4 Discussions
In this study, the features of vascular endothelial glycocalyx under
stimulation of heat stress and LPS corroborated vascular inflammatory
injury and coagulation disorder. For the first time, the study showed
that heat stroke triggered endothelial glycocalyx degradation and
further induced oxidative damage and apoptosis of HPMEC, which impaired
its ability of resisting inflammatory injury and maintaining vascular
permeability.
HSPG was a major element of glycocalyx, which played important roles in
endothelial cell homeostatic signal based on their unique structures and
interactions with both the intra- and extracellular environments.
Decreased HSPG prompted endothelial glycocalyx degradation and vascular
disease. 21 In this study, we detected lower HSPG
level in HPMEC under heat stress and LPS compared with controls
(Figure 1A~C ). Heparn sulfate (HS) and
syndecans-1 (SDC-1) were principal components of glycocalyx which
documented the erosion of glycocalyx. 28 Previous
study reported that increased blood levels of SDC-1 as a signal for
glycocalyx degradation. 29 In the present study,
upregulated supernatant levels of SDC-1 and HS(Figure 1D )
derived from cells treated with heat stress and LPS were found, which
suggested that glycocalyx was possibly degraded in heat stroke.
Interestingly, these changes were more obviously in cells treated with
HPSE and UFH, which suggested that greater damage to the glycocalyx
resulted in more severe degradation.
Earlier studies demonstrated that heat stress contributed to injury and
apoptosis of endothelial cells by an activation of inflammation, which
played an important role in organ injury secondary to heat stroke.30 Given this timeline, our results showed that heat
stress and LPS induced endothelial cells apoptosis morphologically,
which were more significantly in HPSE-treated cells (Figure 2 ).
A recent study found that endothelium apoptosis may be induced and
aggravated by endothelial glycocalyx injury31.
Collectively, our results suggested that the activation of endothelium
apoptosis may be highly associated with endothelial glycocalyx damage as
a result of heat stroke.
Inflammation and coagulation played a critical role in
pathophysiological basis of tissues/ organs damage secondary to heat
stroke. 32 Moreover, abnormal structure and function
of endothelium contributed to exacerbate inflammation through increasing
vascular permeability and promoting adhesion of inflammatory cells, as
well as producing massive inflammatory mediators and procoagulants.
However, the development of inflammation and coagulation after heat
stroke-induced glycocalyx injury was still to be elucidated. Given this
association, it will be essential to explore the relationship between
inflammation and glycocalyx damage in addition to endothelium.
TNF-α was known to promote inflammation via upregulating the expression
of adhesion factors on the surface of endothelium, such as VCAM-1 and
ICAM-1. 33 IL-6 played a promoted role in development
of inflammation, which was related to intracellular cascade signaling
transduction induced by binding to specific receptors, and further
magnified the effects of TNF-α. 34, 35 Moreover, IL-6
played strong roles in promoting inflammation in the pathogenesis of
MODS secondary to heat stroke. 35 vWF and ET-1 were
associated with the function of promoting coagulation.36 Endothelium was effective in constricting blood
vessels and reducing the leakage via releasing ET-1.36 In addition, endothelium damage was mediated by
inflammatory factors which contributed to enlarging endothelial space
and further aggravating inflammatory response. 37These may explain why the expression of TNF-α, IL-6, vWF and ET-1 showed
increased actions in glycocalyx injury in combination stimulation of
heat stress and LPS, typically in HPSE-treated cells (Figure
3 ). Taken together, these results might imply that heat stroke-induced
glycocalyx degradation enhanced the activation of inflammation and
coagulation.
E-selectin and VCAM-1 were the important symbols to reflect injury of
endothelial glycocalyx and endothelium, which also played critical roles
in the spread of inflammation via mediating the adhesion between
leukocyte and inflammatory cells. 24, 38, 39 In our
results, increased expressions of VCAM-1 and E-selectin were
significantly observed in endothelium treated with heat stress and LPS,
especially in cells suffered from HPSE (Figure 3C ). These
findings were consistent with those revealed a significant upregulation
of E-selectin in injured vascular endothelial glycocalyx,40, 41, which confirmed a dysfunction of endothelial
cells inhibiting in inflammation. Occludin was effective in preventing
endothelium from leakage, localized in the tight junctions between
cellular preferentially. 25, 26 In the present study,
western blot analysis revealed that the expression of Occludin was
aggravated by injured endothelial glycocalyx in HPMEC (Figure
3B ~C). These data suggested that heat stroke possibly
triggered endothelial glycocalyx damage, which resulted in endothelial
tight junction injury. Decreased Occludin may be associated with
widening gaps between endothelial cells, which aggravated the vascular
leakage possibly. Our data indicated that heat stroke-induced glycocalyx
damage enhanced adhesion proteins expression, and further led to
pathophysiological changes including increased vascular permeability and
leukocyte adhesion. 42 It is consequently hypothesized
that endothelial glycocalyx damage may be th key pathophysiological
basic of dysfunction of endothelium and inflammatory damages induced by
heat stroke.
ROS was widely involved in signaling transduction and life process of
cells, however, excessive ROS may lead to diseases via inducing
oxidative stress in mitochondria. 43 Further studies
showed that under heat stroke, increased inflammatory factors mediated
iNOS to generate NO, and further generated excessive ROS, which was
associated with heat stroke-induced ALI. 44 In the
present study, the relationship between intracellular oxidative stress
and heat stroke was confirmed by an overproduction of ROS under heat
stress, which may be associated with the extent of severity of
glycocalyx damage (Figure 4B ). Our results suggested that heat
stroke contributed to severe oxidative stress injury of vascular
endothelial cells, which might be activated and aggravated by glycocalyx
degradation.