefore examined whether RA could attenuate acute cardiac transplant rejection in vivo in a mouse model by regulating the reciprocal differentiation of Tregs and Th17 cells. The iTregs and naive T cells were respectively transferred into congenic mice. Two weeks later, the percentages of transferred cells and Forkhead box P3 (FoxP3)+ Tregs were measured in spleen. Mice with cardiac transplants were treated with TGF‐β alone, RA alone, both or none. The percentage of Tregs or Th17 cells in CD4+T cells, the level of FoxP3 protein or serous interleukin (IL)‐17A, or suppressive function of Tregs from recipient mice were assessed. The percentage of Th17 cells and level of serum IL‐17A both increased significantly during acute rejection. RA favored differentiation to Tregs over Th17 cells. Unlike naive T cells, only a few transferred iTregs remained after transfer. Treatment with RA plus TGF‐β prolonged graft survival, increased the percentage of Tregs, and decreased the percentage of Th17 cells in peripheral T cells. Tregs from all recipients had normal suppressive function. In conclusion, treatment with RA plus TGF‐β attenuates acute rejection by promoting the differentiation of Tregs and inhibiting the differentiation of Th17 cells.

Impaired Thymic Export and Apoptosis Contribute to Regulatory T-Cell Defects in Patients with Chronic Heart Failure

Objective Animal studies suggest that regulatory T (Treg) cells play a beneficial role in ventricular remodeling and our previous data have demonstrated defects of Treg cells in patients with chronic heart failure (CHF). However, the mechanisms behind Treg-cell defects remained unknown. We here sought to elucidate the mechanism of Treg-cell defects in CHF patients. Methods and Results We performed flow cytometry analysis and demonstrated reduced numbers of peripheral blood CD4+CD25+FOXP3+CD45RO−CD45RA+ naïve Treg (nTreg) cells and CD4+CD25+FOXP3+CD45RO+CD45RA− memory Treg (mTreg) cells in CHF patients as compared with non-CHF controls. Moreover, the nTreg/mTreg ratio (p<0.01), CD4+CD25+FOXP3+CD45RO− CD45RA+CD31+ recent thymic emigrant Treg cell (RTE-Treg) frequency (p<0.01), and T-cell receptor excision circle levels in Treg cells (p<0.01) were lower in CHF patients than in non-CHF controls. Combined annexin-V and 7-AAD staining showed that peripheral Treg cells from CHF patients exhibited increased spontaneous apoptosis and were more prone to interleukin (IL)-2 deprivation- and CD95 ligand-mediated apoptosis than those from non-CHF individuals. Furthermore, analyses by both flow cytometry and real-time polymerase chain reaction showed that Treg-cell frequency in the mediastinal lymph nodes or Foxp3 expression in hearts of CHF patients was no higher than that of the non-CHF controls. Conclusion Our data suggested that the Treg-cell defects of CHF patients were likely caused by decreased thymic output of nascent Treg cells and increased susceptibility to apoptosis in the periphery.

High-mobility group box-1 protein induces osteogenic phenotype changes in aortic valve interstitial cells

OBJECTIVES Calcific aortic valve (AV) disease is known to be an inflammation-related process. High-mobility group box-1 (HMGB1) protein and Toll-like receptor 4 (TLR4) have been reported to participate in several inflammatory diseases. The purpose of the present study was to determine whether the HMGB1-TLR4 axis is involved in calcific AV disease, and to evaluate the effect of HMGB1, and its potential mechanisms, on the pro-osteogenic phenotype change of valvular interstitial cells (VICs). METHODS Expression of HMGB1 and TLR4 in human calcific AVs was evaluated using immunohistochemical staining and immunoblotting. Cultured VICs were used as an in vitro model. The VICs were stimulated with HMGB1 for analysis, with versus without TLR4 small interfering ribonucleic acid (siRNA), c-Jun N-terminal kinase mitogen-activated protein kinase (JNK MAPK), and nuclear factor kappa-B (NF-κB) inhibitors. RESULTS Enhanced accumulation of HMGB1 and TLR4 was observed in calcific valves. Moreover, we found that HMGB1 induced high levels of pro-inflammatory cytokine production and promoted the osteoblastic differentiation and calcification of VICs. In addition, HMGB1 induced phosphorylation of JNK MAPK and NF-κB. However, these effects were markedly suppressed by siRNA silencing of TLR4. In addition, blockade of JNK MAPK and NF-κB phosphorylation prohibited HMGB1-induced production of pro-osteogenic factors, and mineralization of VICs. CONCLUSIONS The HMGB1 protein may promote osteoblastic differentiation and calcification of VICs, through the TLR4-JNK-NF-κB signaling pathway.

