High-throughput profiling of the circulating proteome suggests sexually dimorphic corticosteroid signaling following ischemic stroke

Abstract

Increasing evidence suggests that there are innate differences between sexes with respect to stroke pathophysiology; however, the molecular mechanisms underlying these differences remain unclear. In this investigation, we employed a shotgun approach to broadly profile sex-associated differences in the plasma proteomes of a small group of male (n = 6) and female (n = 4) ischemic stroke patients. Peripheral blood was sampled during the acute phase of care, and liquid chromatography electrospray ionization mass spectrometry was used to quantify plasma proteins. We observed widespread differences in plasma composition, as 77 out of 294 detected proteins were significantly differentially expressed between sexes. Corticosteroid-binding globulin (CBG), a negative acute-phase reactant that inversely regulates levels of bioactive free cortisol, was the most dramatically differentially regulated, exhibiting 16-fold higher abundance in plasma from women relative to men. Furthermore, functional annotation analysis revealed that the remaining differentially expressed proteins were significantly enriched for those involved in response to corticosteroid signaling. Plasma CBG levels were further examined in an additional group of male (n = 19) and female (n = 28) ischemic stroke patients, as well as a group of male (n = 13) and female (n = 18) neurologically normal controls. CBG levels were significantly reduced in male stroke patients relative to male controls; however, no differences were observed between female stroke patients and female controls, suggesting that women may exhibit an attenuated cortisol response to stroke. Collectively, our findings reinforce the idea that there are sex-associated differences in stroke pathophysiology and suggest that cortisol signaling should be investigated further as a potential molecular mediator.

BACKGROUND

Stroke has emerged as one of the most prevalent health burdens in modern society, as it is currently the second leading cause of death and third leading cause of disability worldwide (14). It is becoming increasingly evident that there are pathophysiological aspects of stroke that are sexually dimorphic. There are notable differences between sexes with regards to stroke incidence, etiology, severity, and outcome. Women exhibit lower rates of stroke at premenopausal ages than male counterparts, but higher rates of stroke later in life (19, 37). Furthermore, women experience more severe strokes, as well as higher rates of disability and mortality (3); while these differences are partially attributed to the fact that women are typically older at stroke onset (37), several studies have reported these trends independently of age (13, 28, 40), and similar sex-associated effects have been observed in animal models (19, 43).

Biologically, the mechanisms that drive these sex differences are not yet fully understood. While it is believed that these differences are at least in part driven by the direct physiological effects of estrogen (25), the fact that several sex differences persist following menopause (13, 28, 40) and independently of estrogen status (15) suggests additional mechanisms that have yet to be elucidated. Better characterization of these mechanisms has the potential to provide valuable insights into the molecular mediators that modify stroke risk and influence outcome.

As with many disease states, the circulating proteome is intricately involved in stroke pathology. For example, numerous circulating proteases, factors, and cofactors play an integral role in coagulation and fibrinolysis; dysregulation of these hemostatic processes is often the direct driver of acute embolic and thrombotic cerebrovascular events (42). In addition, circulating cytokines, hormones, and acute-phase reactants shape the peripheral immune response to stroke (9), which is becoming increasingly recognized as having a significant influence on outcome; several common secondary complications such as poststroke infection, edema, and hemorrhagic transformation can be linked to underlying immunologic mechanisms (10, 18, 22, 34). Thus, examination of the circulating proteome may be able to provide further insights into the molecular mechanisms that underlie sexually dimorphic aspects of stroke pathophysiology. In this study, we employed shotgun mass spectrometry to broadly profile sex-associated differences in the plasma proteomes of a small group of ischemic stroke patients during the acute phase of care.

METHODS

Experimental design.

