- Email: [email protected]
Disparate effects of catecholamines under stress conditions on endothelial glycocalyx injury: An in vitro model
Accepted Manuscript Disparate effects of catecholamines under stress conditions on endothelial glycocalyx injury: An in vitro model Jonathan V. Martin, David M. Liberati, Lawrence N. Diebel PII:
S0002-9610(17)30514-7
DOI:
10.1016/j.amjsurg.2017.09.018
Reference:
AJS 12539
To appear in:
The American Journal of Surgery
Received Date: 17 March 2017 Revised Date:
1 September 2017
Accepted Date: 20 September 2017
Please cite this article as: Martin JV, Liberati DM, Diebel LN, Disparate effects of catecholamines under stress conditions on endothelial glycocalyx injury: An in vitro model, The American Journal of Surgery (2017), doi: 10.1016/j.amjsurg.2017.09.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title: Disparate effects of catecholamines under stress conditions on endothelial glycocalyx injury: An in vitro model Jonathan V. Martin, MDa; David M. Liberati, MSa; Lawrence N. Diebel, MDa.
Authors Email:
[email protected]; [email protected]; [email protected]
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Authors:
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Authors Affiliation: aMichael and Marian Ilitch Department of Surgery Wayne State University Detroit, MI Jonathan V. Martin, MD Michael and Marian Ilitch Department of Surgery 6C University Health Center 4201 Saint Antoine Street Detroit, MI 48201 USA
Phone:
1-313-577-5005
Fax:
1-313-577-5310
Email:
[email protected]
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Correspondence:
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This study will be presented at the 69th Annual Meeting of the Southwestern Surgical Congress in Maui, HI, April 2-5, 2017.
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Background: Geriatric trauma patients have high circulating norepinephrine (NE) levels but attenuated release of epinephrine (Epi) in response to increasing severity of injury. We hypothesized that NE and
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Epi have different effects on the endothelial and glycocalyx components of the vascular barrier following shock. Methods:
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Human umbilical vein endothelial cells (HUVEC) were treated with varying concentrations of NE or Epi and exposed to simulated shock conditions (HR). Relevant biomarkers were sampled
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to index glycocalyx injury and endothelial cell activation. Results:
NE was associated with significantly greater glycocalyx damage and endothelial activation/injury vs. Epi treatment groups. There were minimal changes in PAI-1 with either NE
Conclusions:
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or Epi ± H/R. However NE ± H/R was associated with significantly higher tPA levels.
NE favors a profibrinolytic state. Our study supports investigating liberal use of the anti-
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fibrinolytic agent tranexamic acid in the severely injured geriatric trauma patient.
ACCEPTED MANUSCRIPT 1 Keywords: norepinephrine; endotheliopathy of trauma; sympathoadrenal activation; geriatric trauma; human umbilical vein endothelial cell (HUVEC); glycocalyx Funding Source:
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This research did not receive any specific grant from funding agencies in the public, commercial,
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or not-for-profit sectors.
ACCEPTED MANUSCRIPT 2 Introduction The endothelial cell and its accompanying glycocalyx are well known contributors to trauma induced coagulopathy as well as sepsis and other shock associated coagulopathies.1,2,3,4
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Biomarkers of degraded glycocalyx and injured endothelium (such as syndecan-1 and soluble thrombomodulin, respectively) have been associated with hypocoagulability in several
prospective trauma studies.5,6,7 The increased production of tPA by endothelial cells has been
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implicated as a primary cause for hyperfibrinolysis following trauma.8,9
In addition to the endothelial cell, age is also being evaluated as an independent
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contributor of trauma outcomes.10,11,12 In a prospective study performed by Johansson et al,12 they noted that in older trauma patients (defined as 66 years old, range of 56-75 years old), there was increased production of norepinephrine and decreased response of epinephrine, leukocytes, and platelets when compared with their younger cohorts (defined as 34 years old, range of 25-40
levels.12,13
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years old). Increased mortality was associated with advanced age and elevated epinephrine
Other groups, such as the Denver group, have also produced work suggestive of age
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associated differences following trauma.14 In their evaluation of fibrinolysis with viscoelastic assays, they noted three distinct profiles; hyperfibrinolysis, physiologic fibrinolysis, and
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fibrinolytic shutdown.15 While physiologic fibrinolysis was associated with increased survival, hyperfibrinolysis produced increased mortality due to hemorrhage and fibrinolytic shutdown increased mortality due to multiple organ failure.15,16 Elderly patients were associated with fibrinolytic shutdown.14 Although the elderly were associated with shutdown, subgroup analysis suggested that empiric treatment with an antifibrinolytic (tranexamic acid) may be warranted, as
ACCEPTED MANUSCRIPT 3 mortality with hyperfibrinolysis was significantly increased compared to shutdown and the risk of hemorrhagic death outweighed the risk of potential thrombosis (unpublished data). In light of these findings, our group elected to study the effects of epinephrine (epi) and
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norepinephrine (NE) on the endothelium and its glycocalyx in an in vitro cell model. Varying concentrations of catecholamine were administered to control groups and simulated shock
groups. Relevant biomarkers were measured to index glycocalyx degradation (syndecan-1),
respectively), and coagulopathy (tPA and PAI-1).
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Methods
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endothelial cell injury and integrity (thrombomodulin and angiopoietin-1/angiopoietin-2 ratios,
Experimental Design
Once confluent HUVEC monolayers are formed, determined by TEER measurement, the culture was exposed to hypoxia followed by reoxygenation. Hypoxic conditions (60 minutes at
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5% O2; 95% N2, 37ºC) were carried out in a hypoxic glove box purchased from Coy Laboratory Products (Ann Arbor, MI), after which cell culture plates were returned to standard conditions (21% O2, 37ºC). Subgroups were challenged with 100 µM hydrogen peroxide (H2O2) and/or
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varying concentrations of norepinephrine (250, 500 and 750 pg/ml) or epinephrine (10-3 µM and 10-6 µM) added to the apical chamber during the hypoxic event. Cell culture supernatants were
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obtained 1 hour after return of cell cultures to standard conditions. Glycocalyx injury was quantified by syndecan-1 release using enzyme-linked immunosorbent assay (ELISA; Abcam, Cambridge, MA). Endothelial cell activation/injury was determined by measuring soluble thrombomodulin (TM; R&D, Minneapolis, MN), plasminogen activator inhibitor-1 (PAI-1) and tissue type plasminogen activator (tPA) concentrations all by ELISA (Abcam). Figure 1 summarizes our experimental timeline.
