CD133+Exosome Treatment Improves Cardiac Function after Stroke in Type 2 Diabetic Mice

Abstract

Cardiac complications post-stroke are common, and diabetes exacerbates post-stroke cardiac injury. In this study, we tested whether treatment with exosomes harvested from human umbilical cord blood derived CD133+ cells (CD133+Exo) improves cardiac function in type 2 diabetes mellitus (T2DM) stroke mice. Adult (3-4 m), male, BKS.Cg-m+/+Lepr^db/J (db/db, T2DM) and non-DM (db+) mice were randomized to sham or photothrombotic stroke groups. T2DM-stroke mice were treated with phosphate-buffered saline (PBS) or CD133+Exo (20 μg, i.v.) at 3 days after stroke. T2DM sham and T2DM+CD133+Exo treatment groups were included as controls. Echocardiography was performed, and mice were sacrificed at 28 days after stroke. Cardiomyocyte hypertrophy, myocardial capillary density, interstitial fibrosis, and inflammatory factor expression were measured in the heart. MicroRNA-126 expression and its target gene expression were measured in the heart. T2DM mice exhibit significant cardiac deficits such as decreased left ventricular ejection fraction (LVEF) and shortening fraction (LVSF), increased left ventricular diastolic dimension (LVDD), and reduced heart rate compared to non-DM mice. Stroke in non-DM and T2DM mice significantly decreases LVEF compared to non-DM and T2DM-sham, respectively. Cardiac dysfunction is worse in T2DM-stroke mice compared to non-DM-stroke mice. CD133+Exo treatment of T2DM-stroke mice significantly improves cardiac function identified by increased LVEF and decreased LVDD compared to PBS treated T2DM-stroke mice. In addition, CD133+Exo treatment significantly decreases body weight and blood glucose but does not decrease lesion volume in T2DM-stroke mice. CD133+Exo treatment of T2DM mice significantly decreases body weight and blood glucose but does not improve cardiac function. CD133+Exo treatment in T2DM-stroke mice significantly decreases myocardial cross-sectional area, interstitial fibrosis, transforming growth factor beta (TGF-β), numbers of M1 macrophages, and oxidative stress markers 4-HNE (4-hydroxynonenal) and NADPH oxidase 2 (NOX2) in heart tissue. CD133+Exo treatment increases myocardial capillary density in T2DM-stroke mice as well as upregulates endothelial cell capillary tube formation in vitro. MiR-126 is highly expressed in CD133+Exo compared to exosomes derived from endothelial cells. Compared to PBS treatment, CD133+Exo treatment significantly increases miR-126 expression in the heart and decreases its target gene expression such as Sprouty-related, EVH1 domain-containing protein 1 (Spred-1), vascular cell adhesion protein (VCAM), and monocyte chemoattractant protein 1 (MCP1) in the heart of T2DM-stroke mice. CD133+Exo treatment significantly improves cardiac function in T2DM-stroke mice. The cardio-protective effects of CD133+Exo in T2DM-stroke mice may be attributed at least in part to increasing miR-126 expression and decreasing its target protein expression in the heart, increased myocardial capillary density and decreased cardiac inflammatory factor expression.

Keywords: CD133; Cardiac function; Echocardiography; Exosomes; Stroke; Type 2 diabetes mellitus.

Fig.1. CD133+Exo treatment reduces body weight and blood glucose in T2DM-stroke mice

a) Time line of experiments. At 28 days after stroke, CD133+Exo treatment significantly b) decreases body weight and blood glucose without altering cholesterol level and c) does not decrease lesion volume when compared to PBS treated T2DM-stroke mice. T2DM-stroke+PBS: n=7; T2DM-stroke+CD133+Exo: n=8.

Fig.2. Stroke and T2DM induce cardiac dysfunction in mice and CD133+Exo treatment improves cardiac function in T2DM-stroke mice

a) Compared to non-DM mice, T2DM mice exhibit significantly reduced left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVSF), heart rate, and increased left ventricular diastolic dimension (LVDD). In non-DM and T2DM mice, stroke significantly decreases LVEF at 1 month compared to non-DM and T2DM sham mice, respectively. T2DM-stroke mice have significantly reduced LVEF, heart rate and increased LVDD compared to non-DM-stroke mice at 1 month after stroke. b) Stroke in T2DM mice significantly decreases LVEF and increases LVDD at 1 month after stroke but not at 3 days after stroke. CD133+Exo treatment significantly increases LVEF and decreases LVDD but does not significantly increase LVSF compared to PBS treated T2DM-stroke mice. There were no significant differences in heart rate between stroke and control or between PBS and CD133+Exo treated T2DM-stroke mice. c) Representative images of M-mode transthoracic echocardiography measurements in short-axis view. Non-DM: n=10; Non-DM-stroke: n=10; T2DM: n=10; T2DM-stroke+PBS: n=7; T2DM+CD133+Exo: n=7.

Fig.3. CD133+Exo treatment reduces body weight and blood glucose but does not improve cardiac function in T2DM mice

a) Representative images of M-mode transthoracic echocardiography measurements in short-axis view. b) CD133+Exo treatment in T2DM mice does not significantly affect cardiac function evaluated by echocardiography at 1 month after treatment. c) CD133+Exo treatment in T2DM mice significantly decreases body weight and blood glucose compared to baseline values as well as T2DM sham mice at 1 month. T2DM: n=10; T2DM+CD133+Exo: n=7

Fig.4. CD133+Exo treatment decreases cardiomyocyte hypertrophy and cardiac fibrosis while increasing myocardial capillary density in T2DM-stroke mice

a) CD133+Exo treatment significantly decreases cardiomyocyte cross section area (MCSA) compared to PBS treated T2DM-stroke mice, indicated by FITC-Peanut agglutinin (FITC-PNA) immunostaining and quantification analysis. b) CD133+Exo treatment of T2DM-stroke mice significantly decreases interstitial fibrosis in the heart indicated by decreased interstitial collagen fraction (ICF) in PicroSirius Red (PSR) immunostaining. c) CD133+Exo treatment significantly increases myocardial capillary density compared to PBS treated T2DM-stroke miceindicated by Rhodamine-labeled Griffoniasimplicifolia lectin (lectin) immunostaining and quantification analysis. T2DM-stroke+PBS: n=7; T2DM-stroke+CD133+Exo: n=8. d) In-vitro, CD133+Exo treatment significantly increases capillary tube formation of mouse brain endothelial cells compared to media control.

Fig.5. CD133+Exo treatment significantly decreases cardiac oxidative stress in T2DM-stroke mice

CD133+Exo treatment in T2DM-stroke mice significantly decreases oxidative stress marker a) NADPH oxidase-2 (NOX2) expression and b) 4-Hydroxynonenal (4-HNE) expression compared to PBS treated T2DM-stroke mice. T2DM-stroke+PBS: n=7; T2DM-stroke+CD133+Exo: n=8.

Fig.6. CD133+Exo treatment significantly decreases cardiac inflammation, increases miR-126 expression and decreases its target genes in T2DM-stroke mice

CD133+Exo treatment significantly decreases cardiac tissue inflammatory factor expression such as a) transforming growth factor-β (TGF-β), b) M1 macrophage (ED1) and c) monocyte chemoattractant protein 1 (MCP1) compared to PBS treated T2DM-stroke mice. d) CD133+Exo have higher miR-126 expression compared to EC-Exo. e) CD133+Exo treatment significantly increases cardiac miR-126 expression and decreases its target gene Spred-1, VCAM and MCP-1 expression measured using PCR analysis. T2DM-stroke+PBS: n=7; T2DM-stroke+CD133+Exo: n=8.