SCCM Resource Library-Estrogen Effects on the Endothelial-Glycocalyx Barrier Under Shock Conditions

Video Transcription

Hello, my name is Michael Cargi. I'm one of the fifth-year general surgery residents at the Detroit Medical Center Wayne State University program. I have no disclosures to report.  The title of our research project is Estrogen Effects on the Endothelial Glycocalyx Barrier Under Shock Conditions.  Regarding some background information on our study, trauma hemorrhagic shock causes a multitude of detrimental physiologic effects, including damage to the microvasculature.  This includes the underlying endothelial barrier and glycocalyx, which then lead to the downstream clinical effects of shock that we see in practice.  The endothelial barrier and glycocalyx also serve as the gatekeepers to these insults. They serve as mediators and also a layer of protection to the underlying endothelium.  Sex hormones have been noted and shown experimentally to modulate these effects in various ways.  We also know clinically that there have been studies showing more favorable outcomes in the female sex when faced with trauma hemorrhagic shock.  This has been particularly true in premenopausal states in which estrogen levels are higher.  This next slide depicts the entity of shock-induced endotheliopathy that was described by Johannesson and colleagues.  In this model, an injurious hit, such as septic shock, or in our model, hemorrhagic shock, leads to endothelial cell activation and subsequent damage.  This leads to capillary leakage via injury of the microvascular system, leading into the inflammatory mediators that then give the clinical effects downstream of shock  that we see in practice. Estrogen in particular has been shown to play a protective role in these states.  It does this in several different ways, including modulating the endothelial layer in multiple end-organ beds.  It also exerts antioxidant effects that protect against reactive oxygen species, thus contributing to the downregulation of inflammation.  It also influences nitric oxide modulation in shock conditions, which then exert effects on the vasculature system. Estrogen is able to do this via genomic and non-genomic pathways.  It has also been noted that there is an association between estrogen concentration and protective  effects as it relates to the estrous cycle, meaning when estrogen levels are higher, there is a greater protective effect.  In this next slide, we present our research question and hypothesis. What is the physiologic effect of estrogen on the microvascular endothelium and glycocalyx  when faced with trauma hemorrhagic shock conditions? We hypothesized that estrogen would have a protective effect on the endothelium and glycocalyx  following biomimetic shock conditions. This next slide depicts our methods. We first established human umbilical vein endothelial cell monolayers and subjected  them to a constant shear flow rate. Each group was then either infused with 20 to 400 picograms per milliliter of estrogen, or DHT at 10 nanograms per milliliter for 24 hours.  This was then followed by controller shock conditions using hypoxia reactionation plus epinephrine for 90 minutes.  After this insult, we then measured the endothelial cell glycocalyx thickness via fluorescent  microscopy as well as collection of the perfusate and measurement of syndactin-1 and hyaluronic acid levels.  We also were able to characterize endothelial cell activation and injury by measuring the perfusate for soluble thrombomodulin.  Finally, we established the coagulative phenotype of each group by collecting the perfusate  and measuring them for either tissue plasminogen activator or plasminogen activator inhibitor 1. The following slides depict our results.  This first figure here shows the effect of estrogen and DHT on glycocalyx shedding. As you can note, under hypoxia reoxygenation plus epinephrine conditions, there is significant  shedding of the glycocalyx products hyaluronic acid and syndactin-1. The levels of shedding remain similar when exposed to DHT, and as you can note, with  varying levels of estrogen in a dose-dependent fashion, the higher the concentration of estrogen, the less shedding of the glycocalyx layer there is.  This next slide depicts the effects of estrogen on the glycocalyx thickness. Over to the left of the figure is our control group.  This shows the glycocalyx thickness that is measured via fluorescent microscopy. When exposed to hemorrhagic shock conditions, the glycocalyx thickness is significantly decreased.  When exposed to estrogen, there is a protective effect of this layer. As you can see, there is a concentration-dependent effect in regards to varying levels of estrogen,  with the most protective effect coming in the 400 picogram per milliliter group. This next slide demonstrates the effects of sex hormones on soluble thrombomodulin.  Again, this surrogate is a measurement of the amount of endothelial cell activation and injury that is present.  As you can see, our control group has low levels of soluble thrombomodulin. When exposed to hypoxia reoxygenation and epinephrine alone, there is a significant  spike in the levels of soluble thrombomodulin. As you can see, these levels persist in the DHT group.  When exposed to varying levels of estrogen, the value of soluble thrombomodulin decreases in a concentration-dependent effect, with, again, the 400 picogram per milliliter group  having the least amount of endothelial cell activation and injury. This next slide depicts the quantification of tissue plasminogen activator.  This serves as a coagulative phenotype surrogate for a hypofibrillinic state. As you can see, our control group has very low levels of TPA expressed.  When exposed to hypoxia reoxygenation plus epinephrine, there is a significant spike in a hyperfibrillinic phenotype that is expressed.  This level persists in the DHT group as well as the lower estrogen group. When exposed to higher levels of estrogen, the hyperfibrillinic phenotype is suppressed.  This final result slide depicts a coagulative phenotype as determined by plasminogen activator inhibitor 1 measurements.  This measurement serves as a surrogate for a prothrombotic phenotype. As you can see, our control values have a high circulating value of plasminogen activator inhibitor 1.  When exposed to hypoxia reoxygenation and epinephrine, these levels significantly decrease. The DHT and low estrogen levels match those that are seen in the hypoxia reoxygenation  and epinephrine group alone. When exposed to higher levels of estrogen, the plasminogen activator inhibitor 1 measurements  persist, thus demonstrating a prothrombotic phenotype when the level of estrogen is increased. This next slide depicts the limitations of our study.  First off, our study is an in vitro study that has several inherent limitations compared to an in vivo setting.  This physiology that is seen in the in vivo setting is much more complex than any one model could account for.  We also used only one cell type, that being of human umbilical vein endothelial cells. However, it has been described in the literature that there are differences between different  endothelial cell types depending on the end organ bed.  There is even differences between veins, arteries, and capillaries in terms of their response to shock conditions.  There also may be genetic variability in response to sex hormones that is different for everyone on an individual basis.  Also in the clinical setting, there has been mixed results in terms of the influence of sex hormones.  This heterogeneity may be due to knowing the influence of the estrous cycle and in turn the hormonal status of the patient in these studies.  There is also varying ways of stratifying patients in these studies, including by their injury severity or various mortality rates.  It is also unclear the interaction of other sex hormones, such as progesterone, that may influence results in our model that we did not account for.  To conclude, trauma hemorrhagic shock conditions induce detrimental physiologic changes to the endothelium and underlying glycocalyx layer.  This is evidenced by endothelial cell activation and injury and subsequent glycocalyx layer shedding.  In our study, we were able to demonstrate that estrogen provided a protective effect to the endothelium and underlying glycocalyx layers in a concentration-dependent fashion.  This was associated with a procoagulant profile as related to plasminogen activator inhibitor one and TPA expression.  Our study also was able to demonstrate the non-genomic effects of estrogen during a shock state.  This information may be used for further clinical studies in regards to the effects of the estrous cycle during shock states.  This also may help to contribute to novel treatments to protect against the effects of trauma-induced coagulopathy and endotheliopathy via the modulation of sex hormones.  Thank you for taking the time to listen to our research study and presentation. I would also like to thank the Society of Critical Care Medicine for the opportunity  to present our research in this forum.