ACCEPTED MANUSCRIPT 4 HUVEC culture Female human umbilical vein endothelial cells (HUVEC) were purchased from Lonza Walkersville, Inc. (Walkersville, MD). Cells were grown in a 75 cm2 flask using complete media
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(EGM-2 BulletKit, Lonza). Time to subculture is 5-9 days with media changes every 2 days. Cells are subcultured at 85% confluence using 2ml of 0.5% trypsin-EDTA (Life Technologies, Carlsbad, CA). A new culture flask is prepared and the remaining cells are used to seed the
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apical chambers of 6 well transwell plates (Corning, Inc. Kennebunk, ME). Transwell plates are monitored for confluence by measuring the transepithelial electrical resistance (TEER) using a
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Millicell electrical resistance system (Millipore, Bedford, MA). Syndecan-1 analysis
Quantitative measurement of Syndecan protein in HUVEC was accomplished using the Syndecan-1 Human ELISA kit (Abcam, Cambridge, MA). Briefly, microtiter strips containing a
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monoclonal antibody specific for syndecan-1 is provided. Unknown supernatants, standards of known syndecan-1 concentrations and a biotinylated monoclonal antibody specific for syndecan1 are simultaneously incubated. After washing, Streptavidin-HRP is added and incubated. A
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TMB substrate is subsequently added and the colored product is recorded using a spectrophotometer (450nm) and the intensity of the colored product is directly proportional to
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the concentration of syndecan-1 present in the samples. Determination of thrombomodulin (TM) levels in HUVEC supernatants Quantikine Human Thrombomodulin Immunoassay was used for the determination of
TM in the supernatant of HUVEC cultures exposed to agents as outlined in experimental design above. The ELISA was performed according to the manufacturer’s instructions (R & D Systems, Inc., Minneapolis MN). Briefly, the precoated plate containing a monoclonal antibody specific
ACCEPTED MANUSCRIPT 5 for human TM is incubated with fifty microliters of culture supernatants or the provided standards. After washing, the plate was further incubated with the TM conjugate and finally the substrate. Finally, the color reaction was stopped using 2N H2SO4 and the optical density
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measured at 450nm using a spectrophotometer.
Quantification of human angiopoietin-1 (APO-1) and angiopoietin-2 (APO-2)
Quantikine Human Angiopoietin -1 Immunoassay kit and Human Angiopoietin-2
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Quantikine ELISA kits from R & D Systems, Inc. Minneapolis, MN were used to measure APO1 and APO-2 in HUVEC culture supernatants. Fifty microliters of culture supernatants were
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transferred directly to microplate test strips of the ELISA plate. Further assay procedures were followed and completed. The absorbance at 450 nm was measured using a spectrophotometer and APO-1 and APO-2 levels were calculated using a standard curve of recombinant human APO-1 and APO-2 provided.
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Human tissue plasminogen activator (tPA) determination
The quantitative measurement of free tPA in HUVEC culture supernatants was determined using the Tissue Plasminogen Activator in vitro simple step ELISA kit from Abcam,
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Cambridge, MA. Fifty microliters of the cell culture supernatants were directly transferred into the test wells of the Enzyme-Linked Immunosorbent Assay (ELISA) plate. Further procedures
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were performed following the manufacturer’s instructions. The absorbance was read at 450 nm in a spectrophotometer and tPA levels were determined using a calibration curve generated from a set of tPA standards provided. Human plasminogen activator inhibitor-1 (PAI-1) determination Levels of free PAI-1 in HUVEC culture supernatants were determined using the PAI-1 Human SimpleStep ELISA kit from Abcam, Cambridge MA. Fifty microliters of the
ACCEPTED MANUSCRIPT 6 supernatants collected were transferred directly into the appropriate microplate strips of the ELISA plate. Further procedures outlined in the protocol were performed. The absorbance at 450
calibration curve using human PAI-1 provided as a standard. Statistical analysis
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nm was measured in a spectrophotometer and free PAI-1 levels were determined with a
An analysis of variance with a post hoc Tukey test was used to analyze the data using
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GraphPad InStat 3 (GraphPad Software, Inc). Statistical significance was inferred at p values of less than 0.05. All data are expressed as mean ± SD. N = 4 for each group.
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Results
For all catecholamines administered, there was a dose dependent response noted. This was demonstrated with epi concentrations of 10-6 and 10-3 µM and NE concentrations of 250, 500, and 750 pg/mL (data not shown). Direct comparison was made between epi at 10-3 µM and
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NE at 250 pg/mL as these were similar concentrations. Syndecan-1
Figure 2 demonstrates that syndecan-1 levels were approximately 2x baseline when
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HUVEC were exposed to either epi or NE, and up to 4x baseline for both after HR + H2O2, (p < 0.05). Statistical difference between NE and epi groups depended on dose; epi at 10-3 µM vs. NE
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at 250 pg/mL was significant only when applied alone to HUVEC, whereas if compared with stress dose NE at 750 pg/mL, NE levels were significantly higher in all subgroups (p < 0.05) (data not shown).
Thrombomodulin
ACCEPTED MANUSCRIPT 7 Levels of thrombomodulin were increased to approximately 4x baseline in both epi and NE and can be seen in Figure 3. In all subgroups except plain HUVEC, NE significantly
Angiopoietin-1/Angiopoietin-2 (APO-1/APO-2) Ratios
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increased thrombomodulin levels compared with epi (p < 0.05).
Levels of both APO-1 and APO-2 were increased in all subsets. APO-2 was often 2-3x higher than baseline, resulting in decreased APO-1/APO-2 ratios as low as 1/5th baseline for epi
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and ½ baseline for NE following HR + H2O2 (Figure 4A). However, NE tended to increase levels of APO-1 more than APO-2 whereas epi produced opposite results. This was observed in
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the overall ratios increasing with severity of insult for the NE groups while ratios decreased in the epi group. Levels of APO-1 were significantly increased in all NE groups compared to epi (p < 0.05), whereas levels of APO-2 were significantly increased compared to NE only in the HR + H2O2 group (p < 0.05) (Figure 4B and 4C).
