Ercc1 DNA repair deficiency results in vascular aging characterized by VSMC phenotype switching, ECM remodeling, and an increased stress response.

Cardiovascular diseases are the number one cause of death globally. The most important determinant of cardiovascular health is a person's age. Aging results in structural changes and functional decline of the cardiovascular system. DNA damage is an important contributor to the aging process, and mice with a DNA repair defect caused by Ercc1 deficiency display hypertension, vascular stiffening, and loss of vasomotor control. To determine the underlying cause, we compared important hallmarks of vascular aging in aortas of both Ercc1Δ/- and age-matched wildtype mice. Additionally, we investigated vascular aging in 104 week old wildtype mice. Ercc1Δ/- aortas displayed arterial thickening, a loss of cells, and a discontinuous endothelial layer. Aortas of 24 week old Ercc1Δ/- mice showed phenotypical switching of vascular smooth muscle cells (VSMCs), characterized by a decrease in contractile markers and a decrease in synthetic markers at the RNA level. As well as an increase in osteogenic markers, microcalcification, and an increase in markers for damage induced stress response. This suggests that Ercc1Δ/- VSMCs undergo a stress-induced contractile-to-osteogenic phenotype switch. Ercc1Δ/- aortas showed increased MMP activity, elastin fragmentation, and proteoglycan deposition, characteristic of vascular aging and indicative of age-related extracellular matrix remodeling. The 104 week old WT mice showed loss of cells, VSMC dedifferentiation, and senescence. In conclusion, Ercc1Δ/- aortas rapidly display many characteristics of vascular aging, and thus the Ercc1Δ/- mouse is an excellent model to evaluate drugs that prevent vascular aging in a short time span at the functional, histological, and cellular level.

Induction of Fenestrae in Human Induced Pluripotent Stem Cell-Derived Endothelial Cells for Disease Modeling.

The endothelial linings of capillaries, such as those in the kidney and small intestines, possess fenestrae that facilitate fluid and exchange of small molecules. Alterations in the size and number of endothelial fenestrae have been implicated in the pathogenesis of various diseases. The re-creation of fenestrated endothelium using human induced pluripotent stem cells (hiPSCs) provides a promising avenue to investigate the involvement of fenestrae in disease mechanisms and pharmacodynamics. In this project, we aim to induce the formation of fenestrae in nonfenestrated hiPSCs-derived endothelial cells (hiPSC-ECs). Vascular endothelial growth factor A (VEGFA) and phorbol myristate acetate (PMA) were used as inducers of fenestrae in hiPSC-ECs. The assessment of fenestrae formation included gene-expression analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and immunofluorescent staining. Endothelial monolayer functionality was evaluated by dextran permeability assays. Stimulation with VEGFA and PMA significantly induced expression of the diaphragmed fenestrae-associated marker, plasmalemmal vesicle-associated protein (PLVAP), in hiPSC-ECs at the mRNA, and protein levels. SEM analysis revealed VEGFA- and PMA-induced fenestrae structures on the cell membrane of hiPSC-ECs. The increased membrane localization of PLVAP visualized by TEM and immunofluorescent staining supported these findings. The induced fenestrated endothelium in hiPSC-ECs demonstrated selective passage of small solutes across a confluent monolayer with intact cell junctions, confirming functional competence. In conclusion, we present a novel methodology for inducing and regulating fenestrated endothelium in hiPSC-ECs. This innovative approach paves the way for the development of fenestrated microvasculature in human organ-on-a-chip systems, enabling complex disease modeling and physiologically relevant investigations of pharmacodynamics.