Peripheral venous blood was sampled from a discovery cohort consisting of 6 male and 4 female ischemic stroke patients during the acute phase of care. Liquid chromatography electrospray ionization mass spectroscopy (LC-ESI-MS) was used for global identification of plasma proteins, and the relative levels of successfully identified proteins were subsequently compared between sexes. Functional annotation enrichment analysis was used to determine whether differentially expressed proteins were enriched for those involved in specific biological processes or signaling pathways. Proteins exhibiting the most dramatic sex associated differences in expression in the discovery cohort were further examined by ELISA in a validation cohort consisting of an additional 19 male and 28 female ischemic stroke patients, as well as 13 male and 18 female neurologically normal controls.

Patients.

All 10 discovery cohort subjects were recruited at Ruby Memorial Hospital (Morgantown, WV); 68 validation cohort subjects were recruited at Ruby Memorial Hospital, while 10 were recruited at University of Cincinnati Medical Center (Cincinnati, OH). All ischemic stroke patients displayed definitive radiographic evidence of vascular ischemic pathology on magnetic resonance imaging or computerized tomography according to the criteria for diagnosis of acute ischemic cerebrovascular syndrome (23). All diagnoses were confirmed by an experienced neurologist. All blood was sampled within 24 h of symptom onset and before the administration of thrombolytics. Injury severity was determined according to the National Institutes of Health stroke scale (NIHSS) at the time of blood draw. Infarct volume was assessed via tracing of neuroradiographic images as described previously (32, 33). Demographic information was collected from either the subject or a significant other by a trained clinician. Procedures were approved by the institutional review boards of West Virginia University, Ruby Memorial Hospital, and University of Cincinnati Medical Center. Written informed consent was obtained from all subjects or their authorized representatives before study procedures.

Blood collection.

Peripheral venous blood samples were collected from subjects via K₂EDTA vacutainer (Becton Dickenson, Franklin Lakes, NJ) and processed within 30 min of blood draw. Vacutainers were spun at 2,000 g for 10 min at room temperature to sediment hemocytes, and the resultant plasma was aliquoted and immediately stored at −80°C.

Sample preparation.

Plasma (10 µl) from each sample was thawed and processed for spectroscopy analysis. To enhance the diversity of detected proteins, 14 of the most highly abundant human plasma proteins were depleted with CaptureSelect immunoaffinity beads (Thermo Fisher, Waltham, MA) according to the manufacturer’s recommended protocol. Following depletion, samples were desalted via centrifugal filtration with a 3 kDa molecular weight cut-off spin filter (Amicon Ultra; Millipore-Sigma, Burlington, MA) and lyophilized. Lyophilized protein was reconstituted in 90 µl of ultrapure water, mixed with 90 µl of pH 5.5 digestion buffer (product # PE-151-5KIT; Protea Biosciences, Morgantown, WV) and incubated for 55°C for 1 h. Following incubation, the pH was adjusted to 8.5 with ammonium bicarbonate; trypsin was added at final concentration of 0.3 µg/µl; and samples were microwaved at 850 watts for 15 min. After digestion, the resultant peptides were lyophilized and reconstituted in 100 µl of ultrapure water containing 0.1% formic acid for downstream LC-ESI-MS analysis.

Mass spectrometry.

LC-ESI-MS was performed using an Acquity ultrahigh-performance liquid chromatography unit (Waters, Milford MA) coupled with a Q-Exactive mass spectrometer (Thermo Fisher). The Xcalaber 2.2 software package was used for instrument control and data acquisition (Thermo Fisher). Peptides were separated on an Acquity UPLC BEH C18 analytical column (Waters) at 40°C and a 100 µl/min flow rate using the gradient outline in Table 1. Eluted peptides were ionized in positive ion mode with a spray voltage of 3,800 V, and detection was performed in data-dependent mode; the 10 most intense multiply charged ions in each scan were subjected to analyses. Data were searched against the Uniprot human protein database (41) via the Proteome Discoverer 1.4 software package (Thermo Fisher) using the Sequest algorithm to identify peptides. The search was performed according to the following parameters: trypsin cleavage constraints allowing for up to two missed cleavage sites, variable oxidation on methionine, static carboxyamidomethylation of cysteine, 10 ppm peptide mass tolerance, and 20 ppm fragment mass tolerance. Postsearch analysis was performed with the Scaffold 4 software package (Proteome Software, Portland, OR) with peptide false discovery rate set to 5.0%, and protein probability thresholds set to 99% with two peptides required for identification; spectral counts associated with positively identified proteins were normalized on default settings and subsequently used for intergroup comparison.