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tPA and PAI-1
Overall, NE stimulated production of tPA, nearly 3x more than baseline in the HR+H2O2 group, with all NE groups significantly higher than baseline and their epi counterparts (p < 0.05).
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This is in contrast to epi which produced only mild increases in tPA or, in case of epi administration alone, decreased tPA (p < 0.05). This can be seen in Figure 5. PAI-1 levels, on
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the other hand, tended to decrease in subgroups devoid of catecholamine (Figure 6). Catecholamine treated groups tended to resemble baseline status, except for epi + HR + H2O2; this was higher than baseline. Discussion
The results of our study suggest two major differences between the endothelial response to epi and NE, primarily regarding endothelial integrity and fibrinolysis. Both epi and NE are
ACCEPTED MANUSCRIPT 8 implicated in glycocalyx degradation and endothelial injury as noted by shedding of syndecan-1 and thrombomodulin. Yet while both instigate a measure of dysfunctional cellular integrity as demonstrated by decreased APO-1/APO-2 ratios and increased APO-2 when compared to
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baseline, NE appears to have an ameliorative effect by stimulating significantly more APO-1 (a biomarker associated with increased endothelial stability and survival in critically ill patients) than APO-2 (a biomarker associated with opposite effects of APO-1).17 This effect is even more
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pronounced with higher stress doses of NE (Figures 4B and 4C). Older in vitro studies
conducted by Bottaro et al18 noted that endothelial cells treated with NE had significantly less
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permeability to albumin compared with controls and suggested that NE may indeed promote cellular integrity by stimulating production of intercellular adhesion molecules. This would bear further investigation in our model.
Regarding fibrinolysis, the decreased production of tPA and increased production of PAI-
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1 associated with epi is also somewhat intriguing. Observations by Johansson et al19 suggest that in vivo, low doses of catecholamine are actually associated with mildly hypercoagulable state. Another possible mechanism to explain the fibrinolytic difference between epi and NE may be
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selective β-receptor response. A recent study conducted on critically ill patients with septic shock and tachycardia noted decreased mortality if patients were treated with a short-acting β -
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blocker during their ICU course.20 Further investigations with different epi concentrations and selective β-blockade may bear some light in our model. Clinically, advanced age is independently associated with the development of vascular
endothelial dysfunction.21 This may be associated with plasma norepinephrine concentrations.22 Our study has shown that there is increased production of tPA by NE compared with epi. As there are increased levels of NE in elderly trauma patients, it is prudent to evaluate this as a
ACCEPTED MANUSCRIPT 9 potential mechanism for development of hyperfibrinolysis in the elderly and suggests a more liberal use of tranexamic acid in this patient population.23 Given that this is only an in vitro study, data should be interpreted cautiously and further investigation in human trials would be
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needed to confirm observations. Conclusion
In summary, NE induces a fibrinolytic state with elevated tPA levels compared to epi.
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While both degrade the glycocalyx and damage the endothelium, epi appears to produce more severe endothelial instability as demonstrated by increased APO-2 levels. Both produce
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responses in a dose dependent fashion. With the above findings of increased tPA due to NE and the knowledge that elderly trauma patients tend to produce increased NE, it would not be unreasonable to further investigate early tranexamic acid therapy for the elderly trauma patient.
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Acknowledgements:
LND and DML made substantial contributions to the conception or design of the work; they also conceived and designed the experiments. LND, JVM, and DML analyzed the data. JVM,
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DLM, and LND drafted the manuscript. LND, JVM, and DML critically revised the manuscript
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for intellectual content. All authors provided final approval of the version to be published.
ACCEPTED MANUSCRIPT 10 References 1.
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ACCEPTED MANUSCRIPT 12 Sauaia A. Hyperfibrinolysis, physiologic fibrinolysis, and fibrinolysis shutdown: the spectrum of postinjury fibrinolysis and relevance to antifibrinolytic therapy. J Trauma Acute Care Surg. 2014;77(6):811-7; discussion 7. Moore EE, Moore HB, Gonzalez E, Chapman MP, Hansen KC, Sauaia A, Silliman CC,
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ACCEPTED MANUSCRIPT 13 vascular endothelial function with aging in healthy women. J Appl Physiol (1985). 2011;111(5):1416-21. 23.
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Figure 1: Diagram showing transwell plate set-up and timeline of experiment, starting with hypoxia and other insults for 1 hour followed by reoxygenation, with sampling of supernatants 2 hours after initial insult.
