Chromatin-modifying agents convert fibroblasts to OCT4+ and VEGFR-2+ capillary tube-forming cells

Rationale The human epigenome is plastic. The goal of this study was to address if fibroblast cells can be epigenetically modified to promote neovessel formation. Methods and results Here, we used highly abundant human adult dermal fibroblast cells (hADFCs) that were treated with the chromatin-modifying agents 5-aza-2\u27-deoxycytidine and trichostatin A, and subsequently subjected to differentiation by activating Wnt signaling. Our results show that these epigenetically modified hADFCs increasingly expressed β-catenin, pluripotency factor octamer-binding transcription factor-4 (OCT4, also known as POU5F1), and endothelial cell (EC) marker called vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Fetal Liver Kinase-1). In microscopic analysis, β-catenin localized to cell-cell contact points, while OCT4 was found to be localized primarily to the nucleus of these cells. Furthermore, in a chromatin immunoprecipitation experiment, OCT4 bound to the VEGFR-2/FLK1 promoter. Finally, these modified hADFCs also transduced Wnt signaling. Importantly, on a two-dimensional (2D) gel substrate, a subset of the converted cells formed vascular network- like structures in the presence of VEGF. Conclusion Chromatin-modifying agents converted hADFCs to OCT4+ and VEGFR-2+ capillary tubeforming cells in a 2D matrix in VEGF-dependent manner

The Role of KLF4 and KLF2 Crosstalk in Endothelial to Mesenchymal Transition and Pulmonary Fibrosis

Transcription factors KLF4 and KLF2 are both known to be highly expressed in the vasculature and described to play a role in pulmonary physiology. However, the mechanism by which KLF4 and KLF2 might regulate each other has not been investigated. To address this gap, the relationship of Klf4 with Klf2 was examined using the in vitro and in vivo loss-of-function approaches as they relate to overall lung architecture and lung physiology. Key findings include the upregulation and autoregulation of KLF2 in the absence of KLF4, as well as loss of endothelial cell (EC) identity and transition of these cells into mesenchymal cell state, accompanied by fibrosis, alveolar enlargement, and alterations in lung physiology. Importantly, intravenous administration of alpha-1-antitrypsin (A1AT) ameliorated this pathological phenotype in the mouse model. Together, these results suggest a novel mechanism by which KLF4 depletion causes pathological EndoMT, dysregulation leading to fibrotic state, as well as a potential therapeutic approach

Chromatin-modifying agents convert fibroblasts to OCT4+ and VEGFR-2+ capillary tube-forming cells

<div><p>Rationale</p><p>The human epigenome is plastic. The goal of this study was to address if fibroblast cells can be epigenetically modified to promote neovessel formation.</p><p>Methods and results</p><p>Here, we used highly abundant human adult dermal fibroblast cells (hADFCs) that were treated with the chromatin-modifying agents 5-aza-2'-deoxycytidine and trichostatin A, and subsequently subjected to differentiation by activating Wnt signaling. Our results show that these epigenetically modified hADFCs increasingly expressed β-catenin, pluripotency factor octamer-binding transcription factor-4 (OCT4, also known as POU5F1), and endothelial cell (EC) marker called vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Fetal Liver Kinase-1). In microscopic analysis, β-catenin localized to cell-cell contact points, while OCT4 was found to be localized primarily to the nucleus of these cells. Furthermore, in a chromatin immunoprecipitation experiment, OCT4 bound to the <i>VEGFR-2/FLK1</i> promoter. Finally, these modified hADFCs also transduced Wnt signaling. Importantly, on a two-dimensional (2D) gel substrate, a subset of the converted cells formed vascular network-like structures in the presence of VEGF.</p><p>Conclusion</p><p>Chromatin-modifying agents converted hADFCs to OCT4+ and VEGFR-2+ capillary tube-forming cells in a 2D matrix in VEGF-dependent manner.</p></div

DataSheet2_A requirement for Krüppel Like Factor‐4 in the maintenance of endothelial cell quiescence.PDF

Rationale and Goal: Endothelial cells (ECs) are quiescent and critical for maintaining homeostatic functions of the mature vascular system, while disruption of quiescence is at the heart of endothelial to mesenchymal transition (EndMT) and tumor angiogenesis. Here, we addressed the hypothesis that KLF4 maintains the EC quiescence.Methods and Results: In ECs, KLF4 bound to KLF2, and the KLF4-transctivation domain (TAD) interacted directly with KLF2. KLF4-depletion increased KLF2 expression, accompanied by phosphorylation of SMAD3, increased expression of alpha-smooth muscle actin (αSMA), VCAM-1, TGF-β1, and ACE2, but decreased VE-cadherin expression. In the absence of Klf4, Klf2 bound to the Klf2-promoter/enhancer region and autoregulated its own expression. Loss of EC-Klf4 in RosamT/mG::Klf4fl/fl::Cdh5CreERT2 engineered mice, increased Klf2 levels and these cells underwent EndMT. Importantly, these mice harboring EndMT was also accompanied by lung inflammation, disruption of lung alveolar architecture, and pulmonary fibrosis.Conclusion: In quiescent ECs, KLF2 and KLF4 partnered to regulate a combinatorial mechanism. The loss of KLF4 disrupted this combinatorial mechanism, thereby upregulating KLF2 as an adaptive response. However, increased KLF2 expression overdrives for the loss of KLF4, giving rise to an EndMT phenotype.</p

Human gene specific primers used for q-RT-PCR data shown in S1 Fig.