Table 1.

HPLC gradient parameters

Time, min	Mobile Phase A,* %	Mobile Phase B,† %	Flow rate, µl/min
1	95	5	100
34	65	35	100
37	20	80	100
39	20	80	100
39.1	95	5	100
42	95	5	100

^*0.1% formic acid in water;

^†0.1% formic acid in acetonitrile.

Functional annotation enrichment analysis.

Functional annotation enrichment analysis was performed using the “topGO” package (2) for R (R project for statistical computing) (21). Biological process ontology annotations associated with detected proteins were retrieved from the Gene Ontology Consortium database via Uniprot accession number using the Uniprot ID mapping tool (4). Differentially expressed proteins were submitted as a query list, and all proteins detected via LC-ESI-MS were submitted as background; Fisher’s exact test was used to identify biological processes enriched in the query list over background via the runtest() function defaults.

ELISA.

Proteins of interest were quantified in EDTA-treated plasma with commercially available colorimetric ELISA kits according to manufacturer’s recommended protocol (product #RD192234200R; BioVendor, Asheville, NC). Absorbance values were obtained with a Synergy HT multimode plate reader (Biotek, Winooski, VT).

Statistics.

Sample sizes were arbitrarily determined based on the availability of subjects. All statistics were performed by R 3.3. Fisher’s exact test was used for comparison of dichotomous variables, while t-test or two-way ANOVA was used for the comparison of continuous variables where appropriate. Strength and significance of correlational relationships were assessed via Spearman’s rho. In the case of all statistical testing, the null hypothesis was rejected when P < 0.05. In the case of multiple comparisons, P values were adjusted via the Benjamini-Hochberg method (7). The parameters of all statistical tests performed are outlined in detail within the figure legends.

RESULTS

Discovery cohort clinical and demographic characteristics.

Male and female stroke patients in the discovery cohort were relatively well matched with respect to age, the prevalence of cardiovascular disease risk factors, and medication status. Furthermore, sexes were well matched in terms of the time from symptom onset to blood draw, and stroke severity as determined by the NIHSS and infarct volume (Table 2).

Table 2.

Discovery cohort clinical and demographic characteristics

	Men (n = 6)	Women (n = 4)	P Value
*Age, yr, mean ± SD	82.5 ± 8.9	82.0 ± 6.9	0.923
†African American, n (%)	0 (0.0)	0 (0.0)	1.000
†Caucasian, n (%)	6 (100.0)	4 (100.0)	1.000
*NIHSS, mean ± SD	10.8 ± 11.5	15.0 ± 9.5	0.551
*Infarct volume, cm3, mean ± SD	5.6 ± 7.5	5.8 ± 4.6	0.973
*Time to blood draw, min, mean ± SD	374 ± 231	336 ± 258	0.817
†Hypertension, n (%)	6 (100.0)	4 (100.0)	1.000
†Dyslipidemia, n (%)	5 (83.3)	4 (100.0)	1.000
†Diabetes, n (%)	2 (33.3)	2 (50.0)	1.000
†Atrial fibrillation, n (%)	3 (50.0)	3 (75.0)	0.571
†Hypertension medication, n (%)	4 (67.7)	4 (100.0)	0.466
†Diabetes medication, n (%)	2 (33.3)	2 (50.0)	1.000
†Cholesterol medication, n (%)	3 (50.0)	3 (75.0)	0.571
†Anticoagulant or antiplatelet, n (%)	4 (67.7)	2 (50.0)	1.000
†Current smoker, n (%)	0 (0.0)	0 (0.0)	1.000

NIHSS, National Institutes of Health stroke scale; SD, standard deviation.