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Figure 2: Effects of Epi vs. NE on Syndecan-1. Results of endothelial exposure to simulated shock and epi 10-3 µM and NE 250 pg/mL. HUVEC = human umbilical vein endothelial cell, HR = hypoxia-reoxygenation, Epi = epinephrine, NE = norepinephrine
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Figure 3: Effects of Epi vs. NE on Thrombomodulin. Results of endothelial exposure to simulated shock and epi 10-3 µM and NE 250 pg/mL. HUVEC = human umbilical vein endothelial cell, HR = hypoxia-reoxygenation, Epi = epinephrine, NE = norepinephrine, TM = thrombomodulin
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Figure 4A: Effects of Epi vs. NE on APO-1/APO-2 Ratios. Results of endothelial exposure to simulated shock with epi 10-3 µM or NE 250 pg/mL. HUVEC = human umbilical vein endothelial cell, HR = hypoxia-reoxygenation, Epi = epinephrine, NE = norepinephrine, APO = angiopoietin
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Figure 4B: Effects of Epi vs. NE on APO-1. Results of endothelial exposure to simulated shock with epi 10-3 µM, NE 250 pg/mL, or NE 750 pg/mL. HUVEC = human umbilical vein endothelial cell, HR = hypoxia-reoxygenation, Epi = epinephrine, NE = norepinephrine, APO = angiopoietin
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Figure 4C: Effects of Epi vs. NE on APO-2. Results of endothelial exposure to simulated shock with epi 10-3 µM, NE 250 pg/mL, or NE 750 pg/mL. HUVEC = human umbilical vein endothelial cell, HR = hypoxia-reoxygenation, Epi = epinephrine, NE = norepinephrine, APO = angiopoietin
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Figure 5: Effects of Epi vs. NE on tPA. Results of endothelial exposure to simulated shock with epi 10-3 µM or NE 250 pg/mL. HUVEC = human umbilical vein endothelial cell, HR = hypoxia-reoxygenation, Epi = epinephrine, NE = norepinephrine, tPA = tissue plasminogen activator
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Figure 6: Effects of Epi vs. NE on PAI-1. Results of endothelial exposure to simulated shock with epi 10-3 µM or NE 250 pg/mL. HUVEC = human umbilical vein endothelial cell, HR = hypoxia-reoxygenation, Epi = epinephrine, NE = norepinephrine, PAI-1 = plasminogen activator inhibitor-1
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The work of Chapman et al8 and Cardenas et al9 have demonstrated that a key development in hyperfibrinolysis is the development of an imbalance between tPA and PAI-1 (plasminogen activator inhibitor-1), with overproduction of tPA and minimal
improved outcomes and decreased mortality.9
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change in PAI-1. Patients that maintained a relative balance of tPA to PAI-1 had
Relevant biomarkers were measured at 2 hours after insult to index glycocalyx
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degradation (syndecan-1), endothelial cell injury and integrity (thrombomodulin and
angiopoietin-1/angiopoietin-2 ratios, respectively), and coagulopathy (tPA and PAI-1).
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Once confluent HUVEC monolayers are formed, determined by TEER measurement, the culture was exposed to hypoxia followed by reoxygenation. Hypoxic conditions (1 hour at 5% O2; 95% N2, 37ºC) were carried out in a hypoxic glove box purchased from Coy Laboratory Products (Ann Arbor, MI), after which cell culture plates
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were returned to standard conditions (21% O2, 37ºC) for an additional 1 hour. Subgroups were challenged with 100 µM hydrogen peroxide (H2O2) and/or varying concentrations of epi (10-3 µM and 10-6 µM) or NE (250, 500 and 750 pg/ml) were added to the apical
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chamber during the hypoxic event. Concentrations for H2O2 and epi/NE were based upon prior experiments performed in our lab, as well as evaluation of other endothelial cell
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models.17 Data from Johansson and Ostrowski(3,7,12,13) regarding levels of epi and NE in trauma patients suggested that a range of 200-500 pg/mL and 300-1000 pg/mL would be reasonable concentrations to evaluate in our model. We compared specifically epi 10-3 µM (240 pg/mL) with NE 250 pg/mL as these were similar in concentration. Cell culture supernatants were sampled 2 hours after initial cellular insult, allowing 1 hour for initial hypoxia/injury and an additional 1 hour for reoxygenation, as
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had been done in previous experiments. Glycocalyx injury was quantified by syndecan-1 release using enzyme-linked immunosorbent assay (ELISA; Abcam, Cambridge, MA). Endothelial cell activation/injury was determined by measuring soluble thrombomodulin
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(TM; R&D, Minneapolis, MN), plasminogen activator inhibitor-1 (PAI-1) and tissue type plasminogen activator (tPA) concentrations all by ELISA (Abcam). Figure 1 summarizes our experimental timeline.
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For all catecholamines administered, there was a dose dependent response noted. This was demonstrated with epi concentrations of 10-6 and 10-3 µM and NE
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concentrations of 250, 500, and 750 pg/mL (data not shown). Direct comparison was made between epi at 10-3 µM and NE at 250 pg/mL as these were similar concentrations. Regarding cellular viability, in prior HUVEC experiments with epi, we noted the rate of necrosis for control; hypoxia; hypoxia + epi; and hypoxia + epi + H2O2 to be 2.2%,
37.9%, respectively.
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5.2%, 11.3%, and 12.4%, respectively. Rates of apoptosis were 4.7%, 8.9%, 20.1%, and
). A similar observation regarding the levels of APO-1&2 was noted in a study by
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Johansson et al19 to evaluate the effects of endogenously produced NE on septic shock patients. A small cohort was given a dose of NE and were subsequently analyzed.
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Although elevated endogenous NE production was associated with increased glycocalyx degradation and mortality, the extra exogenous levels did not appear to have any adverse effect; any beneficial effect was speculated to be due to increased recruitment of platelets. Older in vitro studies conducted by Bottaro et al20 noted that endothelial cells treated with NE had significantly less permeability to albumin compared with controls and suggested
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that NE may indeed promote cellular integrity by stimulating production of intercellular adhesion molecules. This would bear further investigation in our model. The degradation of the glycocalyx associated with catecholamine exposure may in
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part be explained by increased activity of membrane-anchored proteins such as the
matrix-metalloproteinases (MMPs) and ADAMs (a disintigrin and metalloproteinase)
family of proteases. MMPs and ADAMs are capable of degrading extracellular matrix
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proteins and are known to be stimulated by catecholamines.21,22 Prior work in our lab has shown that exposure to epi increased the activity of these proteases with subsequent
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shedding of glycocalyx components such as syndecan-1 and hyaluronic acid. Evaluation of the effect of NE on these proteases is warranted. 17.
Gill RR, Gbur CJ Jr, Fisher BJ, Hess ML, Fowler AA 3rd, Kukreja RC, Sholley
MM. Heat Shock Provides Delayed Protection against Oxidative Injury in Culture
19.
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Human Umbilical Vein Endothelial Cells.” J Mol Cell Cardiol. 1998;30(12):2739-2749. Johansson PI, Haase N, Perner A, Ostrowski SR. Association between
sympathoadrenal activation, fibrinolysis, and endothelial damage in septic patients: A
21.
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prospective study. J Crit Care. 2014;29(3):327-33. Reitz A, Spiers JP. The relationship between the MMP system, adrenoceptors and
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phosphoprotein phosphatases. Br J Pharmacol. 2012;166(4):1225-43. 22.
Peng Z, Ban K, LeBlanc A, Kozar RA. Intraluminal tranexamic acid inhibits
intestinal sheddases and mitigates gut and lung injury and inflammation in a rodent model of hemorrhagic shock. J Trauma Acute Care Surg. 2016 Aug;81(2):358-365.
