Biology subjects

Lytvyn, M.

Publications and source records attributed to Lytvyn, M..

4 recordsLinked to original sources

bioRxiv2025

Genetic mosaics reveal mechanisms of resistance to VEGF signaling loss during angiogenesis

The VEGF ligand and its main receptor VEGFR2 are considered to be essential for endothelial differentiation, proliferation, sprouting and survival. Blood vessels cannot form in growing embryos or tissues when VEGF/VEGFR2 signalling is compromised in all cells. The Anti-VEGF blocking antibody is one of the most widely used antibodies in the clinics, blocking angiogenesis in cancer, wound healing or in ischemic diseases. However, vascular resistance to Anti-VEGF has been reported. Here we used iFlpMosaics and iSuRe-HadCre to induce and track genetic mosaics of endothelial cells (ECs) lacking VEGFR2 during the entire embryonic and postnatal development. Surprisingly, Vegfr2KOECs adapt, proliferate normally and compete with wild-type cells over time, ultimately forming a substantial portion of the capillary network in most organs. We found that Vegfr2KO ECs are not able to sprout during the first wave of angiogenesis in most tissues, because it is highly VEGF-dependent and favours the growth and mobilization of wild-type ECs. However, due to their inability to respond to VEGF, over time Vegfr2KOECs become dominant in veins, which provide for a long term and continuous source of ECs for subsequent waves of VEGF-independent angiogenesis. Comparative scRNAseq analysis of ECs with acute and long-term loss of VEGFR2, revealed both common and organ-specific molecular mechanisms of adaptation and resistance to VEGF signalling loss. This included significant endothelial venousization and the upregulation of ligands for VEGFR1 and VEGFR3. VEGFR1 only partially compensated for VEGFR2 loss in capillary ECs, whereas VEGFR3 only compensated in arterial ECs. Loss of the three VEGF receptors did not compromise venous growth. This work changes our understanding of the role of VEGF signaling and its receptors in angiogenesis and reveals mechanisms of adaptation and resistance to their loss.

developmental biology↗

bioRxiv2023

iFlpMosaics: A method for the ratiometric induction and high-throughput comparative analysis of mutant and wildtype cells

To understand gene function, it is necessary to compare cells carrying the mutated target gene with normal cells. In most biomedical studies, the cells being compared are in different mutant and control animals and therefore do not experience the same epigenetic changes and tissue microenvironment. The experimental induction of genetic mosaics is essential to determine a gene cell-autonomous function and to model the etiology of diseases caused by somatic mutations. Current technologies used to induce genetic mosaics in mice lack either accuracy, throughput or barcoding diversity. Here, we present a large set of new genetic tools and mouse lines that enable Flp recombinase-dependent ratiometric induction and single-cell clonal tracking of multiple fluorescently labeled wildtype and Cre-mutant cells within the same time window and tissue microenvironment. The labeled cells can be profiled by multispectral imaging or by FACS and scRNA-seq. This technology facilitates the induction and analysis of genetic mosaics in any cell type and for any given single or combination of floxed genes. iFlpMosaics enables a more accurate understanding of how induced genetic mutations affect the biology of single cells during tissue development, homeostasis, and disease.

cell biology↗

bioRxiv2023

Incongruence between transcriptional and vascular pathophysiological cell states

The Notch pathway is a major regulator of transcriptional specification and vascular biology. Previous studies have suggested that targeting the ligand Dll4 or the Notch-receptors results in similar molecular and angiogenesis outcomes. Here, we analyzed single and compound genetic mutants for all Notch signaling members and found very significant differences in the way ligands and receptors regulate vascular homeostasis. Loss of Notch receptors, leads to minor vascular pathology featuring hypermitogenic MAPK-driven cell-cycle arrest and senescence. In contrast, loss of Dll4 triggers a strong Myc-driven switch towards cell proliferation and sprouting and major organ pathology. Targeting of Myc completely suppressed the proliferative and tip-cell angiogenic states induced by Dll4 loss-of-function, however, this did not avoid vascular pathology. Only VEGF blockade prevented the pathology induced by Dll4 loss, but without fully suppressing its transcriptional and metabolic programs. This study shows incongruence between single-cell transcriptional states and adult vascular phenotypes and related pathophysiology.

physiology↗

bioRxiv2020

SARS-CoV-2 protein Nsp1 alters actomyosin cytoskeleton and phenocopies arrhythmogenic cardiomyopathy-related PKP2 mutant

Mutations in desmosomal Plakophilin-2 (PKP2) are the most prevalent drivers of arrhythmogenic cardiomyopathy (ACM) and a common cause of sudden cardiac death in young athletes. However, partner proteins that elucidate PKP2 cellular mechanism to understand cardiac dysfunction in ACM are mostly unknown. Here we identify the actin-based motor proteins Myh9 and Myh10 as key PKP2 interactors, and demonstrate that the expression of the ACM-related PKP2 mutant R735X alters actin fiber organization and cell mechanical stiffness. We also show that SARS-CoV-2 Nsp1 protein acts similarly to this known pathogenic R735X mutant, altering the actomyosin component distribution on cardiac cells. Our data reveal that the viral Nsp1 hijacks PKP2 into the cytoplasm and mimics the effect of delocalized R735X mutant. These results demonstrate that cytoplasmic PKP2, wildtype or mutant, induces the collapse of the actomyosin network, since shRNA-PKP2 knockdown maintains the cell structure, validating a critical role of PKP2 localization in the regulation of actomyosin architecture. The fact that Nsp1 and PKP2 mutant R735X share similar phenotypes also suggests that direct SARS-CoV-2 heart infection could induce a transient ACM-like disease in COVID-19 patients, which may contribute to right ventricle dysfunction, observed in patients with poor survival prognosis. HighlightsThe specific cardiac isoform Plakophilin-2a (PKP2) interacts with Myh9 and Myh10. PKP2 delocalization alters actomyosin cytoskeleton component organization. SARS-CoV-2 Nsp1 protein hijacks PKP2 from the desmosome into the soluble fraction where it is downregulated. Viral Nsp1 collapses the actomyosin cytoskeleton and phenocopies the arrhythmogenic cardiomyopathy-related mutant R735X.

molecular biology↗