Rapamycin and Suramin Effects on TNF‐⍺‐Mediated Mast Cell and Brain Microvascular Endothelial Cell Dysfunction

Chronic blood-brain barrier (BBB) disruption due to impaired function of brain microvascular endothelial cells (BMECs) is commonly observed in neuroinflammatory and neurodegenerative conditions. Current treatment approaches are generally limited in their capacity to reduce this dysfunction, with the Akt/mTOR/GSK pathway modulator rapamycin showing recent promise in ameliorating neuroinflammatory BBB dysfunction. Understanding the role of early, cellular level BMEC dysfunction, particularly in the context of interplay with immune cells involved in neuroinflammation, such as mast cells (MCs), is important for identifying targets for therapeutic intervention related to BBB disruption. In the present work, we investigate primary human BMECs and human MC line HMC-1.2 dysfunction in response to inflammatory insult with TNF-α and paracrine interactions, with an emphasis on the Akt/mTOR/GSK pathway-an upstream regulator of angiogenesis and MC activation-and associated extracellular and intracellular cytokine production and oxidative stress. We further compare alterations in BMEC-MC paracrine inflammatory crosstalk in response to Akt/mTOR/GSK pathway modulator suramin (100 µM) relative to rapamycin (250 nM). In monoculture, TNF-α stimulation significantly increased oxidative stress-assessed through measuring PGE2-extracellularly in BMECs. Similarly, proangiogenic and pro-inflammatory cytokine and chemokine secretion was increased in both TNF-α stimulated BMEC and MC monocultures. Additionally, TNF-α stimulation increased BMEC levels of the Akt/mTOR/GSK pathway intermediates p-p70S6K and p-RPS6 and MC levels of p-GSK3α and p-GSK3β. Coculture of TNF-α stimulated BMECs and MCs resulted in a modest increase in extracellular PGE2, and effects on extracellular cytokine/chemokine levels were primarily limited to increases in pro-inflammatory CCL2, CCL3 and CCL5 relative to TNF-α-stimulated BMEC monoculture. In contrast, the levels of intracellular cytokines in MCs increased 2-100 fold with TNF-α-stimulated coculture, concomitant with a decrease in MC p-p70S6K levels. Rapamycin treatment of TNF-⍺-stimulated cocultures resulted a modest increase in extracellular PGE2 as well as decreases in extracellular chemokines CCL2 and CCL3. In contrast, suramin treatment significantly decreased extracellular PGE2, GM-CSF, CCL2, and CCL5 while markedly increasing the BBB-stabilizing PDGF-BB. However, suramin also increased intracellular BMEC levels of multiple pro-inflammatory cytokines. Neither rapamycin nor suramin improved the intracellular inflammatory profile of cocultured MCs, indicating that MC activation had not been resolved by either treatment.