S0002-9610(17)30514-7
DOI:
10.1016/j.amjsurg.2017.09.018
Reference:
AJS 12539
To appear in:
The American Journal of Surgery
Received Date: 17 March 2017 Revised Date:
1 September 2017
Accepted Date: 20 September 2017
Please cite this article as: Martin JV, Liberati DM, Diebel LN, Disparate effects of catecholamines under stress conditions on endothelial glycocalyx injury: An in vitro model, The American Journal of Surgery (2017), doi: 10.1016/j.amjsurg.2017.09.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title: Disparate effects of catecholamines under stress conditions on endothelial glycocalyx injury: An in vitro model Jonathan V. Martin, MDa; David M. Liberati, MSa; Lawrence N. Diebel, MDa.
Authors Email:
[email protected]; [email protected]; [email protected]
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Authors:
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Authors Affiliation: aMichael and Marian Ilitch Department of Surgery Wayne State University Detroit, MI Jonathan V. Martin, MD Michael and Marian Ilitch Department of Surgery 6C University Health Center 4201 Saint Antoine Street Detroit, MI 48201 USA
Phone:
1-313-577-5005
Fax:
1-313-577-5310
Email:
[email protected]
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Correspondence:
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This study will be presented at the 69th Annual Meeting of the Southwestern Surgical Congress in Maui, HI, April 2-5, 2017.
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Background: Geriatric trauma patients have high circulating norepinephrine (NE) levels but attenuated release of epinephrine (Epi) in response to increasing severity of injury. We hypothesized that NE and
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Epi have different effects on the endothelial and glycocalyx components of the vascular barrier following shock. Methods:
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Human umbilical vein endothelial cells (HUVEC) were treated with varying concentrations of NE or Epi and exposed to simulated shock conditions (HR). Relevant biomarkers were sampled
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to index glycocalyx injury and endothelial cell activation. Results:
NE was associated with significantly greater glycocalyx damage and endothelial activation/injury vs. Epi treatment groups. There were minimal changes in PAI-1 with either NE
Conclusions:
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or Epi ± H/R. However NE ± H/R was associated with significantly higher tPA levels.
NE favors a profibrinolytic state. Our study supports investigating liberal use of the anti-
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fibrinolytic agent tranexamic acid in the severely injured geriatric trauma patient.
ACCEPTED MANUSCRIPT 1 Keywords: norepinephrine; endotheliopathy of trauma; sympathoadrenal activation; geriatric trauma; human umbilical vein endothelial cell (HUVEC); glycocalyx Funding Source:
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This research did not receive any specific grant from funding agencies in the public, commercial,
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or not-for-profit sectors.
ACCEPTED MANUSCRIPT 2 Introduction The endothelial cell and its accompanying glycocalyx are well known contributors to trauma induced coagulopathy as well as sepsis and other shock associated coagulopathies.1,2,3,4
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Biomarkers of degraded glycocalyx and injured endothelium (such as syndecan-1 and soluble thrombomodulin, respectively) have been associated with hypocoagulability in several
prospective trauma studies.5,6,7 The increased production of tPA by endothelial cells has been
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implicated as a primary cause for hyperfibrinolysis following trauma.8,9
In addition to the endothelial cell, age is also being evaluated as an independent
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contributor of trauma outcomes.10,11,12 In a prospective study performed by Johansson et al,12 they noted that in older trauma patients (defined as 66 years old, range of 56-75 years old), there was increased production of norepinephrine and decreased response of epinephrine, leukocytes, and platelets when compared with their younger cohorts (defined as 34 years old, range of 25-40
levels.12,13
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years old). Increased mortality was associated with advanced age and elevated epinephrine
Other groups, such as the Denver group, have also produced work suggestive of age
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associated differences following trauma.14 In their evaluation of fibrinolysis with viscoelastic assays, they noted three distinct profiles; hyperfibrinolysis, physiologic fibrinolysis, and
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fibrinolytic shutdown.15 While physiologic fibrinolysis was associated with increased survival, hyperfibrinolysis produced increased mortality due to hemorrhage and fibrinolytic shutdown increased mortality due to multiple organ failure.15,16 Elderly patients were associated with fibrinolytic shutdown.14 Although the elderly were associated with shutdown, subgroup analysis suggested that empiric treatment with an antifibrinolytic (tranexamic acid) may be warranted, as
ACCEPTED MANUSCRIPT 3 mortality with hyperfibrinolysis was significantly increased compared to shutdown and the risk of hemorrhagic death outweighed the risk of potential thrombosis (unpublished data). In light of these findings, our group elected to study the effects of epinephrine (epi) and
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norepinephrine (NE) on the endothelium and its glycocalyx in an in vitro cell model. Varying concentrations of catecholamine were administered to control groups and simulated shock
groups. Relevant biomarkers were measured to index glycocalyx degradation (syndecan-1),
respectively), and coagulopathy (tPA and PAI-1).
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Methods
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endothelial cell injury and integrity (thrombomodulin and angiopoietin-1/angiopoietin-2 ratios,
Experimental Design
Once confluent HUVEC monolayers are formed, determined by TEER measurement, the culture was exposed to hypoxia followed by reoxygenation. Hypoxic conditions (60 minutes at
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5% O2; 95% N2, 37ºC) were carried out in a hypoxic glove box purchased from Coy Laboratory Products (Ann Arbor, MI), after which cell culture plates were returned to standard conditions (21% O2, 37ºC). Subgroups were challenged with 100 µM hydrogen peroxide (H2O2) and/or
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varying concentrations of norepinephrine (250, 500 and 750 pg/ml) or epinephrine (10-3 µM and 10-6 µM) added to the apical chamber during the hypoxic event. Cell culture supernatants were
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obtained 1 hour after return of cell cultures to standard conditions. Glycocalyx injury was quantified by syndecan-1 release using enzyme-linked immunosorbent assay (ELISA; Abcam, Cambridge, MA). Endothelial cell activation/injury was determined by measuring soluble thrombomodulin (TM; R&D, Minneapolis, MN), plasminogen activator inhibitor-1 (PAI-1) and tissue type plasminogen activator (tPA) concentrations all by ELISA (Abcam). Figure 1 summarizes our experimental timeline.
