Endothelial-specific telomerase inactivation causes telomere-independent cell senescence and multi-organ dysfunction characteristic of aging

Abstract

It has remained unclear how aging of endothelial cells (EC) contributes to pathophysiology of individual organs. Cell senescence results in part from inactivation of telomerase (TERT). Here, we analyzed mice with Tert knockout specifically in EC. Tert loss in EC induced transcriptional changes indicative of senescence and tissue hypoxia in EC and in other cells. We demonstrate that EC-Tert-KO mice have leaky blood vessels. The blood-brain barrier of EC-Tert-KO mice is compromised, and their cognitive function is impaired. EC-Tert-KO mice display reduced muscle endurance and decreased expression of enzymes responsible for oxidative metabolism. Our data indicate that Tert-KO EC have reduced mitochondrial content and function, which results in increased dependence on glycolysis. Consistent with this, EC-Tert-KO mice have metabolism changes indicative of increased glucose utilization. In EC-Tert-KO mice, expedited telomere attrition is observed for EC of adipose tissue (AT), while brain and skeletal muscle EC have normal telomere length but still display features of senescence. Our data indicate that the loss of Tert causes EC senescence in part through a telomere length-independent mechanism undermining mitochondrial function. We conclude that EC-Tert-KO mice is a model of expedited vascular senescence recapitulating the hallmarks aging, which can be useful for developing revitalization therapies.

Keywords: accelerated aging; endothelial; hypoxia; knockout; metabolism; mitochondrial disease; senescence; telomerase.

FIGURE 1

Endothelial cells (EC) Tert KO results in AT EC telomere attrition and senescence. (a) q‐RT‐PCR reveals Tert expression reduction in mG+ cells FACS‐sorted from SAT of EC‐Tert‐KO mice (8 months old). (b) Telo‐FISH reveals shorter telomeres (lower red TelC‐Cy3 signal) in mG+ cells (green outline arrow) from VAT of EC‐Tert‐KO mice (12 months old), whereas nuclear TelC‐Cy3 signal in mT+ cells (insets) is comparable. (c) q‐PCR on DNA from VAT and SAT mG+ lineage cells reveals shorter telomeres in HFD‐fed EC‐Tert‐KO mice at 8 months of age. Real‐time PCR data are normalized to data for a single copy gene. (d) SA‐β‐gal staining reveals senescence in VAT of EC‐Tert‐KO mice (8 months old). (e) SA‐β‐gal staining reveals senescence in cultured mG+ cells from SAT of EC‐Tert‐KO mice (8‐month‐old). Left: plate wells; middle: brightfield micrograph; right: fluorescence of the same area. EC colonies (mG+) are intermixed with stromal cells (mT+), which results in yellow color. (f) q‐RT‐PCR reveals higher Cdkn2a expression (normalized to 18S RNA) in mG+ cells FACS‐sorted from SAT of EC‐Tert‐KO mice (8 months old). (g) Flow cytometry on SVF recovered 3 days after EdU injection comparing incorporation into mG+ and mT+ cells in VAT, and SAT of HFD‐fed mice at 9 months of age. EdU fluorescence: 647 nm channel, side scatter is used for separation. (h) Primary culture of SVC from VAT and SAT of 7‐month‐old mice 2 days after plating at identical density. Note reduced proliferation and the large size of mG+ cells from EC‐Tert‐KO mice. For all data, shown are mean ± SEM (error bars). *p < 0.05 (two‐sided Student's t‐test). Scale bar = 100 μm.

FIGURE 2

Tert KO causes endothelial cells (EC) dysfunction, mis‐differentiation, and vessel leakiness. (a) Anti‐GFP IF analysis in tissue sections, with IB4 counterstaining EC, reveals a lack of mG+ cells in blood vessels in VAT of EC‐Tert‐KO mice fed HFD (9 months). (b) Data quantification from (a) based on IB4 binding and mG expression in the vasculature. (c) Self‐organization of SAT mG+/mT+ SVC into stromal‐vascular networks showing a defect of Tert‐KO EC. (d) Whole mount of SAT showing increased frequency of EC‐derived adipocytes in AT of EC‐Tert‐KO mice. Graph: Data quantification. (e) Primary culture of SVC from SAT after adipogenesis induction showing increased differentiation of Tert‐KO mG+ cells into adipocytes containing lipid droplets. Graph: Data quantification. (f) VAT 0.5 h after intravenous injection of Evans blue and subsequent systemic perfusion showing increased dye retention in EC‐Tert‐KO mice. Graph: Dye retention quantification. (g) q‐RT‐PCR reveals higher Hif1a expression (normalized to 18S RNA) in mG+ cells FACS‐sorted from SAT of EC‐Tert‐KO mice (8 months old). (h) Extracts from VAT 0.5 h after injection of hypoxyprobe analyzed by PAGE. β‐Actin immunoblotting: Loading control. Note increased hypoxyprobe retention in VAT of EC‐Tert‐KO males (M) and females (F). Graph: Data quantification: Mean ± SEM. For all data, shown are mean ± SEM (error bars). *p < 0.05 (two‐sided Student's t‐test). Scale bar = 50 μm.

