DYRK1B inhibition exerts senolytic effects on endothelial cells and rescues endothelial dysfunctions.

Aging is the major risk factor for chronic disease development. Cellular senescence is a key mechanism that triggers or contributes to age-related phenotypes and pathologies. The endothelium, a single layer of cells lining the inner surface of a blood vessel, is a critical interface between blood and all tissues. Many studies report a link between endothelial cell senescence, inflammation, and diabetic vascular diseases. Here we identify, using combined advanced AI and machine learning, the Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1B (DYRK1B) protein as a possible senolytic target for senescent endothelial cells. We demonstrate that upon induction of senescence in vitro DYRK1B expression is increased in endothelial cells and localized at adherens junctions where it impairs their proper organization and functions. DYRK1B knock-down or inhibition restores endothelial barrier properties and collective behavior. DYRK1B is therefore a possible target to counteract diabetes-associated vascular diseases linked to endothelial cell senescence.

PIP4K2B is mechanoresponsive and controls heterochromatin-driven nuclear softening through UHRF1.

Phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) are stress-regulated phosphoinositide kinases able to phosphorylate PtdIns5P to PtdIns(4,5)P2. In cancer patients their expression is typically associated with bad prognosis. Among the three PIP4K isoforms expressed in mammalian cells, PIP4K2B is the one with more prominent nuclear localisation. Here, we unveil the role of PIP4K2B as a mechanoresponsive enzyme. PIP4K2B protein level strongly decreases in cells growing on soft substrates. Its direct silencing or pharmacological inhibition, mimicking cell response to softness, triggers a concomitant reduction of the epigenetic regulator UHRF1 and induces changes in nuclear polarity, nuclear envelope tension and chromatin compaction. This substantial rewiring of the nucleus mechanical state drives YAP cytoplasmic retention and impairment of its activity as transcriptional regulator, finally leading to defects in cell spreading and motility. Since YAP signalling is essential for initiation and growth of human malignancies, our data suggest that potential therapeutic approaches targeting PIP4K2B could be beneficial in the control of the altered mechanical properties of cancer cells.

Anisotropic topographies restore endothelial monolayer integrity and promote the proliferation of senescent endothelial cells.

Thrombogenicity remains a major issue in cardiovascular implants (CVIs). Complete surficial coverage of CVIs by a monolayer of endothelial cells (ECs) prior to implantation represents a promising strategy but is hampered by the overall logistical complexity and the high number of cells required. Consequently, extensive cell expansion is necessary, which may eventually lead to replicative senescence. Considering that micro-structured surfaces with anisotropic topography may promote endothelialization, we investigated the impact of gratings on the biomechanical properties and the replicative capacity of senescent ECs. After cultivation on gridded surfaces, the cells showed significant improvements in terms of adherens junction integrity, cell elongation, and orientation of the actin filaments, as well as enhanced yes-associated protein nuclear translocation and cell proliferation. Our data therefore suggest that micro-structured surfaces with anisotropic topographies may improve long-term endothelialization of CVIs.

The transcription factor PREP1(PKNOX1) regulates nuclear stiffness, the expression of LINC complex proteins and mechanotransduction.

Mechanosignaling, initiated by extracellular forces and propagated through the intracellular cytoskeletal network, triggers signaling cascades employed in processes as embryogenesis, tissue maintenance and disease development. While signal transduction by transcription factors occurs downstream of cellular mechanosensing, little is known about the cell intrinsic mechanisms that can regulate mechanosignaling. Here we show that transcription factor PREP1 (PKNOX1) regulates the stiffness of the nucleus, the expression of LINC complex proteins and mechanotransduction of YAP-TAZ. PREP1 depletion upsets the nuclear membrane protein stoichiometry and renders nuclei soft. Intriguingly, these cells display fortified actomyosin network with bigger focal adhesion complexes resulting in greater traction forces at the substratum. Despite the high traction, YAP-TAZ translocation is impaired indicating disrupted mechanotransduction. Our data demonstrate mechanosignaling upstream of YAP-TAZ and suggest the existence of a transcriptional mechanism actively regulating nuclear membrane homeostasis and signal transduction through the active engagement/disengagement of the cell from the extracellular matrix.

The Role of Tricellulin in Epithelial Jamming and Unjamming via Segmentation of Tricellular Junctions.

Collective cellular behavior in confluent monolayers supports physiological and pathological processes of epithelial development, regeneration, and carcinogenesis. Here, the attainment of a mature and static tissue configuration or the local reactivation of cell motility involve a dynamic regulation of the junctions established between neighboring cells. Tricellular junctions (tTJs), established at vertexes where three cells meet, are ideally located to control cellular shape and coordinate multicellular movements. However, their function in epithelial tissue dynamic remains poorly defined. To investigate the role of tTJs establishment and maturation in the jamming and unjamming transitions of epithelial monolayers, a semi-automatic image-processing pipeline is developed and validated enabling the unbiased and spatially resolved determination of the tTJ maturity state based on the localization of fluorescent reporters. The software resolves the variation of tTJ maturity accompanying collective transitions during tissue maturation, wound healing, and upon the adaptation to osmolarity changes. Altogether, this work establishes junctional maturity at tricellular contacts as a novel biological descriptor of collective responses in epithelial monolayers.