Histone acetylation and methylation significantly change with severity of atherosclerosis in human carotid plaques.

BACKGROUND: The aim of the study was to analyze histone acetylation, methylation, and the expression of their corresponding transferases in atherosclerotic plaques of patients with carotid artery stenosis. METHODS: Atherosclerotic tissue from our biobank (n=80) was divided into various segments covering all plaque stages and classified according to the American Heart Association. The plaques were assigned to early (types I-III) or advanced (types V-VII) stage group of atherosclerosis. Ten healthy carotid arteries from transplant donors served as controls. The expression of histone acetyltransferases (GNAT group: GCN5L, P300/CBP group: P300, MYST group: MYST1 and MYST2) and histone methyltransferases (H3K4: MLL2/4, SET7/9, and hSET1A; H3K9: SUV39H1, SUV39H2, ESET/SETDB1, and EHMT1; H3K27: EZH2 and G9a) was analyzed by SYBR-green-based real-time polymerase chain reaction. Histone acetylation/methylation in the cells within atherosclerotic plaques was determined by immunohistochemistry. RESULTS: Increased histone acetylation was observed on H3K9 and H3K27 in smooth muscle cells (SMCs) in advanced atherosclerotic lesions compared to healthy vessels (P=.002 and .034). H3K9 acetylation in SMCs and macrophages was associated with plaque severity of atherosclerosis (P=.048 and <.001). Expression of GCN5L and MYST1 also correlated with the severity of atherosclerosis (P<.001). Methylation of H3K9 and H3K27 was significantly reduced in atherosclerotic plaques in SMCs and inflammatory cells (P<.001 and .026). Methylation on H3K4 was significantly associated with the severity of atherosclerosis. Expression of methyltransferase MLL2/4 was increased in advanced stages of atherosclerosis (P<.001). CONCLUSIONS: Histone acetylation and methylation seem to play a decisive role in atherosclerosis, showing significant differences between healthy vessels and vessels at different stages of atherosclerosis.

5-azacytidine improves the osteogenic differentiation potential of aged human adipose-derived mesenchymal stem cells by DNA demethylation.

The therapeutic value of adipose-derived mesenchymal stem cells (Ad-MSCs) for bone regeneration is critically discussed. A possible reason for reduced osteogenic potential may be an age-related deterioration of the Ad-MSCs. In long term in vitro culture, epigenomic changes in DNA methylation are known to cause gene silencing, affecting stem cell growth as well as the differentiation potential. In this study, we observed an age-related decline in proliferation of primary human Ad-MSCs. Decreased Nanog, Oct4 and Lin28A and increased Sox2 gene-expression was accompanied by an impaired osteogenic differentiation potential of Ad-MSCs isolated from old donors (>60 a) as compared to Ad-MSCs isolated from younger donors (<45 a). 5-hydroxymethylcytosine (5 hmC) and 5-methylcytonsine (5 mC) distribution as well as TET gene expression were evaluated to assess the evidence of active DNA demethylation. We observed a decrease of 5 hmC in Ad-MSCs from older donors. Incubation of these cells with 5-Azacytidine induced proliferation and improved the osteogenic differentiation potential in these cells. The increase in AP activity and matrix mineralization was associated with an increased presence of 5 hmC as well as with an increased TET2 and TET3 gene expression. Our data show, for the first time, a decrease of DNA hydroxymethylation in Ad-MSCs which correlates with donor-age and that treatment with 5-Azacytidine provides an approach which could be used to rejuvenate Ad-MSCs from aged donors.

Decellularized kidney matrix for perfused bone engineering.

