Journal: Scientific Reports

Article Title: Hox genes are involved in vascular wall-resident multipotent stem cell differentiation into smooth muscle cells

doi: 10.1038/srep02178

Figure Lengend Snippet: (A) Equal cell numbers of VW-MPSCs were seeded and cultured in NGM and transfected with Control (non-silencing) or HOXB7, HOXC6 and HOXC8, siRNA. After 4 days cell numbers were analysed. Data are presented as mean ± SD from 3 independent experiments performed in duplicates each. (B) For analysing the sprouting capacity transfected cells were embedded in GFR-Matrigel as 3D-spheroids. In-gel sprouting was quantified 48 hours after embedding. The data represent the mean cumulative length of all cord-like sprouts growing from 10 individual spheroids per experimental group. The figure shows the results of 1 of 3 independent experiments with similar results. (B) ** p ≤ 0.005.

Article Snippet: Control A and B siRNA, HOXC8, HOXC6 and HOXB7 siRNA were from Santa Cruz (Santa Cruz, USA).

Techniques: Cell Culture, Transfection, Control

Journal: Scientific Reports

Article Title: Hox genes are involved in vascular wall-resident multipotent stem cell differentiation into smooth muscle cells

doi: 10.1038/srep02178

Figure Lengend Snippet: Results are presented as scheme depicting numbers of genes significantly altered upon siRNA silencing (mean signal control (average) compared to mean signal HOXC6 (average) as well as HOXB7 (average) and HOXC8 (average)). For each probe set three (Control, HOXC6, HOXB7) and two (HOXC8) Affymetrix® DNA chips were used. Histone genes as well as TAGLN which were represented on the chips are listened and fold induction (siRNA treatment/control) was calculated. For complete list see online.

Article Snippet: Control A and B siRNA, HOXC8, HOXC6 and HOXB7 siRNA were from Santa Cruz (Santa Cruz, USA).

Techniques: Control

Journal: Scientific Reports

Article Title: Hox genes are involved in vascular wall-resident multipotent stem cell differentiation into smooth muscle cells

doi: 10.1038/srep02178

Figure Lengend Snippet: (A) VW-MPSCs were transfected with Control (non-silencing) or HOXC8, HOXC6 and HOXB7 siRNA in single or in indicated combinations. After 4 days cells were harvested and subjected for mRNA isolation and Real-Time RT-PCR quantification of histone gene expression. Data are presented as mean ± SEM from 4 independent experiments performed in duplicates each. (B) Histone H1, H2B, H2A protein levels were detected by Western blot. Equal protein amounts (50 μg, whole cell lysate) were loaded. Beta-actin was included as a loading control. (C) Double-immunofluorescence analyses on freshly isolated CD44(+) cells plated on glass cover slips were performed for HOXC6 and HOXC8 (green) and H1 (red). H1 was detected in the nuclei of cultured VW-MPSC and was found to be partially co-localized with the HOXC6 and HOXC8 protein. Hoechst 33242 iodide was used for nuclei staining.

Article Snippet: Control A and B siRNA, HOXC8, HOXC6 and HOXB7 siRNA were from Santa Cruz (Santa Cruz, USA).

Techniques: Transfection, Control, Isolation, Quantitative RT-PCR, Gene Expression, Western Blot, Immunofluorescence, Cell Culture, Staining

Journal: Scientific Reports

Article Title: Hox genes are involved in vascular wall-resident multipotent stem cell differentiation into smooth muscle cells

doi: 10.1038/srep02178

Figure Lengend Snippet: (A) VW-derived MPSCs were transfected with Control (non-silencing) or HOXB7, HOXC6 and HOXC8, siRNA. After 4 days cells were harvested and subjected for mRNA isolation and qRT-PCR quantification of the late SMC differentiation marker TAGLN. (B) Increased TAGLN as well as CNN1 expression were confirmed at the protein level after gene silencing of HOXB7, HOXC6 and HOXC8 in VW-MPSCs. (C, D) Silencing H1 mRNA expression directly decreases gene expression of TAGLN and CNN1. Data are presented as mean ± SEM from 4 independent experiments performed in duplicates each * p ≤ 0.05.

Article Snippet: Control A and B siRNA, HOXC8, HOXC6 and HOXB7 siRNA were from Santa Cruz (Santa Cruz, USA).

