BACKGROUND: Endothelial cells (ECs) are primed to respond to various signaling cues. For example, TGF (transforming growth factor)-β has major effects on EC function and phenotype by driving ECs towards a more mesenchymal state (ie, triggering endothelial to mesenchymal activation), a dynamic process associated with cardiovascular diseases. Although transcriptional regulation triggered by TGF-β in ECs is well characterized, post-transcriptional regulatory mechanisms induced by TGF-β remain largely unknown. METHODS: Using RNA interactome capture, we identified global TGF-β driven changes in RNA-binding proteins in ECs. We investigated specific changes in the RNA-binding patterns of hnRNP H1 (heterogeneous nuclear ribonucleoprotein H1) and Csde1 (cold shock domain containing E1) using RNA immunoprecipitation and overlapped this with RNA-sequencing data after knockdown of either protein for functional insight. Using a modified proximity ligation assay, we visualized the specific interactions between hnRNP H1 and Csde1 and target RNAs in situ both in vitro and in mouse heart sections. RESULTS: Characterization of TGF-β–regulated RBPs (RNA-binding proteins) revealed hnRNP H1 and Csde1 as key regulators of the cellular response to TGF-β at the post-transcriptional level, with loss of either protein-promoting mesenchymal activation in ECs. We found that TGF-β drives an increase in binding of hnRNP H1 to its target RNAs, offsetting mesenchymal activation, but a decrease in Csde1 RNA-binding, facilitating this process. Both, hnRNP H1 and Csde1, dynamically bind and regulate specific subsets of mRNAs related to mesenchymal activation and endothelial function. CONCLUSIONS: Together, we show that RBPs play a key role in the endothelial response to TGF-β stimulation at the post-transcriptional level and that the RBPs hnRNP H1 and Csde1 serve to maintain EC function and counteract mesenchymal activation. We propose that TGF-β profoundly modifies RNA-protein interaction entailing feedback and feed-forward control at the post-transcriptional level, to fine-tune mesenchymal activation in ECs.

Cardiac fibrosis plays a crucial role in heart failure, but research in this field remains challenging due to heterogeneity of cardiac fibroblast populations. In the past years, several Cre-drivers have been proposed for cardiac fibroblast-specific deletion of floxed genes in adult mouse research models. Here, we evaluated whether Tcf21-iCre, a tamoxifen-inducible Cre recombinase (iCre) under the endogenous Tcf21 promoter, was able to successfully target two floxed loci (Hypoxia inducible factor 1a (Hif1a) and the fluorescent reporter locus mTmG) after intraperitoneal tamoxifen injections. Six-week-old mice received intraperitoneal tamoxifen injections on five consecutive days to induce Cre activity, and the recombination efficiency was measured in isolated fibroblasts via qPCR, Western Blot and fluorescence microscopy. Recombination efficiency was very low, with minimal reduction in expression of Hif1a. For the mTmG locus, both bi-allelic and mono-allelic recombination was detected, with mono-allelic events suggesting low Cre activity even within Tcf21-expressing fibroblasts. The addition of experimental models of heart failure with reduced or preserved ejection fraction as pro-fibrotic stimuli or an inclusion of an additional round or higher doses of tamoxifen injections did not improve recombination efficiency of the targeted alleles. Thus, Tcf21-iCre targets only a small proportion of fibrogenic fibroblasts following i.p. tamoxifen injection, limiting its usability in loss-of-function studies that require broad fibroblast coverage and highlighting the need for alternative Cre-drivers or delivery strategies to facilitate antifibrotic therapy development.