<p>Human gene specific primers used for q-RT-PCR data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176496#pone.0176496.s001" target="_blank">S1 Fig</a>.</p

DataSheet1_A requirement for Krüppel Like Factor‐4 in the maintenance of endothelial cell quiescence.PDF

Rationale and Goal: Endothelial cells (ECs) are quiescent and critical for maintaining homeostatic functions of the mature vascular system, while disruption of quiescence is at the heart of endothelial to mesenchymal transition (EndMT) and tumor angiogenesis. Here, we addressed the hypothesis that KLF4 maintains the EC quiescence.Methods and Results: In ECs, KLF4 bound to KLF2, and the KLF4-transctivation domain (TAD) interacted directly with KLF2. KLF4-depletion increased KLF2 expression, accompanied by phosphorylation of SMAD3, increased expression of alpha-smooth muscle actin (αSMA), VCAM-1, TGF-β1, and ACE2, but decreased VE-cadherin expression. In the absence of Klf4, Klf2 bound to the Klf2-promoter/enhancer region and autoregulated its own expression. Loss of EC-Klf4 in RosamT/mG::Klf4fl/fl::Cdh5CreERT2 engineered mice, increased Klf2 levels and these cells underwent EndMT. Importantly, these mice harboring EndMT was also accompanied by lung inflammation, disruption of lung alveolar architecture, and pulmonary fibrosis.Conclusion: In quiescent ECs, KLF2 and KLF4 partnered to regulate a combinatorial mechanism. The loss of KLF4 disrupted this combinatorial mechanism, thereby upregulating KLF2 as an adaptive response. However, increased KLF2 expression overdrives for the loss of KLF4, giving rise to an EndMT phenotype.</p

Chromatin modification strategy, timeline of experiments, and gene expression analyses.

<p><b>A</b>) The strategy and timeline used for all the experiments described in this study. <b>B</b>) Descriptions of the four experimental groups: (<b>a</b>) day-2 (d2) dish is control untreated cells; (<b>b</b>) day-3 (d3) dish received one dose of Aza + TSA for 24 hours before the cells were used; (<b>c</b>) day-4 (d4) received a second dose of Aza + TSA for 24 hours before use; (d) day-5 (d5) received a third dose of Aza + TSA and TDG 24 hours before use. <b>C</b>) Total mRNAs prepared from these cells were subjected to q-RT-PCR using gene-specific primers for <i>18S RNA</i> (control), <i>VEGFR-2/FLK1</i>, <i>OCT4</i>, and <i>LPP3</i>. Experiments were carried out three times, with triplicate mRNAs. * p<0.05 or ** p<0.01 vs. control <i>18S RNA</i> from same group.</p

A model of the conversion process of fibroblast cells into tube-forming cells.

<p>hADFCs are mature differentiated cells with highly condensed chromatin containing histones tightly bound to DNAs. However, upon treatment with Aza and TSA for two or three cycles, histone acetylation increases, DNA methylation decreases, the chromatin becomes relaxed, so that the transcription factors could activate the expression of mesodermal-endothelial genes. These events mediate the formation of tube-like structures in 2D Matrigel in a VEGF-dependent manner.</p

Microscopic analysis of β-catenin, OCT4, N-cadherin and vWF in epigenetically modified cells.

<p>hADFCs were plated on coverslips, left untreated or treated with epigenetic modifiers as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176496#pone.0176496.g001" target="_blank">Fig 1A</a>, and stained with anti-β-catenin (A-D, green) or anti-OCT4 (E-P, green) or anti-vWF (Q-T, red). Representative microscopic images of: <b>A&E</b>) d2 control untreated cells; <b>B&F</b>) d3 cells treated once with Aza + TSA; <b>C&G</b>) d4 cells received two doses of Aza + TSA; <b>D&H</b>) d5, treated with a third dose of Aza + TSA and TDG. White arrowheads indicate β-catenin; magenta arrowheads show nuclear accumulation of OCT4. <b>I-L</b>) d5 cells treated with a third dose of Aza + TSA and TDG were stained with DAPI (blue), TRITC-phalloidin (red), and OCT4 (green). Magenta arrows (<b>L</b>) indicate nuclear localization. <b>M-P</b>) d5 cells treated with a third dose of Aza + TSA and TDG were stained with DAPI (blue), OCT4 (green), and N-cadherin (red). <b>Q-T</b>) d2, d3, d4 and d5 hADFC were stained with DAPI (blue) and vWF (red). Magnification is as shown.</p