^*Means compared via two-sample two-tailed t-test;

^†Proportions compared via 2×2 Fisher’s exact test.

Differential expression of plasma proteins.

We observed widespread differences in the composition of plasma samples from male and female stroke patients in the discovery cohort. We successfully detected and identified 297 plasma proteins via mass spectroscopy; of these, 77 exhibited statistically significant differences in expression between sexes after we controlled for multiple comparisons. Interestingly, a majority of significantly differentially expressed proteins were upregulated in females relative to males (Fig. 1). Corticosteroid-binding globulin (CBG), a negative acute-phase reactant that inversely regulates levels of bioactive free cortisol, was the most dramatically differentially regulated, exhibiting 16-fold higher abundance in plasma from women relative to men (Fig. 2). Furthermore, functional annotation enrichment analysis revealed that proteins differentially expressed between sexes were 3.6-fold enriched for those involved in corticosteroid signaling over background. Other significantly enriched biological processes included several involved in thrombosis such as fibrinolysis, coagulation, and platelet activation (Fig. 3).

Fig. 1.

Differential expression of plasma proteins in the discovery cohort. Volcano plot depicting the magnitude and statistical significance of differential expression between male and female stroke patients for all plasma proteins detected via mass spectrometry in the discovery cohort. Fold difference indicates protein abundance in female samples relative to male. Protein abundance was compared between sexes by two-sample two-tailed t-tests, and P values were adjusted for multiple comparisons by the Benjamini-Hochberg method.

Fig. 2.

Expression levels of differentially regulated plasma proteins. Expression levels of plasma proteins differentially regulated between male and female stroke patients in the discovery cohort. Uniprot human protein database accession numbers corresponding with each protein are listed. Fold difference indicates protein abundance in female samples relative to male. Protein abundance was compared between sexes by two-sample two-tailed t-tests, and P values were adjusted for multiple comparisons by the Benjamini-Hochberg method.

Fig. 3.

Biological processes enriched among differentially expressed plasma proteins. Biological processes enriched among plasma proteins differentially expressed between male and female stroke patients in the discovery cohort. Gene Ontology Consortium database accession numbers associated with each process are listed. Significance of enrichment was assessed via Fisher’s exact test, and P values were adjusted for multiple comparisons by the Benjamini-Hochberg method.

Validation cohort clinical and demographic characteristics.

Plasma CBG levels were further analyzed via ELISA in the validation cohort. Irrespective of sex, validation cohort stroke patients displayed a significantly higher prevalence of cardiovascular disease risk factors relative to control subjects, and a higher percentage were medicated with antihypertensives and anticoagulants. With respect to sex, male and female patients within the stroke group were relatively well matched in terms of clinical and demographic factors, as were male and female subjects within the control group (Table 3).

Table 3.

