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.