Mild Heat Pretreatment and Serum From Sedentary Humans Subjected to Passive Heat Therapy Protect Endothelial Cells Against Stress from Hypoxia‐Reoxygenation
Authors: Brunt Vienna E , Needham Karen Weidenfeld , Comrada Lindan N , Minson Christopher T 
Published: November 2023

Impaired stress resistance is a hallmark of disease and dysfunction in many clinical populations, including primary aging and cardiovascular diseases. Repeated exposure to passive heat stress (‘heat therapy’) has widespread physiological benefits, including cellular protection against novel stressors. Both increased heat shock protein (HSP) expression as a direct effect of increases in body core temperature and upregulation of circulating factors may impart this protection.

Purpose

We tested the ability of both mild heat pre‐treatment and serum from human subjects who had undergone 8 weeks of heat therapy to protect against cellular stress following hypoxia‐reoxygenation (H/R), a model of ischemic events, in cultured endothelial cells.

Methods

Serum was collected from 10 young, sedentary subjects before (0wk) and after 8 weeks of passive heat therapy (immersion in 40.5°C bath 4–5× per week to increase rectal temperature to 38.5–39.0°C for 60 min per session). Purchased human umbilical vein endothelial cells were incubated for 24h at 37°C (control), 39°C (heat pre‐treatment), or at 37°C with 10% serum from heat therapy subjects. Cells were collected before and after incubation at 1% O2 for 16h (hypoxia), followed by 20% O2 for 4h (reoxygenation) and assessed for markers of cell stress. Data are presented as mean±S.E. % change from basal conditions (before H/R).

Results

In control cells, H/R increased nuclear NFkB p65 protein (i.e. activation) by +106±38%, increased IL‐6 release into the supernatant by +37±8% (normalized to total protein), and increased superoxide production by +272±45% (cell permeable fluorescent stain). Heat pre‐treatment prevented H/R‐induced NFkB activation (−3±12%, p=0.04 vs. control) and attenuated superoxide production (+141±11%, p=0.01), but had no effect on IL‐6 production (+40±14%, p=0.85). Exposure to sera from human subjects who had undergone heat therapy prevented H/R‐induced NFkB activation (0wk: +301±125% vs. 8wk: +13±31%, p=0.045) and attenuated IL‐6 release (0wk: 172±57 pg/mg total protein vs. 8wk: 110±40 pg/mg, p=0.048), but had no effect on superoxide production (0wk: +47±14% vs. 8wk: +124±70%, p=0.24). H/R also decreased cytoplasmic hemeoxygenase‐1 (HO‐1) protein by −25±8%, a heat shock protein (Hsp32) that can suppress NFkB activation. Interestingly, HO‐1 protein increased following H/R in cells pre‐treated with heat (+45±27%, p=0.02) or exposed to heat therapy sera (0wk: −11±15% vs. 8wk: +38±24%, p=0.06).

Conclusions

Markers of cellular oxidative and inflammatory stress associated with H/R were attenuated by both increases in temperature (to levels similar to core temperature increases observed in humans undergoing heat therapy) and heat therapy‐induced changes in circulating factors. These data suggest heat therapy is capable of imparting stress resistance via both direct heat (presumably via HSPs) and humoral factors.

Support or Funding Information

Supported by AHA Grant #14PRE20380300 and the Eugene and Clarissa Evonuk Memorial Foundation