Video Summary

In this video, Dr. Michael Cargi discusses a research project focused on the effects of estrogen on the endothelial glycocalyx barrier under shock conditions. Shock, such as trauma hemorrhagic shock, can cause damage to the microvasculature, specifically the endothelial barrier and glycocalyx. Previous studies have suggested that estrogen may have a protective effect in these conditions, particularly in premenopausal women. The research used human umbilical vein endothelial cells and observed the impact of estrogen on glycocalyx shedding, thickness, endothelial cell activation and injury, and coagulative phenotype. The study found that estrogen had a protective effect on the endothelium and glycocalyx in a dose-dependent manner. This information may contribute to further studies and the development of treatments for trauma-induced coagulopathy and endotheliopathy.

Asset Subtitle

Shock Non Sepsis, Trauma, 2022

Asset Caption

INTRODUCTION: Clinical and animal studies have shown a protective effect of the female sex on outcomes following trauma hemorrhagic shock (T/HS) and other inflammatory conditions. This has been attributed to estrogen (E2) and its menstrual related concentrations. Estrogen impacts endothelial function via membrane and cellular receptors. The endothelial glycocalyx (EGL) layer is a critical component of the endothelial vascular barrier. The impact of estrogen on endothelial and glycocalyx barriers after T/HS are unknown.
METHODS: Human umbilical vein endothelial cell monolayers (HUVEC) were established in microfluidic well plates and subjected to flow. HUVEC were then treated with E2 at variable concentrations (20, 200 or 400 pg/ml), testosterone (DHT, 10 ng/ml) or media alone followed by exposure to hypoxia and epinephrine and reoxygenation (HR + epi). Endothelial activation was indexed by soluble thrombomodulin (sTM); coagulation phenotype by tissue type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1) in the perfusate. Glycocalyx integrity was indexed by shedding of syndecan-1 (syn-1) and hyaluronic acid (HLA) into the perfusate. Glycocalyx thickness was determined using fluorescent imaging and XYZ stack analysis.
RESULTS: HR + epi increased sTM and tPA but decreased PAI-1 (p < 0.05) vs. control. There was an E2 concentration effect that returned these parameters to control values. DHT had no effect. HR + epi increased EGL shedding and diminished its thickness vs. control (p < 0.05). E2 but not DHT protected the EGL barrier in a concentration dependent fashion (p < 0.05 vs. HR + epi, p = ns vs. control at the highest E2 concentration).
CONCLUSIONS: E2 has a concentration dependent protective effect on endothelial and glycocalyx derangement following biomimetic shock conditions. This was associated with a procoagulant profile as it relates to relative tPA and PAI-1 concentrations. These data suggest E2 may protect against trauma induced endotheliopathy.

Meta Tag

Content Type Presentation

Knowledge Area Trauma

Knowledge Area Shock Non Sepsis

Knowledge Level Advanced

Membership Level Select

Tag Shock

Tag Hemorrhage

Tag Surgery

Tag Burns

Year 2022

Keywords

Dr. Michael Cargi

estrogen

endothelial glycocalyx barrier

shock conditions

trauma-induced coagulopathy

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