FIGURE 3

High calorie diet reveals EC‐Tert‐KO WAT abnormality and metabolism dysfunction. (a) The time course of body weight change in mice fed HFD. (b) Body composition measured in males fed chow or HFD for 3‐months. (c) AT resected depots from female mice, whole and minced, show reduced SAT and VAT adiposity of EC Tert KO. (d) Daily food consumption of mice on normal chow and HFD. (e) Plasma‐free fatty acid concentration increase after isoproterenol administration, not observed in EC‐Tert‐KO mice. (f) Plasma insulin concentration is lower in EC‐Tert‐KO mice. (g) Plasma glucose concentration in mice fed chow or HFD, lower in EC‐Tert‐KO mice. (h) Cold tolerance test: body temperature measured after placement at 4°C. (i) Calorimetric measurement of energy expenditure and respiratory exchange ratio (RER), calculated as VCO₂/VO₂, over 2 days. (j) Mean values for all timepoints in (i). (k) Glucose tolerance test (GTT) after 6 months of chow and HFD feeding. (l) Insulin tolerance test (ITT) after 6 months of chow and HFD feeding. For all data, shown are mean ± SEM (error bars). *p < 0.05 (two‐sided Student's t‐test). Scale bar = 50 μm.

FIGURE 4

Tert KO impairs brain ECs and cognitive function without telomere attrition. (a) q‐PCR on DNA from brain mG+ lineage cells of EC‐Tert‐KO mice at 10 months of age. Real‐time PCR data are normalized to data for a single copy gene. (b) Telo‐FISH reveals comparable telomere length (red TelC‐Cy3 signal) in mG+ cells (green outline arrow) from EC‐Tert‐KO and WT mice. (c) mG and mT fluorescence in hypothalamus sections reveals normal vasculature in EC‐Tert‐KO mice. (d) Primary culture of cells from the brains 2 days after plating at identical density. Note reduced proliferation and larger size of mG+ EC‐Tert‐KO cells. (e) Open field test does not reveal behavioral abnormality in EC‐Tert‐KO mice. (f) Novel object recognition test (NORT) reveals memory impairment in EC‐Tert‐KO males and females. Lipopolysaccharide (LPS) injection, impairing memory, was used as a positive control. (g) Brains 0.5 h after tail vein injection of Evans blue and subsequent systemic perfusion showing increased dye retention in EC‐Tert‐KO mice. Graph: calorimetric Evans blue quantification. For all data, shown are mean ± SEM (error bars). *p < 0.05 (two‐sided Student's t‐test). Scale bar = 50 μm.

FIGURE 5

Tert KO impairs muscle ECs and function without telomere attrition. (a) q‐PCR on DNA from combined quadriceps (Quad) and gastrocnemius (GM) muscle mG+ lineage cells of EC‐Tert‐KO mice at 10 months of age. Real‐time PCR data are normalized to data for a single copy gene. (b) Flow cytometry on hindlimb skeletal muscle cells recovered 3 days after EdU injection comparing incorporation into mG+ and mT+ cells. (c) Fluorescence analysis of mG+ and mT+ cells in Quad muscle. Graph: data quantification based on vascular mG expression. (d) Grip strength, measured for forelimb and hindlimb of EC‐Tert‐KO and WT littermates (N = 5). (e) Quantification of data from e for 10 daily runs of N = 5 WT and N = 5 EC‐Tert‐KO mice, showing reduced physical endurance of EC‐Tert‐KO mice, reflected in Joules of work performed (weight (kg) × speed (m/min) × time (min) × incline (degree) × 9.8 m/s²). (f) A snapshot of WT and EC‐Tert‐KO littermates running on a treadmill (arrows: direction) illustrating reduced fatigue resistance capacity in EC‐Tert‐KO mice. (g) IF analysis of muscle fiber types in GM muscle. (h) Immunohistochemistry on GM and Quad reveals decreased succinate dehydrogenase (SDH1) expression in the muscle fibers of EC‐Tert‐KO mice. (i) SDH1 expression quantification from (h). For all data, shown are mean ± SEM (error bars). *p < 0.05 (two‐sided Student's t‐test). Scale bar = 50 μm.

FIGURE 6

Tert‐KO endothelial cells (EC) have reduced mitochondrial function and increased glycolysis. (a) Primary culture of mixed mT/mG SVC from SAT of 12‐month‐old mice 2 days after plating at identical density stained with Mitotracker (red). Note that intracellular Mitotracker signal (arrow) is reduced in mG+ Tert‐KO EC. Graph: data quantification. (b) Mitochondrial DNA content, based on the ratio of ND1 to HK2 gene expression measured by q‐RT‐PCR in mG+ cells FACS‐sorted from SAT of EC‐Tert‐KO mice (8 months old). (c) q‐RT‐PCR reveals lower expression of mitochondrial respiration genes PGC1a (normalized to 18S RNA). (d) q‐RT‐PCR reveals higher expression of glycolysis effectors HK1 (p = 0.03) and LDH (p = 0.009) (normalized to 18S RNA). (e) IF analysis of GLUT1 expression showing its induction colocalized with mG+ blood vessels in SAT and skeletal muscle sections of EC‐Tert‐KO mice. Graphs: data quantification. (f) Seahorse XF Cell Mito Stress Assay revealing decreased basal and induced oxygen consumption rate (OCR). (g) Increased lactate concentration in plasma of EC‐Tert‐KO mice. For all data, shown are mean ± SEM (error bars). *p < 0.05 (two‐sided Student's t‐test). Scale bar = 50 μm.