Fabrication of a novel hybrid scaffold for tissue engineered heart valve

The aim of this study was to fabricate biomatrix/polymer hybrid scaffolds using an electrospinning technique. Then tissue engineered heart valves were engineered by seeding mesenchymal stromal cells (MSCs) onto the scaffolds. The effects of the hybrid scaffolds on the proliferation of seed cells, formation of extracellular matrix and mechanical properties of tissue engineered heart valves were investigated. MSCs were obtained from rats. Porcine aortic heart valves were decellularized, coated with poly(3-hydroxybutyrate-co-4-hydroxybutyrate) using an electrospinning technique, and reseeded and cultured over a time period of 14 days. In control group, the decellularized valve scaffolds were reseeded and cultured over an equivalent time period. Specimens of each group were examined histologically (hematoxylin-eosin [HE] staining, immunohistostaining, and scanning electron microscopy), biochemically (DNA and 4-hydroxyproline) and mechanically. The results showed that recellularization was comparable to the specimens of hybrid scaffolds and controls. The specimens of hybrid scaffolds and controls revealed comparable amounts of cell mass and 4-hydroxyproline (P>0.05). However, the specimens of hybrid scaffolds showed a significant increase in mechanical strength, compared to the controls (P<0.05). This study demonstrated the superiority of the hybrid scaffolds to increase the mechanical strength of tissue engineered heart valves. And compared to the decellularized valve scaffolds, the hybrid scaffolds showed similar effects on the proliferation of MSCs and formation of extracellular matrix. It was believed that the hybrid scaffolds could be used for the construction of tissue engineered heart valves.SummaryThe aim of this study was to fabricate biomatrix/polymer hybrid scaffolds using an electrospinning technique. Then tissue engineered heart valves were engineered by seeding mesenchymal stromal cells (MSCs) onto the scaffolds. The effects of the hybrid scaffolds on the proliferation of seed cells, formation of extracellular matrix and mechanical properties of tissue engineered heart valves were investigated. MSCs were obtained from rats. Porcine aortic heart valves were decellularized, coated with poly(3-hydroxybutyrate-co-4-hydroxybutyrate) using an electrospinning technique, and reseeded and cultured over a time period of 14 days. In control group, the decellularized valve scaffolds were reseeded and cultured over an equivalent time period. Specimens of each group were examined histologically (hematoxylin-eosin [HE] staining, immunohistostaining, and scanning electron microscopy), biochemically (DNA and 4-hydroxyproline) and mechanically. The results showed that recellularization was comparable to the specimens of hybrid scaffolds and controls. The specimens of hybrid scaffolds and controls revealed comparable amounts of cell mass and 4-hydroxyproline (P>0.05). However, the specimens of hybrid scaffolds showed a significant increase in mechanical strength, compared to the controls (P<0.05). This study demonstrated the superiority of the hybrid scaffolds to increase the mechanical strength of tissue engineered heart valves. And compared to the decellularized valve scaffolds, the hybrid scaffolds showed similar effects on the proliferation of MSCs and formation of extracellular matrix. It was believed that the hybrid scaffolds could be used for the construction of tissue engineered heart valves.