The mitotic regulator PLK1 modulates mouse endothelial homeostasis and in vitro angiogenesis

Trabajo presentado en la Endothelial Cell Phenotypes in Health and Disease Gordon Research Conference, celebrada en Castelldefels (España) el 26 de junio de 2022.Pioneering studies in the 70¿ and 80¿ of the last century showed that the endothelium of higher organisms is a very low proliferative tissue. Moreover, even during angiogenesis, despite implying the formation of new tissue, cell proliferation has to be tightly controlled. A higher proliferation rate in ECs leads to aberrant angiogenesis processes. Likewise, it has been also shown that hypoxia-induced angiogenesis leads to EC proliferation downregulation. This dichotomy, new tissue formation with reduced levels of proliferation, indicates that cell cycle and division genes need to be tightly regulated in their expression and function in ECs. How cell cycle and mitotic genes interfere in angiogenesis is yet unknown, and might open new avenues to understand tumoral angiogenesis, and the endothelial impact of the newly described therapeutic strategies that target the cell cycle. Our laboratory tries to address this question by evaluating the role of the mitotic gene PLK1 in ECs function and during angiogenesis. Plk1 kinase is a master regulator of the cell cycle, considered an oncogene and a bona fide cancer target, as its inhibition leads to cell death. As there are already Plk1 inhibitors in advanced clinical trials, it is important to address any possible physiological role of Plk1 in higher organisms, and putative pathology-associated toxicities upon Plk1 inhibition. Although not explored yet, we believe that Plk1 can modulate angiogenesis based on the following facts: (i) PLK1 gene expression can be regulated by hypoxia, a critical molecular signaling for angiogenesis. (ii) PLK1, despite being a proliferative gene, can be expressed in vascular tissues such as arteries, modulating the actomyosin cytoskeleton activity of VSMC, and controlling vascular homeostasis. (iii) Plk1 interacts, in tumoral cells, with important angiogenic factors such as PTEN, thus might modulate EC proliferation/differentiation in a PTEN-dependent manner. We have performed in vivo studies, showing that mice with a precise Plk1 conditional genetic depletion in endothelial cells, die within a few weeks harboring severe alterations in kidney and liver tissue. In addition, by performing in vitro angiogenesis assays using mouse embryoid bodies, we have preliminary data showing that PLK1 inhibition alters the angiogenesis process

Metabolic labelling of RNA uncovers the contribution of transcription and decay rates on hypoxia-induced changes in RNA levels

© 2020 Tiana et al.Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. However, most transcriptomic studies do not distinguish the relative contribution of transcription, RNA processing and RNA degradation processes to cellular homeostasis. Here we used metabolic labeling followed by massive parallel sequencing of newly transcribed and preexisting RNA fractions to simultaneously analyze RNA synthesis and decay in primary endothelial cells exposed to low oxygen tension. We found that changes in transcription rates induced by hypoxia are the major determinant of changes in RNA levels. However, degradation rates also had a significant contribution, accounting for 24% of the observed variability in total mRNA. In addition, our results indicated that hypoxia led to a reduction of the overall mRNA stability from a median half‐life in normoxia of 8.7 hours, to 5.7 hours in hypoxia. Analysis of RNA content per cell confirmed a decrease of both mRNA and total RNA in hypoxic samples and that this effect is dependent on the EGLN/HIF/TSC2 axis. This effect could potentially contribute to fundamental global responses such as inhibition of translation in hypoxia. In summary, our study provides a quantitative analysis of the contribution of RNA synthesis and stability to the transcriptional response to hypoxia and uncovers an unexpected effect on the latter.This work was supported by Ministerio de Ciencia e Innovación (Spanish Ministry of Science and Innovation, MICINN) [SAF2014-53819-R to Luis del Peso and Benilde Jimenez; SAF2017-88771-R to Luis del Peso and Benilde Jimenez].Peer reviewe

The SIN3A histone deacetylase complex is required for a complete transcriptional response to hypoxia

Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. To identify genes regulated by hypoxia at the transcriptional level, we pulse-labeled HUVEC cells with 4-thiouridine and sequenced nascent transcripts. Then, we searched genome-wide binding profiles from the ENCODE project for factors that correlated with changes in transcription and identified binding of several components of the Sin3A co-repressor complex, including SIN3A, SAP30 and HDAC1/2, proximal to genes repressed by hypoxia. SIN3A interference revealed that it participates in the downregulation of 75% of the hypoxia-repressed genes in endothelial cells. Unexpectedly, it also blunted the induction of 47% of the upregulated genes, suggesting a role for this corepressor in gene induction. In agreement, ChIP-seq experiments showed that SIN3A preferentially localizes to the promoter region of actively transcribed genes and that SIN3A signal was enriched in hypoxia-repressed genes, prior exposure to the stimulus. Importantly, SINA3 occupancy was not altered by hypoxia in spite of changes in H3K27ac signal. In summary, our results reveal a prominent role for SIN3A in the transcriptional response to hypoxia and suggest a model where modulation of the associated histone deacetylase activity, rather than its recruitment, determines the transcriptional output.Ministerio de Ciencia e Innovacion (Spanish Ministry of Science and Innovation, MICINN) [SAF2011 24225 to L.d.P., SAF2014–53819-R to L.d.P., B.J.]; Canadian Institutes of Health Research (CIHR) [MOP-82875 to W.W.).]; Natural Sciences and Engineering Research Council of Canada (NSERC) [RGPIN355532–10 to W.W.W.]; National Institutes of Health [1R01GM084875 to W.W.W.]; CIHR Fellowship (to R.W.H.); Michael Smith Foundation for Health Research Fellowship (to R.W.H.); Caja Madrid Foundation for Visiting Professor Fellowship (to L.d.P). Funding for open access charge: Spanish Ministry of Science and Innovation, MICINN, [SAF2014-53819-R].Peer reviewe

Hypoxia compensates cell cycle arrest with progenitor differentiation during angiogenesis

Angiogenesis, the main mechanism that allows vascular expansion for tissue regeneration or disease progression, is often triggered by an imbalance between oxygen consumption and demand. Here, by analyzing changes in the transcriptomic profile of endothelial cells (ECs) under hypoxia we uncovered that the repression of cell cycle entry and DNA replication stand as central responses in the early adaptation of ECs to low oxygen tension. Accordingly, hypoxia imposed a restriction in S‐phase in ECs that is mediated by Hypoxia‐Inducible Factors. Our results indicate that the induction of angiogenesis by hypoxia in Embryoid Bodies generated from murine Stem Cells is accomplished by the compensation of decreased S‐phase entry in mature ECs and differentiation of progenitor cells. This conditioning most likely allows an optimum remodeling of the vascular network. Identification of the molecular underpinnings of cell cycle arrest by hypoxia would be relevant for the design of improved strategies aimed to suppress angiogenesis in pathological contexts where hypoxia is a driver of neovascularization.This work was supported by Ministerio de Ciencia e Innovación (Spanish Ministry of Science and Innovation, MICINN) SAF2014‐53819‐R to Luis del Peso and Benilde Jimenez; SAF2017‐ 88771‐R to Luis del Peso and Benilde Jimenez and SAF2015‐71381‐R to Maria C. Marin and by Junta de Castilla y Leon LE021P17 to Maria C. Marin. Laura Maeso‐Alonso is supported by a predoctoral scholarship from the Asociación Española contra el Cáncer (AECC).Peer reviewe