Effects of Senescent Human Endothelial Cells on Neuronal Viability and Neurite Outgrowth Are Not IL‐8‐ or Serpin E1‐Dependent

Abstract

It is estimated that more than 50% of adults over 80 will develop dementia, including Alzheimer’s disease (AD), and the primary risk factor for dementia is aging. Aging provides the substrate for disease through increased inflammation and cellular dysfunction, which in turn sets the stage for neurodegenerative disease. In a previous study we validated replicative senescence, a model of cell senescence that permits the study of cells as they progress to senescence, in human coronary artery endothelial cells (HCAEC). Here, we tested the hypothesis that senescent endothelial cells, which accumulate with aging and release senescence‐associated secretory phenotype (SASP) and extracellular vesicles, create a pro‐inflammatory environment that adversely affects the viability and morphology of neurons, which are endpoints implicated in AD. We categorized the phenotypes of HCAEC by passage number and identified them as EP‐ early passage (non‐senescent), ES‐ early senescence and LS‐ late senescence. HCAEC at these varying stages of senescence were co‐cultured with LUHMES cells, a human neuronal cell line. Co‐culturing HCAEC LS with LUHMES significantly decreased LUHMES viability, while co‐culturing HCAEC ES with LUHMES decreased the number of neurites extended by LUHMES in 24h and decreased the length of LUHMES’ neurites in 48h, when compared to LUHMES grown in the absence of HCAEC for the same period of time. HCAEC EP or LS co‐cultured with LUHMES did not affect the number or length of the LUHMES’ neurites, when compared to LUHMES grown in the absence of HCAEC. Using a cytokine array containing probes for 105 analytes, we detected 17 inflammatory mediators released by the co‐cultures of HCAEC (EP, ES and LS) with LUHMES. Of these 17 mediators, 14 were also found at supernatants of HCAEC (EP, ES and LS) grown in the absence of LUHMES, and none of the analytes were detected in supernatants from LUHMES grown in the absence of HCAEC. Statistical analysis of the intensity of the positive reaction for inflammatory mediators in the array was run for all the 17 positive analytes, and only IL‐8 and Serpin E1 release into the supernatants of the co‐cultures were significantly increased in co‐cultures of LUHMES and senescent HCAEC (ES or LS), when compared to the levels of the same mediators released by co‐cultures of LUHMES and EP HCAEC. However, exposing LUHMES to IL‐8 and Serpin E1, in the absence of HCAEC, did not affect LUHMES viability, the number or length of LUHMES’ neurites or LUHMES’ NF‐kB activation. The results from the LUHMES and HCAEC co‐cultures study supports a role for aging endothelial cells in the toxicity and morphological remodeling of the surrounding neurons, which may contribute to the development and progression of neurodegenerative diseases. However, our data exclude IL‐8 and Serpin E1 as direct independent mediators of senescent HCAEC on neuronal cytotoxicity and neurite remodeling.