Validation cohort clinical and demographic characteristics

	Stroke (S, n = 47)		Normal (N, n = 31)		P Values
	Male (SM; n = 19)	Female (SF; n = 28)	Male (NM; n = 13)	Female (NF; n = 18)	Main Test	S vs. N	SM vs. SF	NM vs. NF
aAge, mean ± SD	71.3 ± 15	72.3 ± 18.1	60.2 ± 11.1	57.2 ± 9.4	<0.001*	<0.001*	0.804	0.427
bAfrican American, n (%)	1 (5.3)	1 (3.6)	3 (23.1)	3 (16.7)	0.143
bCaucasian, n (%)	18 (94.7)	27 (96.4)	10 (76.9)	15 (83.3)	0.143
cNIHSS, mean ± SD	7.7 ± 6.9	8.9 ± 7.4			0.597
cInfarct volume, cm3, mean ± SD	23.4 ± 20.9	16.8 ± 21.2			0.413
cTime to blood draw, min, mean ± SD	584 ± 397	530 ± 398			0.661
bHypertension, n (%)	13 (68.4)	23 (82.1)	4 (30.8)	4 (22.2)	<0.001*	<0.001*	0.312	0.689
bDyslipidemia, n (%)	10 (52.6)	13 (46.4)	2 (15.4)	5 (27.8)	0.111
bDiabetes, n (%)	5 (26.3)	6 (21.4)	3 (23.1)	1 (5.6)	0.376
bAtrial fibrillation, n (%)	4 (21.1)	9 (32.1)	1 (7.7)	1 (5.6)	0.108
bHypertension medication, n (%)	11 (57.9)	20 (71.4)	4 (30.8)	4 (22.2)	0.004*	0.001*	0.365	0.689
bDiabetes medication, n (%)	4 (21.1)	6 (21.4)	3 (23.1)	0 (0)	0.126
bCholesterol medication, n (%)	7 (36.8)	10 (35.7)	2 (15.4)	4 (22.2)	0.468
bAnticoagulant or antiplatelet, n (%)	11 (57.9)	14 (50)	1 (7.7)	2 (11.1)	<0.001*	<0.001*	0.766	1.000
bCurrent smoker, n (%)	6 (31.6)	6 (21.4)	2 (15.4)	0 (0)	0.057

NIHSS, National Institutes of Health stroke scale; SD, standard deviation.

^aMeans compared via one-way ANOVA with subsequent planned group-wise comparisons by two-sample two-tailed t-test;

^bProportions compared via 2×4 Fisher’s exact test with subsequent planned group-wise comparisons by 2×2 Fisher’s exact test;

^cMeans compared via two-sample two-tailed t-test.

^*Statistically significant.

Differential expression of corticosteroid binding globulin levels.

Consistent with the role of CBG as a negative acute-phase reactant, plasma CBG levels were lower in male stroke patients compared with male controls in the validation cohort. However, this effect was not observed in women, as there were no substantial differences in CBG levels between female stroke patients and female controls. This phenomenon was statistically significant, as two-way ANOVA produced a stroke-sex interaction when we controlled for age, hypertension, and anticoagulatory medication (Fig. 4). Collectively, this suggests circulating levels of CBG are less responsive to stroke in women compared with men.

Fig. 4.

Validation cohort plasma corticosteroid-binding globulin levels. Plasma concentrations of corticosteroid-binding globulin compared between validation cohort male and female ischemic stroke patients and neurologically normal controls. Boxplots indicate mean and SD. Significance was assessed via two-way ANOVA controlling for age, hypertension, and anticoagulatory medication.

CBG levels and stroke severity.

In validation cohort male stroke patients, plasma CBG levels were negatively associated with injury severity as assessed by both NIHSS (Fig. 5A) and infarct volume (Fig. 5D), suggesting that circulating CBG levels are directly responsive to stroke pathology in men. In validation cohort female stroke patients, no significant correlations were seen between plasma CBG levels and either NIHSS (Fig. 5B) or infarct volume (Fig. 5E), further suggesting that the response of CBG to stroke is either attenuated or absent in women.

Fig. 5.

Relationship between corticosteroid-binding globulin (CBG) levels and stroke severity. Associations between plasma CBG levels and National Institutes of Health stroke scale (NIHSS) in male (A), female (B), and all validation cohort stroke patients (C). Associations between plasma CBG levels and infarct volume in male (D), female (E), and all validation cohort stroke patients (F). Strength and significance of correlations were assessed via Spearman’s rho.

DISCUSSION

There are notable differences between sexes with regards to ischemic stroke incidence, etiology, severity, and outcome; however, the molecular basis for these differences is not fully understood. In this study, we broadly profiled the circulating proteomes of male and female ischemic stroke patients to identify potential molecular mediators that could contribute to sex-associated differences in stroke pathophysiology. Our findings infer that women may exhibit an attenuated cortisol response to stroke through differential regulation of CBG, a key regulator of cortisol bioactivity. Interestingly, cortisol signaling is involved in several facets of the acute physiological response to stroke; thus, it is possible that differential cortisol signaling between sexes may contribute to the sexual dimorphism observed in stroke pathophysiology.