The vascularization of tissue-engineered constructs is yet an unsolved problem. Here, recent work on the decellularization of whole organs has opened new perspectives on tissue engineering. However, existing decellularization protocols last several days and derived biomatrices have only been reseeded with cells from the same tissue origin or stem cells differentiating into these types of tissue. Within the present work, we demonstrate a novel standardized, time-efficient, and reproducible protocol for the decellularization of solid tissues to derive a ready to use biomatrix within only 5 h. Furthermore, we prove that biomatrices are usable as potential scaffolds for tissue engineering of vascularized tissues, even beyond tissue and maybe even species barriers. To prove this, we seeded human primary osteoblasts into a rat kidney bioscaffold. Here, seeded cells spread homogeneously within the matrix and proliferate under dynamic culture conditions. The cells do not only maintain their original phenotype within the matrix, they also show a strong metabolic activity and remodel the biomatrix toward a bone-like extracellular matrix. Thus, the decellularization technique has the ability to become a platform technology for tissue engineering. It potentially offers a universally applicable and easily producible scaffold that addresses the yet unsolved problem of vascularization.

Endothelial differentiation of adipose-derived mesenchymal stem cells is improved by epigenetic modifying drug BIX-01294.

Chromatin remodeling plays an essential role in regulation of gene transcription. Consequently, targeted changes in chromatin may also augment pluripotency of somatic cells. The aim of the present study was to evaluate the effect of epigenetic drug BIX-01294 (BIX), a histone G9a inhibitor, on DNA methylation, expression of pluripotency genes POU5F1 (isoform a), NANOG, KLF4, and CMYC in mesenchymal stem cells, and the ability to increase their differentiation potential into endothelial cells (ECs). Human adipose-derived mesenchymal stem cells (AdMSCs) were isolated from abdominal adipose tissue. Cells were pre-treated with BIX for 48h and further differentiated in endothelial medium for 7 and 14 days. Global DNA methylation was determined by MethyLight application, expression of genes for pluripotency, endothelial and angiogenic markers by SYBRGreen-based real-time PCR, immunocytochemistry, and immunobloting. Following treatment with BIX, DNA methylation status of AdMSCs was significantly reduced by 53% (p=0.008), the expression of POU5F1 and NANOG was increased by 2.2-fold (p=0.016) and 1.5-fold (p<0.001), respectively. Furthermore, BIX pre-treatment improved the differentiation capacity of AdMSCs into ECs and significantly increased expression of several endothelial markers and factors involved in blood vessel formation: VCAM-1, PECAM-1, von Willebrand factor, VEGFR-2, PDGF, and ANG-1 in comparison with AdMSCs without BIX pre-treatment. In the present study we demonstrate that epigenetic modifying drug BIX-01294 is able to increase the ability of AdMSCs to differentiate into ECs engaging DNA and histone methylation. Hence, BIX-01294 might serve as a simple tool to increase the differentiation potential of AdMSCs.

Impact of oxLDL and LPS on C-type natriuretic peptide system is different between THP-1 cells and human peripheral blood monocytic cells.

BACKGROUND: The C-type natriuretic peptide (CNP) has anti-inflammatory, anti-proliferative, and anti-migratory properties. The purpose of this study was to investigate the occurrence of CNP and its receptors (NPR2 and NPR3) in a human monocytic cell line (THP-1 cells) as well as in peripheral blood monocytic cells (PBMC). Impact of both, LPS and human oxLDL on expression pattern of CNP and its receptors shall be studied. METHODS: Cells were cultured in standard medium with or without LPS or oxLDL. Expression levels of CNP, NPR2, NPR3, TNF-α, IL-1ß, IL-6, CD14 and CD68 were measured at baseline, 24h, and 48h. RESULTS: Baseline expression of all analysed genes was significantly higher in PBMC compared to THP-1 cells (all p<0.05). Expression levels of CNP, IL-1ß, IL-6, and CD14 were significantly increased in PBMC following stimulation with LPS. In contrast, in THP-1 cells stimulated by LPS, significant increase in expression was found only for IL-6 (p=0.007). In THP-1 cells, oxLDL increased the expression levels of NPR3, TNF-α, IL-1ß, IL-6, CD14, and CD68 significantly. In contrast, expression level of NPR2 was diminished by oxLDL (p=0.007). In PBMC NPR3 was significantly down-regulated (p=0.002). Treatment with oxLDL for 48h increased NPR2/3-ratio significantly in PBMC (22.5 vs. 4.8, p=0.010). In contrast, in THP-1 cells, NPR2/3-ratio was lowered significantly by oxLDL (0.31 vs. 17.0, p=0.008). CONCLUSION: Treatment with LPS or oxLDL leads to diverging changes in gene expression PBMC and THP-1 cells. With respect to CNP and its receptors, data gained from THP-1 cells should be further validated using naive human peripheral blood monocytes. However, THP-1 cells can serve as a negative control for e.g. future signalling pathway studies related to oxLDL effect on CNP system in monocytes/macrophages.

