Endothelial JCAD Worsens Acute Ischemic Stroke Outcomes by Enhancing Inflammation in Response to Ischemia/Reperfusion

Abstract

The role of junctional protein associated with coronary artery disease (JCAD) in acute ischemic stroke (AIS) has not been investigated yet. To investigate its potential as a therapeutic target, transient middle cerebral artery occlusion was induced in JCAD knockout mice, with improvement of stroke outcome and reduced blood-brain barrier permeability and expression of vascular cell adhesion molecule (VCAM)-1. JCAD plays a deleterious role in ischemia/reperfusion cerebral damage and associates with higher 90-day mortality in patients with AIS. JCAD may thus represent a novel prognostic biomarker for patients with AIS, as well as a therapeutic target.

Keywords: acute ischemic stroke; blood-brain barrier; endothelial cells; hypoxia/reoxygenation; inflammation.

Graphical abstract

Figure 1

Role of JCAD Expression in a Murine Model of Acute Brain Ischemia/Reperfusion by Transient Middle Cerebral Artery Occlusion Effect of total JCAD knockout (ko) (Jcad^−/−) on ischemic volume (A and B), motor coordination, assessed by RotaRod test (C), and neurological status, assessed by Bederson scale score (D) Wild-type (Jcad^+/+) littermates were used as control mice; n = 7 Jcad^−/− mice; n = 6 Jcad^+/+ mice. Effect of endothelial-specific JCAD knockout (eJcad^−/−) on ischemic volume (E and F), motor coordination (G), and neurological status (H). Jcad “floxed” (Jcad^fl/fl) littermates were used as control mice; n = 9 eJcad^−/− mice; n = 7 Jcad^fl/fl mice. Effect of postischemic JCAD silencing by small interfering RNA (siJCAD) on ischemic volume (I and J), motor coordination (K), and neurological status (L). RNA scramble (siSCR)-treated littermates were used as control mice; n = 7 siJCAD mice; n = 7 siSCR mice. Data are presented as mean ± SEM. Student’s t-test (A, E, I) and 2-way repeated measures analysis of variance with Sidak’s correction for multiple comparisons (C, D, G,H, K, and L); ∗P < 0.05; ∗∗P < 0.01; and ∗∗∗P < 0.001.

Figure 2

Effect of H/R Injury in Human Brain Microvascular Endothelial Cells After JCAD Silencing Cells were exposed to normoxia (A, C, and E) or hypoxia/reoxygenation (H/R) injury (B, D, and F). Cell death rate, assessed as lactic dehydrogenase (LDH) release in silenced (siJCAD) and unsilenced (siSCR) cells (A and B). Endothelial monolayer integrity, assessed as trans-endothelial electric resistance, in silenced and unsilenced cells (C and D, and E and F). Data are presented as mean ± SEM. n = 6, Student’s t-test (A, B, E, and F) and 2-way repeated measures analysis of variance with Sidak’s correction for multiple comparisons (C and D); ∗P < 0.05; ∗∗P < 0.01; and ∗∗∗P < 0.001. AUC = area under the curve; other abbreviations as in Figure 1.

Figure 3

Molecular Effects of JCAD Silencing in Human Brain Microvascular Endothelial Cells Exposed to Nx or H/R Injury Protein expression was assessed by Western Blot. Vascular endothelial cadherin (VE-cadherin) (A), occludin (B), claudin 5 (C), vascular cell adhesion molecule (VCAM)-1 (D), and intercellular adhesion molecule (ICAM)-1 (E). Data are presented as mean ± SEM. n = 6, 1-way analysis of variance with Tukey’s correction for multiple comparisons; ∗P < 0.05; ∗∗P < 0.01; and ∗∗∗P < 0.001. Nx = normoxia; other abbreviations as in Figures 1 and 2.

Figure 4

Role of Endothelial JCAD Expression in the Pathophysiology of Acute Brain Ischemia Acute brain ischemia has been reproduced in mice with endothelial-specific JCAD deletion (eJcad^−/−) by transient middle cerebral artery occlusion. Effect of endothelial JCAD deletion on blood-brain barrier integrity, assessed by extravascular IgG immunostaining (A and B) after transient middle cerebral artery occlusion. Effect of endothelial JCAD deletion on endothelial inflammation, assessed by immunostaining for the vascular endothelial cell adhesion molecule (VCAM)-1 and the endothelial marker CD31 (C and D). Jcad “floxed” (Jcad^fl/fl) littermates were used as control mice. Data are presented as mean ± SEM. Student’s t-test, n = 8 eJcad^−/− mice; n = 7 Jcad^fl/fl mice. ∗P < 0.05; ∗∗P < 0.01; and ∗∗∗P < 0.001. ko = knockout.

Figure 5

Role of the PI3K/Akt Signaling in Mediating the Physiological Effect of JCAD in Acute Brain Ischemia Human brain microvascular endothelial cells were exposed to Nx or H/R injury. Protein expression was assessed by Western blot analysis. Phosphorylation of Akt (A), ERK (B), and p38 (C) in siJCAD and siSCR human brain microvascular endothelial cells; n = 6, 1-way analysis of variance. Endothelial monolayer integrity, assessed as transendothelial electric resistance, in unsilenced cells, silenced cells, and silenced cells treated with the PI3K/Akt inhibitor wortmannin (D to G); n = 6, 2-way analysis of variance (D and F) and Student’s t-test (E and G). Acute brain ischemia has been reproduced in Jcad “floxed” (Jcad^fl/fl), endothelial-specific JCAD knockout mice (eJcad^−/−), and eJcad^−/− mice treated with wortmannin by transient middle cerebral artery occlusion (H and I). Data are presented as mean ± SEM. One-way analysis of variance with Tukey’s correction for multiple comparisons (A to C, E, G, and H) and 2-way repeated measures analysis of variance with Sidak’s correction for multiple comparisons (D and F); n = 8 eJcad^−/− + wortmannin mice; n = 7 eJcad^−/− + vehicle; n = 7 Jcad^fl/fl mice. ∗P < 0.05; ∗∗P < 0.01; and ∗∗∗P < 0.001. Abbreviations as in Figure 1, Figure 2, Figure 3.

Figure 6

Circulating Levels of JCAD in Patients With Acute Ischemic Stroke Circulating levels of JCAD assessed in control subjects (n = 18) and in patients with acute ischemic stroke (n = 23) 6 and 24 hours after the onset of symptoms. Data are presented as median, maximum, minimum, and Q1-Q3. Kruskal-Wallis test with Dunn’s correction was used for multiple comparisons; ∗P < 0.05; ∗∗P < 0.01; and ∗∗∗P < 0.001 (A). Nonlinear relationship between circulating JCAD levels and 90-day mortality. The dashed horizontal line indicates no effect with median JCAD values serving as reference. For visual clarity, the dose-response curve has been truncated at the 90th percentile of the JCAD distribution. The model has been adjusted for age, National Institutes of Health Stroke Scale (NIHSS) at admission, CRP, and history of heart disease. Covariates are all dichotomized (B). Kaplan-Meier curve depicting the survival of patients with high vs low circulating levels of JCAD (C). Linear relationship between JCAD and CRP. Linear regression was adjusted for NIHSS (dichotomized), history of heart disease, and heart rate (D).