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Cardiac fibroblasts support endothelial cell proliferation and sprout formation but not the development of multicellular sprouts in a fibrin gel co-culture model.

ABSTRACT: A primary impediment to cardiac tissue engineering lies in the inability to adequately vascularize the constructs to optimize survival upon implantation. During normal angiogenesis, endothelial cells (ECs) require a support cell to form mature patent lumens and it has been demonstrated that pericytes, vascular smooth muscle cells and mesenchymal stem cells (MSCs) are all able to support the formation of mature vessels. In the heart, cardiac fibroblasts (CFs) provide important electrical and mechanical functions, but to date have not been sufficiently studied for their role in angiogenesis. To study CFs role in angiogenesis, we co-cultured different concentrations of various cell types in fibrin hemispheres with appropriate combinations of their specific media, to determine the optimal conditions for EC growth and sprout formation through DNA analysis, flow cytometry and immunohistology. ECs proliferated best when co-cultured with CFs and analysis of immunohistological images demonstrated that ECs formed the longest and most numerous sprouts with CFs as compared to MSCs. However, ECs were able to produce more multicellular sprouts when in culture with the MSCs. Moreover, these effects were dependent on the ratio of support cell to EC in co-culture. Overall, CFs provide a good support system for EC proliferation and sprout formation; however, MSCs allow for more multicellular sprouts, which is more indicative of the in vivo process.

SUBMITTER: Twardowski RL

PROVIDER: S-EPMC3969781 | biostudies-other | 2014 May

REPOSITORIES: biostudies-other

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Cardiac fibroblasts support endothelial cell proliferation and sprout formation but not the development of multicellular sprouts in a fibrin gel co-culture model.

Twardowski Rachel L RL Black Lauren D LD

Annals of biomedical engineering 20140117 5

A primary impediment to cardiac tissue engineering lies in the inability to adequately vascularize the constructs to optimize survival upon implantation. During normal angiogenesis, endothelial cells (ECs) require a support cell to form mature patent lumens and it has been demonstrated that pericytes, vascular smooth muscle cells and mesenchymal stem cells (MSCs) are all able to support the formation of mature vessels. In the heart, cardiac fibroblasts (CFs) provide important electrical and mech ...[more]

PMID: 24435656

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Project description:Purpose:To identify transcriptional changes in human endothelial cells (HUVEC) both during normal sprouting angiogenesis (different time points) and with Slug overexpression (different Slug expression), in a 3D fibrin-gel bead angiogenesis Methods: Angiogenic endothelial cell with different level of Slug expression (normal and overexpressing) at different stage (early/sprouting and late/lumen formation) were harvested from a 3D fibrin-gel bead angiogenesis assay. Their transcriptom profile was generated by Next Generation RNA-sequencing as single reads, performed on an Illumina HiSeq 2500 platform, using three donor HUVEC lines.At least 30 million reads per sample were sequenced. Quality control was performed on data using FASTQC(v. 0.11.2), and reads were trimmed using Trimmomatic(v.0.32) with Illumina TruSeq adapter sequences using a PHRED quality score 15 and minimum length 20 bases. Trimmed reads were then aligned to the human hg19 reference genome using the Ensembl GRCh37 annotations and post processed using Tophat2(v.2.0.12), Bowtie2(v.2.2.3) and Samtools(v.0.1.19). Expression levels were quantified both with FPKM (Fragment per kilobase per million mapped reads) using Cufflinks(v. 2.1.1) and with raw counts using HTSeq(v.0.6.1p1.). Differential analysis was done using DESeq2 and significant genes were considered (p-value < 0.05, 2 fold change). K-means clustering was performed to extract dynamic gene clusters based on differential expression between GFP and SLUG knockdown time-points. Gene set enrichment analysis (GSEA) was performed on expressed genes to identify significant gene sets across sample comparisons. Additional gene ontology analysis was performed for each cluster and reported using Metascape. Clustering was performed in R and heatmaps were generated using JavaTree. Results: Using several different gene expression analysis platforms, we found consistent and significant changes, during both normal angiogenesis and with Slug overexpression, in genes related to proliferation, migraion, cell morphology/polarity and junction regulations, all of which are consistent with a partial-EMT program. We also found changes in Notch signaling components in Slug overexpressing cells. Changes in specific genes related to EMT and Notch signaling were validated with both qPCR and WB. Conclusion: This is the first comprehensive transcriptome analysis in human angiogenic EC during normal and excessive sprouting induced by Slug expression in an in vitro 3D angiogenesis assay system. We conclude that a partial-EndoMT is necessary for normal angiogenesis and that endothelial Slug expresion level regulates Notch signaling to determines at least partly the extent of EC's commitment towards a mesenchymal identity.

Dataset Information

Cardiac fibroblasts support endothelial cell proliferation and sprout formation but not the development of multicellular sprouts in a fibrin gel co-culture model.

Publications

Cardiac fibroblasts support endothelial cell proliferation and sprout formation but not the development of multicellular sprouts in a fibrin gel co-culture model.

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