ACCEPTED MANUSCRIPT 4 HUVEC culture Female human umbilical vein endothelial cells (HUVEC) were purchased from Lonza Walkersville, Inc. (Walkersville, MD). Cells were grown in a 75 cm2 flask using complete media
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(EGM-2 BulletKit, Lonza). Time to subculture is 5-9 days with media changes every 2 days. Cells are subcultured at 85% confluence using 2ml of 0.5% trypsin-EDTA (Life Technologies, Carlsbad, CA). A new culture flask is prepared and the remaining cells are used to seed the
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apical chambers of 6 well transwell plates (Corning, Inc. Kennebunk, ME). Transwell plates are monitored for confluence by measuring the transepithelial electrical resistance (TEER) using a
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Millicell electrical resistance system (Millipore, Bedford, MA). Syndecan-1 analysis
Quantitative measurement of Syndecan protein in HUVEC was accomplished using the Syndecan-1 Human ELISA kit (Abcam, Cambridge, MA). Briefly, microtiter strips containing a
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monoclonal antibody specific for syndecan-1 is provided. Unknown supernatants, standards of known syndecan-1 concentrations and a biotinylated monoclonal antibody specific for syndecan1 are simultaneously incubated. After washing, Streptavidin-HRP is added and incubated. A
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TMB substrate is subsequently added and the colored product is recorded using a spectrophotometer (450nm) and the intensity of the colored product is directly proportional to
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the concentration of syndecan-1 present in the samples. Determination of thrombomodulin (TM) levels in HUVEC supernatants Quantikine Human Thrombomodulin Immunoassay was used for the determination of
TM in the supernatant of HUVEC cultures exposed to agents as outlined in experimental design above. The ELISA was performed according to the manufacturer’s instructions (R & D Systems, Inc., Minneapolis MN). Briefly, the precoated plate containing a monoclonal antibody specific
ACCEPTED MANUSCRIPT 5 for human TM is incubated with fifty microliters of culture supernatants or the provided standards. After washing, the plate was further incubated with the TM conjugate and finally the substrate. Finally, the color reaction was stopped using 2N H2SO4 and the optical density
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measured at 450nm using a spectrophotometer.
Quantification of human angiopoietin-1 (APO-1) and angiopoietin-2 (APO-2)
Quantikine Human Angiopoietin -1 Immunoassay kit and Human Angiopoietin-2
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Quantikine ELISA kits from R & D Systems, Inc. Minneapolis, MN were used to measure APO1 and APO-2 in HUVEC culture supernatants. Fifty microliters of culture supernatants were
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transferred directly to microplate test strips of the ELISA plate. Further assay procedures were followed and completed. The absorbance at 450 nm was measured using a spectrophotometer and APO-1 and APO-2 levels were calculated using a standard curve of recombinant human APO-1 and APO-2 provided.
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Human tissue plasminogen activator (tPA) determination
The quantitative measurement of free tPA in HUVEC culture supernatants was determined using the Tissue Plasminogen Activator in vitro simple step ELISA kit from Abcam,
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Cambridge, MA. Fifty microliters of the cell culture supernatants were directly transferred into the test wells of the Enzyme-Linked Immunosorbent Assay (ELISA) plate. Further procedures
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were performed following the manufacturer’s instructions. The absorbance was read at 450 nm in a spectrophotometer and tPA levels were determined using a calibration curve generated from a set of tPA standards provided. Human plasminogen activator inhibitor-1 (PAI-1) determination Levels of free PAI-1 in HUVEC culture supernatants were determined using the PAI-1 Human SimpleStep ELISA kit from Abcam, Cambridge MA. Fifty microliters of the
ACCEPTED MANUSCRIPT 6 supernatants collected were transferred directly into the appropriate microplate strips of the ELISA plate. Further procedures outlined in the protocol were performed. The absorbance at 450
calibration curve using human PAI-1 provided as a standard. Statistical analysis
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nm was measured in a spectrophotometer and free PAI-1 levels were determined with a
An analysis of variance with a post hoc Tukey test was used to analyze the data using
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GraphPad InStat 3 (GraphPad Software, Inc). Statistical significance was inferred at p values of less than 0.05. All data are expressed as mean ± SD. N = 4 for each group.
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Results
For all catecholamines administered, there was a dose dependent response noted. This was demonstrated with epi concentrations of 10-6 and 10-3 µM and NE concentrations of 250, 500, and 750 pg/mL (data not shown). Direct comparison was made between epi at 10-3 µM and
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NE at 250 pg/mL as these were similar concentrations. Syndecan-1
Figure 2 demonstrates that syndecan-1 levels were approximately 2x baseline when
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HUVEC were exposed to either epi or NE, and up to 4x baseline for both after HR + H2O2, (p < 0.05). Statistical difference between NE and epi groups depended on dose; epi at 10-3 µM vs. NE
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at 250 pg/mL was significant only when applied alone to HUVEC, whereas if compared with stress dose NE at 750 pg/mL, NE levels were significantly higher in all subgroups (p < 0.05) (data not shown).
Thrombomodulin
ACCEPTED MANUSCRIPT 7 Levels of thrombomodulin were increased to approximately 4x baseline in both epi and NE and can be seen in Figure 3. In all subgroups except plain HUVEC, NE significantly
Angiopoietin-1/Angiopoietin-2 (APO-1/APO-2) Ratios
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increased thrombomodulin levels compared with epi (p < 0.05).
Levels of both APO-1 and APO-2 were increased in all subsets. APO-2 was often 2-3x higher than baseline, resulting in decreased APO-1/APO-2 ratios as low as 1/5th baseline for epi
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and ½ baseline for NE following HR + H2O2 (Figure 4A). However, NE tended to increase levels of APO-1 more than APO-2 whereas epi produced opposite results. This was observed in
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the overall ratios increasing with severity of insult for the NE groups while ratios decreased in the epi group. Levels of APO-1 were significantly increased in all NE groups compared to epi (p < 0.05), whereas levels of APO-2 were significantly increased compared to NE only in the HR + H2O2 group (p < 0.05) (Figure 4B and 4C).