Neovascularization and angiogenic factors in advanced human carotid artery stenosis.

BACKGROUND: Most atherosclerotic lesions are vascularized, so neovessels may also contribute to plaque progression and vulnerability, but their precise role of neovessels in atherosclerosis is still unknown. The aim of this study was to analyze the possible relationships among neovascularization, relevant angiogenic factors, and plaque vulnerability in patients with advanced carotid artery stenosis. METHODS AND RESULTS: The study group comprised 56 patients (stable: n=28, unstable: n=28) with advanced carotid artery stenosis (>70%). Immunohistochemistry was performed for smooth muscle, endothelial, and inflammatory cells, macrophages, vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), platelet-derived growth factor (PDGF), and angiopoietin-1,-2 (Ang-1,-2). Furthermore, the concentrations of angiogenic factors were measured in serum. Quantitative expression analysis was performed by SYBR-Green-based real-time polymerase chain reaction. Compared with stable carotid lesions, unstable carotid lesions showed 1.8-fold increase in neovascularization (P=0.013), which significantly correlated with accumulation of inflammatory cells (factor 1.9, P<0.001). In unstable lesions, compared with stable lesions, VEGF was 1.7-fold increased (P=0.032) and Ang-1 was 1.9-fold reduced (P=0.029). Furthermore, VEGF was higher in the blood of patients with unstable plaques than in stable plaques (0.32 ± 0.22 vs. 0.22 ± 0.16 ng/ml; P=0.002). Significant correlations were observed between plaque vulnerability, VEGF, neovascularization and inflammatory cells. CONCLUSIONS: Our results show a close association between neovascularization, expression of angiogenic factors, inflammation, and plaque vulnerability in patients with advanced carotid stenosis.

Hepatotropic growth factors protect hepatocytes during inflammation by upregulation of antioxidative systems.

AIM: To investigate effects of hepatotropic growth factors on radical production in rat hepatocytes during sepsis. METHODS: Rat hepatocytes, isolated by collagenase perfusion, were incubated with a lipopolysaccharide (LPS)-containing cytokine mixture of interleukin-1ß, tumor necrosis factor-α and interferon-γ to simulate sepsis and either co-incubated or pre-incubated with hepatotropic growth factors, e.g. hepatocyte growth factor, epidermal growth factor and/or transforming growth factor-α. Cells were analyzed for glutathione levels. Culture supernatants were assayed for production of reactive oxygen intermediates (ROIs) as well as NO(2) (-), NO(3) (-) and S-nitrosothiols. To determine cellular damage, release of aspartate aminotransferase (AST) into the culture medium was analyzed. Activation of nuclear factor (NF)-κB was measured by electrophoretic mobility shift assay. RESULTS: Rat hepatocytes treated with the LPS-containing cytokine mixture showed a significant increase in ROI and nitrogen oxide intermediate formation. AST leakage was not significantly increased in cells treated with the LPS-containing cytokine mixture, independent of growth-factor co-stimulation. However, pretreatment with growth factors significantly reduced AST leakage and ROI formation while increasing cellular glutathione. Application of growth factors did not result in increased NF-κB activation. Pretreatment with growth factors further increased formation of NO(2) (-), NO(3) (-) and S-nitrosothiols in hepatocytes stimulated with LPS-containing cytokine mixture. Thus, we propose that, together with an increase in glutathione increased NO(2) (-), NO(3) (-) formation might shift their metabolism towards non-toxic products. CONCLUSION: Our data suggest that hepatotropic growth factors positively influence sepsis-induced hepatocellular injury by reducing cytotoxic ROI formation via induction of the cellular protective antioxidative systems.