On the biocompatibility of graphene oxide towards vascular smooth muscle cells.

Graphene and its derivatives have shown fascinating potential in biomedical applications. However, the biocompatibility of graphene with vascular smooth muscle cells (VSMCs) and applications to vascular engineering have not been explored extensively. Using a rat aortic smooth muscle cell line, A7r5, as a VSMC model, we have explored the effects of graphene oxide (GO) on the growth and behaviours of VSMCs. Results demonstrated that GO had no obvious toxicity to VSMCs. Cells cultured on GO retained the expression of smooth muscle cell-specific markers CNN1, ACTA2 and SMTN, on both mRNA and protein levels. A wound healing assay demonstrated no effect of GO on cell migration. We also found that small-flaked GO favoured the proliferation of VSMCs, suggesting a potential of using surface chemistry or physical properties of GO to influence cell growth behaviour. These results provide insight into the suitability of GO as a scaffold for vascular tissue engineering.

Patient-Specific iPSC Model of a Genetic Vascular Dementia Syndrome Reveals Failure of Mural Cells to Stabilize Capillary Structures.

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is the most common form of genetic stroke and vascular dementia syndrome resulting from mutations in NOTCH3. To elucidate molecular mechanisms of the condition and identify drug targets, we established a patient-specific induced pluripotent stem cell (iPSC) model and demonstrated for the first time a failure of the patient iPSC-derived vascular mural cells (iPSC-MCs) in engaging and stabilizing endothelial capillary structures. The patient iPSC-MCs had reduced platelet-derived growth factor receptor ß, decreased secretion of the angiogenic factor vascular endothelial growth factor (VEGF), were highly susceptible to apoptotic insults, and could induce apoptosis of adjacent endothelial cells. Supplementation of VEGF significantly rescued the capillary destabilization. Small interfering RNA knockdown of NOTCH3 in iPSC-MCs revealed a gain-of-function mechanism for the mutant NOTCH3. These disease mechanisms likely delay brain repair after stroke in CADASIL, contributing to the brain hypoperfusion and dementia in this condition, and will help to identify potential drug targets.

[5-aza-2'-deoxycytidine-induced inhibition of CDH13 expression and its inhibitory effect on methylation status in human colon cancer cells in vitro and on growth of xenograft in nude mice].

OBJECTIVE: To determine the inhibitory effect of 5-aza-2'-deoxycytidine (5-Aza-CdR) on the growth of human colon carcinoma cells and xenografts in nude mice, to observe its effect on CDH13 gene expression and methylation in the xenografts, and to explore the possible mechanisms. METHODS: Human colon carcinoma cell line HCT116 cells were treated with 5-Aza-CdR, and the cell morphology was observe by phase contrast microscopy. The cell growth was assessed by MTT assay. A tumor-bearing mouse model was generated by subcutaneous inoculation of human colon carcinoma HCT116 cells into nude mice. The tumor growth in the nude mice was observed, the CDH13 gene expression and its methylation status in the tumors were detected using methylation specific PCR (MSP), RT-PCR, Western blotting and immunohistochemistry. RESULTS: After treatment with 5-Aza-CdR, the inhibition rate of the growth of cultured HCT116 cells was increased as the concentration was increasing. The growth of the xenografts in nude mice was significantly inhibited, and the methylated CDH13 gene was reactivated. After 4 weeks of 5-Aza-CdR treatment, no significant difference was found between the body weights of nude mice in the 5-Aza-CdR group [(18.06 ± 1.29) g] and control group [(17.07 ± 0.84) g], (P > 0.10), and the average volume of xenografts of the 5-Aza-CdR group was (907.00 ± 87.29) mm(3), significantly smaller than the (1370.93 ± 130.20) mm(3) in the control group (P < 0.005). No expression of CDH13 gene was found in the control group. The expression of CDH13 gene in the 5-Aza-CdR group was increased along with the increasing concentration of 5-Aza-CdR. CONCLUSIONS: 5-Aza-CdR inhibits the growth of human colon cancer cells in culture and in nude mice, and induces the cancer cells to re-express CDH13 in nude mice. Its mechanism may be that demethylation of the methylated CDH13 promoter induced by 5-Aza-CdR restores CDH13 expression and thus inhibits the tumor growth in nude mice.

Correlation between T-cadherin gene expression and aberrant methylation of T-cadherin promoter in human colon carcinoma cells.

Previous researches showed T-cadherin (CDH13) expression was downregulated in colon cancer tissues and was associated with increase of invasive and metastatic potential. This research was to observe the mechanisms responsible for inactivation of T-cadherin gene in colon carcinoma; we investigated the methylation status around the 5' promoter region of T-cadherin gene of Hct116 colon cancer cell line by methylation-specific polymerase chain reaction (MSP), also detected the expression change of T-cadherin mRNA and protein in Hct116 cell line after 5-Aza-CdR treatment by reverse transcriptase polymerase chain reaction and Western blotting, and compared the T-cadherin methylation status with T-cadherin mRNA and protein expression. We found that hypermethylation of T-cadherin was involved in Hct116 cell line, while T-cadherin mRNA and protein expression was almost lost or downregulated in Hct116 cell line. Therefore, methylation of the T-cadherin promoter region was correlated with the loss or downregulation of T-cadherin mRNA and protein expression in Hct116 colon cancer cell line. Treatment of T-cadherin-negative carcinoma cells with the demethylating agent, 5-aza-2'-deoxycytidine, induced re-expression of this gene. Our findings demonstrate that 5' CpG island methylation is common in colon carcinoma and may play an important role in the inaction of T-cadherin. Our results also suggest that demethylation of the T-cadherin gene may be a potential therapeutic strategy for colon carcinoma.

[Effect of 5-Aza-CdR on expression and methylation of E-cadherin gene in human colon carcinoma cells].

BACKGROUND AND OBJECTIVE: Colon cancer is one of the most common malignant tumors, and its pathogenesis is not fully understood. Transcriptional silencing by DNA methylation is believed to be an important mechanism of carcinogenesis. E-cadherin can suppress tumor cell invasion and metastasis, and is considered as an invasion/metastasis suppressor gene. Inactivation of E-cadherin gene often occurs in colon carcinoma. This study was to investigate the correlation between E-cadherin gene expression and the methylation status of E-cadherin 5' CpG islands in human colon carcinoma cell line HT-29, and to explore the mechanism of carcinogenesis of colon cancer. METHODS: Immunocytochemical dicho-step method and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expression of E-cadherin protein and mRNA in HT-29 cells after 5-Aza-CdR treatment; methylation specific PCR was used to analyze the methylation status at promoter of E-cadherin gene. RESULTS: The expression of E-cadherin gene could be restored by 5-Aza-CdR treatment, immunocytochemical staining showed the positive expression ratio of E-cadherin increased from (21+/-7)% (1 micromol/L) to (39+/-13)% (5 micromol/L); E-cadherin genes were methylated and not expressed in HT-29 cells in the colon carcinoma. CONCLUSIONS: E-cadherin methylation plays an important role in the carcinogenesis of colon carcinoma cells and can re-express after the treatment with 5-Aza-CdR.