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tPA and PAI-1
Overall, NE stimulated production of tPA, nearly 3x more than baseline in the HR+H2O2 group, with all NE groups significantly higher than baseline and their epi counterparts (p < 0.05).
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This is in contrast to epi which produced only mild increases in tPA or, in case of epi administration alone, decreased tPA (p < 0.05). This can be seen in Figure 5. PAI-1 levels, on
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the other hand, tended to decrease in subgroups devoid of catecholamine (Figure 6). Catecholamine treated groups tended to resemble baseline status, except for epi + HR + H2O2; this was higher than baseline. Discussion
The results of our study suggest two major differences between the endothelial response to epi and NE, primarily regarding endothelial integrity and fibrinolysis. Both epi and NE are
ACCEPTED MANUSCRIPT 8 implicated in glycocalyx degradation and endothelial injury as noted by shedding of syndecan-1 and thrombomodulin. Yet while both instigate a measure of dysfunctional cellular integrity as demonstrated by decreased APO-1/APO-2 ratios and increased APO-2 when compared to
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baseline, NE appears to have an ameliorative effect by stimulating significantly more APO-1 (a biomarker associated with increased endothelial stability and survival in critically ill patients) than APO-2 (a biomarker associated with opposite effects of APO-1).17 This effect is even more
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pronounced with higher stress doses of NE (Figures 4B and 4C). Older in vitro studies
conducted by Bottaro et al18 noted that endothelial cells treated with NE had significantly less
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permeability to albumin compared with controls and suggested that NE may indeed promote cellular integrity by stimulating production of intercellular adhesion molecules. This would bear further investigation in our model.
Regarding fibrinolysis, the decreased production of tPA and increased production of PAI-
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1 associated with epi is also somewhat intriguing. Observations by Johansson et al19 suggest that in vivo, low doses of catecholamine are actually associated with mildly hypercoagulable state. Another possible mechanism to explain the fibrinolytic difference between epi and NE may be
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selective β-receptor response. A recent study conducted on critically ill patients with septic shock and tachycardia noted decreased mortality if patients were treated with a short-acting β -
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blocker during their ICU course.20 Further investigations with different epi concentrations and selective β-blockade may bear some light in our model. Clinically, advanced age is independently associated with the development of vascular
endothelial dysfunction.21 This may be associated with plasma norepinephrine concentrations.22 Our study has shown that there is increased production of tPA by NE compared with epi. As there are increased levels of NE in elderly trauma patients, it is prudent to evaluate this as a
ACCEPTED MANUSCRIPT 9 potential mechanism for development of hyperfibrinolysis in the elderly and suggests a more liberal use of tranexamic acid in this patient population.23 Given that this is only an in vitro study, data should be interpreted cautiously and further investigation in human trials would be
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needed to confirm observations. Conclusion
In summary, NE induces a fibrinolytic state with elevated tPA levels compared to epi.
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While both degrade the glycocalyx and damage the endothelium, epi appears to produce more severe endothelial instability as demonstrated by increased APO-2 levels. Both produce
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responses in a dose dependent fashion. With the above findings of increased tPA due to NE and the knowledge that elderly trauma patients tend to produce increased NE, it would not be unreasonable to further investigate early tranexamic acid therapy for the elderly trauma patient.
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Acknowledgements:
LND and DML made substantial contributions to the conception or design of the work; they also conceived and designed the experiments. LND, JVM, and DML analyzed the data. JVM,
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DLM, and LND drafted the manuscript. LND, JVM, and DML critically revised the manuscript
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for intellectual content. All authors provided final approval of the version to be published.
ACCEPTED MANUSCRIPT 10 References 1.
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ACCEPTED MANUSCRIPT 12 Sauaia A. Hyperfibrinolysis, physiologic fibrinolysis, and fibrinolysis shutdown: the spectrum of postinjury fibrinolysis and relevance to antifibrinolytic therapy. J Trauma Acute Care Surg. 2014;77(6):811-7; discussion 7. Moore EE, Moore HB, Gonzalez E, Chapman MP, Hansen KC, Sauaia A, Silliman CC,
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aging endothelial cells. J Mol Cell Cardiol. 2015;89(Pt B):122-35. 22.
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ACCEPTED MANUSCRIPT 13 vascular endothelial function with aging in healthy women. J Appl Physiol (1985). 2011;111(5):1416-21. 23.
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Figure 1: Diagram showing transwell plate set-up and timeline of experiment, starting with hypoxia and other insults for 1 hour followed by reoxygenation, with sampling of supernatants 2 hours after initial insult.
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Figure 2: Effects of Epi vs. NE on Syndecan-1. Results of endothelial exposure to simulated shock and epi 10-3 µM and NE 250 pg/mL. HUVEC = human umbilical vein endothelial cell, HR = hypoxia-reoxygenation, Epi = epinephrine, NE = norepinephrine
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Figure 3: Effects of Epi vs. NE on Thrombomodulin. Results of endothelial exposure to simulated shock and epi 10-3 µM and NE 250 pg/mL. HUVEC = human umbilical vein endothelial cell, HR = hypoxia-reoxygenation, Epi = epinephrine, NE = norepinephrine, TM = thrombomodulin
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Figure 4A: Effects of Epi vs. NE on APO-1/APO-2 Ratios. Results of endothelial exposure to simulated shock with epi 10-3 µM or NE 250 pg/mL. HUVEC = human umbilical vein endothelial cell, HR = hypoxia-reoxygenation, Epi = epinephrine, NE = norepinephrine, APO = angiopoietin
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Figure 4B: Effects of Epi vs. NE on APO-1. Results of endothelial exposure to simulated shock with epi 10-3 µM, NE 250 pg/mL, or NE 750 pg/mL. HUVEC = human umbilical vein endothelial cell, HR = hypoxia-reoxygenation, Epi = epinephrine, NE = norepinephrine, APO = angiopoietin
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Figure 4C: Effects of Epi vs. NE on APO-2. Results of endothelial exposure to simulated shock with epi 10-3 µM, NE 250 pg/mL, or NE 750 pg/mL. HUVEC = human umbilical vein endothelial cell, HR = hypoxia-reoxygenation, Epi = epinephrine, NE = norepinephrine, APO = angiopoietin
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Figure 5: Effects of Epi vs. NE on tPA. Results of endothelial exposure to simulated shock with epi 10-3 µM or NE 250 pg/mL. HUVEC = human umbilical vein endothelial cell, HR = hypoxia-reoxygenation, Epi = epinephrine, NE = norepinephrine, tPA = tissue plasminogen activator
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Figure 6: Effects of Epi vs. NE on PAI-1. Results of endothelial exposure to simulated shock with epi 10-3 µM or NE 250 pg/mL. HUVEC = human umbilical vein endothelial cell, HR = hypoxia-reoxygenation, Epi = epinephrine, NE = norepinephrine, PAI-1 = plasminogen activator inhibitor-1
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The work of Chapman et al8 and Cardenas et al9 have demonstrated that a key development in hyperfibrinolysis is the development of an imbalance between tPA and PAI-1 (plasminogen activator inhibitor-1), with overproduction of tPA and minimal
improved outcomes and decreased mortality.9
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change in PAI-1. Patients that maintained a relative balance of tPA to PAI-1 had
Relevant biomarkers were measured at 2 hours after insult to index glycocalyx
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degradation (syndecan-1), endothelial cell injury and integrity (thrombomodulin and
angiopoietin-1/angiopoietin-2 ratios, respectively), and coagulopathy (tPA and PAI-1).