CBG, also known as transcortin, is a 405-amino acid alpha globulin produced in the liver that acts as a negative acute-phase reactant and peripheral blood transport protein for steroid-based hormones (31). While CBG has the ability to bind a wide range of hormones including aldosterone, progesterone, and testosterone, it exhibits significantly higher affinity for cortisol, and up to 95% of peripheral blood cortisol circulates bound to CBG (39). Cortisol itself is a pleotropic stress hormone that modulates a wide variety of biological processes such as hemostasis, metabolism, and immunity during both chronic and acute sympathetic nervous system responses (11). CBG-bound cortisol is believed to be biologically inactive; cleavage of CBG at a protease bait domain by enzymes such as neutrophil elastase and chymotrypsin triggers a confirmation change and release of free active cortisol (31). Expectedly, circulating CBG levels drop rapidly in response to a wide variety of acute pathophysiologic stressors such as burns, surgery, and septic shock to accommodate an increase in cortisol activity (6, 8, 16).

Prior studies suggest that cortisol levels rise within hours of stroke onset and can remain elevated for up to a week following the acute event (5); thus, it was unsurprising to observe a decrease in plasma CBG levels in male subjects in response to stroke. However, CBG levels appeared unaffected by stroke in female subjects, indirectly inferring that the cortisol response to stroke may be attenuated in women. Such a phenomenon would be consistent with previous studies in other areas, which have reported that females exhibit attenuated cortisol responses to a variety of both psychological and physiological stressors (17, 24, 26, 27, 46). Because cortisol signaling is broadly involved in the physiological response to stroke, it is possible that differential cortisol signaling between sexes could contribute to clinically observed differences in outcome.

While cortisol signaling is believed to drive the alterations in blood pressure and blood glucose often observed in response to stroke (1, 35), perhaps the most notable way cortisol is involved in stroke pathology with respect to outcome is through its modulation of the stroke-induced peripheral immune response. The peripheral adaptive immune system responds to stroke by shifting into a state of suppression; this response reduces the probability of developing detrimental autoimmune complications but also leaves patients susceptible to poststroke infection (9, 10). Consistent with the known role of cortisol as an immunosuppressant, cortisol is believed to be a key mediator of this phenomenon, as cortisol levels are strongly negatively correlated with poststroke lymphocyte counts (45) and are predictive of poststroke immune complications (20). An attenuated cortisol response to stroke in women, such as that suggested by our results, would act to limit the degree of poststroke adaptive immune suppression; this would be highly consistent with a recent study of over 90,000 stroke cases that revealed that women exhibit lower rates of poststroke infection than men (12). Furthermore, such an effect could also explain preclinical and clinical studies that suggest that females may exhibit stronger autoantigen responses following stroke relative to male counterparts (30, 44).

In addition to CBG, we observed sex-associated differential regulation of numerous plasma proteins involved in a wide array of biological processes in the discovery cohort. The fact that over 25% of proteins successfully identified by mass spectrometry were significantly differentially regulated between sexes reinforces the importance of considering the effect of sex when examining pathophysiological mechanisms in stroke. Interestingly, differentially expressed proteins were substantially enriched for those involved in pathways that are linked to thrombosis such as fibrinolysis, coagulation, and platelet activation. These proteins were almost exclusively upregulated in women relative to men, which is consistent with epidemiological studies suggesting a higher risk of stroke in older females (4a, 29, 36). However, a higher frequency of women in the discovery cohort presented with atrial fibrillation than men, indicating that discovery cohort women may have experienced a greater percentage of cardioembolic strokes, making it difficult to definitively determine whether the observed differential regulation of these pathways was associated with inherent sex differences or instead differences in stroke etiology. Due to the small sample size and such potential confounds, definitive conclusions regarding the effects of sex on these pathways cannot be drawn without further investigation in a larger group of patients.