Techniques: Derivative Assay, Transfection, Control, Isolation, Quantitative RT-PCR, Marker, Expressing, Gene Expression

Journal: Scientific Reports

Article Title: Hox genes are involved in vascular wall-resident multipotent stem cell differentiation into smooth muscle cells

doi: 10.1038/srep02178

Figure Lengend Snippet: VW-derived MPSCs were transfected with Control (non-silencing, HOXC8, HOXC6, HOXB7 or H1 siRNA alone or in indicated combinations and subjected for immunofluorescence staining for the proliferation marker Ki67 (A). Cell cycle distribution was analysed by Western blot analysis of Cyclin B1 and D1 expression (B) as well as by Nicoletti staining (C). Two-color flow cytometric analysis was used to quantify cells actively synthesizing DNA and their position in G1, G0/1, S, G2/M phase of the cell cycle. Representative images for three independent experiments are shown. Data are presented as mean from 3 independent experiments.

Article Snippet: Control A and B siRNA, HOXC8, HOXC6 and HOXB7 siRNA were from Santa Cruz (Santa Cruz, USA).

Techniques: Derivative Assay, Transfection, Control, Immunofluorescence, Staining, Marker, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Hox genes are involved in vascular wall-resident multipotent stem cell differentiation into smooth muscle cells

doi: 10.1038/srep02178

Figure Lengend Snippet: Genomic DNA was isolated from control (non-silencing siRNA) and silencing siRNA (HOXC8, HOXC6, HOXB7) transfected VW-MPSC, subjected for bisulfite conversion and finally sequenced using specific primers for the modified DNA which do not contain any CpG sites in their sequence and were generated around the predicted CpG islands. Predicted promoter regions of the human gene TAGLN (ENSG00000149591) were emphasized. Org: Original sequence; BSC bisulfite modified sequence (MethPrimer results); HOXB7, HOXC6, HOXC8, Con (non-silencing control) sequence analysis after bisulfite conversion of siRNA treated genomic DNA isolates; ++ CpG sites (for display, assume all CpG sites are methylated); non-CpG 'C' converted to ‘T’; * similar to BSC (original); # C vs T; TATA box (at position 368) in bold red; promoter start at position 500 encircled. Arrows indicate positions which were altered in non silencing siRNA treated cells as compared to original sequence (BSC), whereas siRNA (HOXB7 and HOXC6) treatment results in sequences similar to original sequence (BSC). Underlined nucleotides of control sequence indicate additional CpG sites.

Article Snippet: Control A and B siRNA, HOXC8, HOXC6 and HOXB7 siRNA were from Santa Cruz (Santa Cruz, USA).

Techniques: Isolation, Control, Transfection, Modification, Sequencing, Generated, Methylation

Journal: Scientific Reports

Article Title: Hox genes are involved in vascular wall-resident multipotent stem cell differentiation into smooth muscle cells

doi: 10.1038/srep02178

Figure Lengend Snippet: (A) CD44(+) VW-MPSCs are preferentially resident in the adventitial “vasculogenic zone” of the vessel wall. Without mobilization from the niche they express HOXB7 and HOXC6 at high level compared to hAoSMCs. These HOX genes suppress the expression of TAGLN in VW-MPSCs, an essential factor of early SMC differentiation. This mechanism probably accounts for keeping the VW-MPSCs quiescent in the adventitial niche. In contrast, silencing of HOX genes alter the CpG methylation of TAGLN promotor resulting in increased TAGLN expression which induces the VW-MPSC differentiation into SMC/pericytes. HOX gene silencing also results in increased expression of H1 in VW-MPSCs. H1 silencing in turn results in reduced expression of both, TAGLN and CNN1 as well as HOXB7 and HOXC6. Whether H1 directly enhances the expression TAGLN and CNN1 remains to be investigated. (B) Signals released, e.g. by endothelial dysfunction, may hypothetically induce the mobilization of CD44(+) cells from the niche toward the intima accompanied by a modification of HOX gens regulating their differentiation into mature SMC. VW-MPSCs differentiation into SMC by mechanisms identified here might be of clinical relevance contributing to new vessel stabilization, neointima formation and tumor vascularization.

Article Snippet: Control A and B siRNA, HOXC8, HOXC6 and HOXB7 siRNA were from Santa Cruz (Santa Cruz, USA).

Techniques: Expressing, CpG Methylation Assay, Modification