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Once confluent HUVEC monolayers are formed, determined by TEER measurement, the culture was exposed to hypoxia followed by reoxygenation. Hypoxic conditions (1 hour at 5% O2; 95% N2, 37ºC) were carried out in a hypoxic glove box purchased from Coy Laboratory Products (Ann Arbor, MI), after which cell culture plates
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were returned to standard conditions (21% O2, 37ºC) for an additional 1 hour. Subgroups were challenged with 100 µM hydrogen peroxide (H2O2) and/or varying concentrations of epi (10-3 µM and 10-6 µM) or NE (250, 500 and 750 pg/ml) were added to the apical
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chamber during the hypoxic event. Concentrations for H2O2 and epi/NE were based upon prior experiments performed in our lab, as well as evaluation of other endothelial cell
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models.17 Data from Johansson and Ostrowski(3,7,12,13) regarding levels of epi and NE in trauma patients suggested that a range of 200-500 pg/mL and 300-1000 pg/mL would be reasonable concentrations to evaluate in our model. We compared specifically epi 10-3 µM (240 pg/mL) with NE 250 pg/mL as these were similar in concentration. Cell culture supernatants were sampled 2 hours after initial cellular insult, allowing 1 hour for initial hypoxia/injury and an additional 1 hour for reoxygenation, as
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had been done in previous experiments. Glycocalyx injury was quantified by syndecan-1 release using enzyme-linked immunosorbent assay (ELISA; Abcam, Cambridge, MA). Endothelial cell activation/injury was determined by measuring soluble thrombomodulin
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(TM; R&D, Minneapolis, MN), plasminogen activator inhibitor-1 (PAI-1) and tissue type plasminogen activator (tPA) concentrations all by ELISA (Abcam). Figure 1 summarizes our experimental timeline.
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For all catecholamines administered, there was a dose dependent response noted. This was demonstrated with epi concentrations of 10-6 and 10-3 µM and NE
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concentrations of 250, 500, and 750 pg/mL (data not shown). Direct comparison was made between epi at 10-3 µM and NE at 250 pg/mL as these were similar concentrations. Regarding cellular viability, in prior HUVEC experiments with epi, we noted the rate of necrosis for control; hypoxia; hypoxia + epi; and hypoxia + epi + H2O2 to be 2.2%,
37.9%, respectively.
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5.2%, 11.3%, and 12.4%, respectively. Rates of apoptosis were 4.7%, 8.9%, 20.1%, and
). A similar observation regarding the levels of APO-1&2 was noted in a study by
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Johansson et al19 to evaluate the effects of endogenously produced NE on septic shock patients. A small cohort was given a dose of NE and were subsequently analyzed.
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Although elevated endogenous NE production was associated with increased glycocalyx degradation and mortality, the extra exogenous levels did not appear to have any adverse effect; any beneficial effect was speculated to be due to increased recruitment of platelets. Older in vitro studies conducted by Bottaro et al20 noted that endothelial cells treated with NE had significantly less permeability to albumin compared with controls and suggested
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that NE may indeed promote cellular integrity by stimulating production of intercellular adhesion molecules. This would bear further investigation in our model. The degradation of the glycocalyx associated with catecholamine exposure may in
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part be explained by increased activity of membrane-anchored proteins such as the
matrix-metalloproteinases (MMPs) and ADAMs (a disintigrin and metalloproteinase)
family of proteases. MMPs and ADAMs are capable of degrading extracellular matrix
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proteins and are known to be stimulated by catecholamines.21,22 Prior work in our lab has shown that exposure to epi increased the activity of these proteases with subsequent
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shedding of glycocalyx components such as syndecan-1 and hyaluronic acid. Evaluation of the effect of NE on these proteases is warranted. 17.
Gill RR, Gbur CJ Jr, Fisher BJ, Hess ML, Fowler AA 3rd, Kukreja RC, Sholley
MM. Heat Shock Provides Delayed Protection against Oxidative Injury in Culture
19.
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Human Umbilical Vein Endothelial Cells.” J Mol Cell Cardiol. 1998;30(12):2739-2749. Johansson PI, Haase N, Perner A, Ostrowski SR. Association between
sympathoadrenal activation, fibrinolysis, and endothelial damage in septic patients: A
21.
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prospective study. J Crit Care. 2014;29(3):327-33. Reitz A, Spiers JP. The relationship between the MMP system, adrenoceptors and
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phosphoprotein phosphatases. Br J Pharmacol. 2012;166(4):1225-43. 22.
Peng Z, Ban K, LeBlanc A, Kozar RA. Intraluminal tranexamic acid inhibits
intestinal sheddases and mitigates gut and lung injury and inflammation in a rodent model of hemorrhagic shock. J Trauma Acute Care Surg. 2016 Aug;81(2):358-365.