Astrocytic cellular senescence in the subfornical organ during Ang-II-induced hypertension
Authors: Dow Samantha A. , Young Colin N. 
Published: May 2023

Hypertension affects nearly half of US adults and is a leading cause for heart attack and stroke. Angiotensin II (Ang-II) is a well-recognized driver of hypertension, particularly through its sympathoexcitatory actions within the central nervous system (CNS). As a peptide hormone, Ang-II is too large to cross into the brain and acts at circumventricular regions lacking a blood-brain-barrier, particularly the forebrain subfornical organ (SFO). While Ang-II has been shown to drive pro-hypertensive cellular stressors in the SFO (i.e. oxidative and endoplasmic reticulum stress), it is unclear how these mechanisms translate into long-term alterations at the cellular level. Intriguingly, stress-associated pathways can induce cellular senescence and the senescence-associated secretory phenotype (SASP). Chronic senescence/SASP leads to detrimental changes in cell metabolism, macromolecule damage, and a pro-inflammatory environment capable of propagating senescence. We previously demonstrated cellular senescence at the mRNA level, including robust upregulation of the key senescence mediator p16 ( CDKN2A), in the SFO during Ang-II-induced hypertension. However, the cell type(s) that undergoes senescence in response to Ang-II remains unknown. Thus, we hypothesized, that Ang-II would elicit cellular senescence at the protein level and furthermore that this would occur in both SFO neurons and astrocytes. To test this, male C57Bl/6J mice were implanted with subcutaneous osmotic minipumps for chronic infusion of Ang II (600 ng/kg/min) for 0 or 14 days (n=4-5/group), and brains were collected and prepared for immunohistochemistry (IHC). Immunohistochemical analysis revealed a marked ~46% increase in p16 intensity in the SFO following 14 days of Ang-II infusion (Integrated density: 1.00±0.08 vs 1.46±0.24, Day 0 vs Day 14, p=0.07). The increase in p16 integrated density occurred throughout rostral to caudal SFO, most notably in the medial region. We next sought to characterize the SFO cell types that undergo cellular senescence using double immunohistochemistry for p16 and either neurons (NeuN) or astrocytes (GFAP). Following 14 days of Ang-II administration and histological procedures, colocalization analysis was performed using IMARIS software. When first examining neuronal populations, extremely low to no co-localization with p16 was found (p16/NeuN: Pearson’s correlation coefficient=-0.074, Mander’s overlap coefficient=0.071). However, p16 was very highly co-localized with astrocytes (p16/GFAP: Pearson’s correlation coefficient=0.545, Mander’s overlap coefficient=0.992). Together, these findings indicate that: 1) p16-associated cellular senescence occurs at the protein level in the SFO during Ang-II-induced hypertension; and 2) p16 is primarily upregulated in SFO astrocytes, but not neurons, during hypertension development. R01DK117007, R01HL141393, F31HL164059 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.