The drivers behind the collective sex differences in the response of the plasma proteome to stroke that we observed in this investigation are unclear. Because a majority of subjects, particularly in the discovery cohort, were older than the average age of menopause, it suggests that these differences occur independently of the direct effects of estrogen. While we did not have specific information regarding menopause status or hormone replacement therapy, menopause status was indirectly controlled for in our validation cohort statistical analysis by controlling for age. Thus, it is likely that these differences are driven by various genetic, epigenetic, and anatomical differences between sexes that persist postmenopause (19). With respect to CBG in particular, this would be consistent with the fact that sex differences in cortisol response to other stressors have been shown to occur independently of estrogen (26, 27). From a broader perspective, this is further consistent with the fact that many of the differences in stroke pathophysiology that are observed clinically cannot be fully explained by the direct effects of sex hormones.

Due to its exploratory nature, this study was not without limitations. In the current study, we did not have the ability to investigate the relationships between circulating CBG levels and clinical outcomes; thus, the clinical significance of a sexually dimorphic cortisol response to stroke remains unclear. Additionally, there were differences between validation cohort stroke and control subjects with respect to factors such as age, hypertension rates, and medication status that could have potentially confounded our results; however these factors were statistically controlled for in our analysis, and we find it unlikely that the difference in CBG response that we observed between sexes was driven by such potential confounds. It is also important to note that we did not directly measure plasma cortisol levels. Therefore, it possible that the sex-associated differences that we observed in CBG response do not manifest in alterations in levels of biologically active cortisol. However, we find this possibility unlikely, as numerous prior investigations have demonstrated CBG is the primary mediator of plasma free cortisol across a broad range of health and disease states (31), and our functional annotation enrichment analysis suggested alterations to cortisol-related pathways. Nonetheless, future studies that look to confirm this CBG-cortisol relationship within the context of stroke and further explore the relationships between cortisol signaling, sex, the peripheral immune response, and clinical outcome are strongly encouraged.

Collectively, our results reinforce the idea that there are there innate differences in stroke pathophysiology between sexes and suggest that investigation of the circulating proteome may be able to provide insight into the molecular mediators of such differences. More specifically, our findings indicate that women may exhibit an attenuated cortisol response to stroke through differential regulation of CBG and that cortisol signaling should be evaluated further as a potential driver of sex-associated differences in pathophysiology.

GRANTS

Work was funded via Robert Wood Johnson Foundation Nurse Faculty Scholar Award 70319 to T. L. Barr, National Institutes of Health Centers of Biomedical Research Excellence (CoBRE) subawards P20 GM-109098 (to T. L. Barr) and P20 GM-109098 (to P. D. Chantler), and Case Western Reserve University Frances Payne Bolton School of Nursing start-up funds issued to G. C. O’Connell.

DISCLOSURES

G.C.O. and T.L.B. have a patent pending re: genomic patterns of expression for stroke diagnosis. T.L.B. serves as chief scientific officer for Valtari Bio Incorporated. Work by G.C.O. is part of a pending licensing agreement with Valtari Bio Incorporated. G.C.O. has received consulting fees from Valtari Bio Incorporated. The remaining authors report no potential conflicts of interest.

AUTHOR CONTRIBUTIONS

G.C.O. and T.L.B. conceived and designed research; G.C.O., O.A., and P.D.C. performed experiments; G.C.O., K.B.W., E.B., and P.D.C. analyzed data; G.C.O. and T.L.B. interpreted results of experiments; G.C.O. prepared figures; G.C.O., K.B.W., O.A., P.D.C., and T.L.B. drafted manuscript; G.C.O., K.B.W., O.A., P.D.C., and T.L.B. edited and revised manuscript; G.C.O., K.B.W., E.B., O.A., and T.L.B. approved final version of manuscript.