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Proper blood vessel formation requires coordinated changes in endothelial cell polarity and rearrangement of cell-cell junctions to form a functional lumen. One important regulator of cell polarity is the centrosome, which acts as a microtubule organizing center. Excess centrosomes perturb aspects of endothelial cell polarity linked to migration, but whether centrosome number influences apical-basal polarity and cell-cell junctions is unknown. Here, we show that excess centrosomes alter the apical-basal polarity of endothelial cells in angiogenic sprouts and disrupt endothelial cell-cell adherens junctions. Endothelial cells with excess centrosomes had narrower lumens in a 3D sprouting angiogenesis model, and zebrafish intersegmental vessels had reduced perfusion following centrosome overduplication. These results indicate that endothelial cell centrosome number regulates proper lumenization downstream of effects on apical-basal polarity and cell-cell junctions. Endothelial cells with excess centrosomes are prevalent in tumor vessels, suggesting how centrosomes may contribute to tumor vessel dysfunction.

Tumor blood vessels support tumor growth and progression. Centrosomes are microtubule organization centers in cells, and often up to 30% of tumor endothelial cells (ECs) acquire excess (>2) centrosomes. Although excess centrosomes can lead to aneuploidy and chromosome instability in tumor cells, how untransformed ECs respond to excess centrosomes is poorly understood. We found that the frequency of primary human ECs with excess centrosomes was quickly reduced in a p53-dependent manner. Excess centrosomes in ECs were associated with p53 phosphorylation at Ser33, increased p21 levels, and decreased cell proliferation and expression of senescence markers, but independent of DNA damage and apoptosis. Aspects of the senescence-associated phenotype were also observed in mouse ECs that were isolated from tumors with excess centrosomes. Primary ECs with excess centrosomes in vascular sprouts also had elevated Ser33 p53 phosphorylation and expressed senescence markers. Our work demonstrates that nontransformed ECs respond differently to excess centrosomes than do most tumor cells-they undergo senescence in vascular sprouts and vessels, which suggests that pathologic outcomes of centrosome overduplication depend on the transformation status of cells.-Yu, Z., Ruter, D. L., Kushner, E. J., Bautch, V. L. Excess centrosomes induce p53-dependent senescence without DNA damage in endothelial cells., (© FASEB.)

Approximately 30% of tumor endothelial cells have over-duplicated (>2) centrosomes, which may contribute to abnormal vessel function and drug resistance. Elevated levels of vascular endothelial growth factor A induce excess centrosomes in endothelial cells, but how other features of the tumor environment affect centrosome over-duplication is not known. To test this, we treated endothelial cells with tumor-derived factors, hypoxia, or reduced p53, and assessed centrosome numbers. We found that hypoxia and elevated levels of bone morphogenetic protein 2, 6 and 7 induced excess centrosomes in endothelial cells through BMPR1A and likely via SMAD signaling. In contrast, inflammatory mediators IL-8 and lipopolysaccharide did not induce excess centrosomes. Finally, down-regulation in endothelial cells of p53, a critical regulator of DNA damage and proliferation, caused centrosome over-duplication. Our findings suggest that some tumor-derived factors and genetic changes in endothelial cells contribute to excess centrosomes in tumor endothelial cells., Competing Interests: The authors have declared that no competing interests exist.

Blood vessel formation requires dynamic movements of endothelial cells (ECs) within sprouts. The cytoskeleton regulates migratory polarity, and centrosomes organize the microtubule cytoskeleton. However, it is not well understood how excess centrosomes, commonly found in tumor stromal cells, affect microtubule dynamics and interphase cell polarity. Here we find that ECs dynamically repolarize during sprouting angiogenesis, and excess centrosomes block repolarization and reduce migration and sprouting. ECs with excess centrosomes initially had more centrosome-derived microtubules but, paradoxically, fewer steady-state microtubules. ECs with excess centrosomes had elevated Rac1 activity, and repolarization was rescued by blockade of Rac1 or actomyosin blockers, consistent with Rac1 activity promoting cortical retrograde actin flow and actomyosin contractility, which precludes cortical microtubule engagement necessary for dynamic repolarization. Thus normal centrosome numbers are required for dynamic repolarization and migration of sprouting ECs that contribute to blood vessel formation., (© 2016 Kushner et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Supernumerary centrosomes contribute to spindle defects and aneuploidy at mitosis, but the effects of excess centrosomes during interphase are poorly understood. In this paper, we show that interphase endothelial cells with even one extra centrosome exhibit a cascade of defects, resulting in disrupted cell migration and abnormal blood vessel sprouting. Endothelial cells with supernumerary centrosomes had increased centrosome scattering and reduced microtubule (MT) nucleation capacity that correlated with decreased Golgi integrity and randomized vesicle trafficking, and ablation of excess centrosomes partially rescued these parameters. Mechanistically, tumor endothelial cells with supernumerary centrosomes had less centrosome-localized γ-tubulin, and Plk1 blockade prevented MT growth, whereas overexpression rescued centrosome γ-tubulin levels and centrosome dynamics. These data support a model whereby centrosome-MT interactions during interphase are important for centrosome clustering and cell polarity and further suggest that disruption of interphase cell behavior by supernumerary centrosomes contributes to pathology independent of mitotic effects., (© 2014 Kushner et al.)