The Effect of Electronic-Cigarette Vaping on Cardiac Function and Angiogenesis in Mice

Abstract

The rapid increase in use of electronic-cigarettes (e-cigarettes), especially among youth, raises the urgency for regulating bodies to make informed decisions, guidance, and policy on these products. This study evaluated cardiac function in an experimental model following exposure to e-cigarettes. We subjected C57BL/6 mice to e-cigarette vaping for 2-weeks, and cardiac function was assessed using echocardiography. Cardiac tissues were collected at the end of e-cigarette exposure for pathological analysis. The experimental data showed that e-cigarette vaping (3 h/day for 14 days) had no significant effect on cardiac contractility as measured by ejection fraction. However, it significantly increased angiogenesis in mouse heart tissue. We found that e-cigarette exposure increased the endothelial cell marker CD31 and CD34 to approximately 2 fold (p < 0.05) in heart tissue from female mice and about 150% (p < 0.05) in male mice. E-cigarette vaping also caused slower weight gain compared to mice exposed to room air. In addition, short-term e-cigarette exposure slightly increased collagen content in heart tissue but did not result in significant tissue fibrosis. These results suggest that short-term exposure to e-cigarettes has no acute effect on cardiac contractile function or tissue fibrosis, but it increases cardiac angiogenesis.

Figure 1

Schematic presentation of the InExpose e-cigarette vaping system.

Figure 2

E-cigarette vaping inhibits mouse bodyweight gain. Mice were exposed to air or e-cigarette vapor for 14 days and bodyweight was measured every two days. The percent change was calculated based on the bodyweight at baseline (one day before exposure). Blood was drawn from heart at the end of last e-cigarette exposure. (A) Plasma concentration of cotinine. (B) Percentage change of bodyweight including both male and female mice. (C) Percentage change of bodyweight in male and female mice, respectively. *Indicates p < 0.05 at the same time point.

Figure 3

Effect of e-cigarette vaping on mouse cardiac function. Echocardiographic measurement was performed at baseline and at the end of the 2^nd week. The heart rate (A), ejection fraction (B) end diastolic dimension (EDD) (C), end systolic dimension (ESD) (D) and aorta dimension (E) were measured and compared between air controls and those subjected to e-cigarette exposure.

Figure 4

Heart weight and heart weight/body weight ratio. (A) The animal heart was collected after euthanization at the end of e-cigarette exposure, and heart weight was measured after removing the aorta and other tissue debris. (B) Heart weight/Body weight (HW/BW) ratio was calculated using the baseline bodyweight.

Figure 5

Effect of e-cigarette exposure on collagen and α-SMA expression in mouse cardiac tissue. Heart tissue homogenates from female (A) and male (B) mice were analyzed using Western blot to probe the collagen I and α-SMA expression. The upper panels were representative Western blot images and the lower panels showed the quantification data.

Figure 6

Effect of e-cigarette exposure on cardiac tissue fibrosis. Formaldehyde fixed and paraffin-embedded heart tissue was cut into 4 µm sections onto a specimen. Trichrome staining was performed and analyzed as described in Method section. Whole slide images were taken by an Olympus whole-slide scanner with a 20x lens.

Figure 7

E-cigarette increases capillary density. (A) Heart tissue sections were immunostained with anti-CD31 antibody and Alexa-594 conjugated secondary anti-rabbit antibody. (B) Heart tissue was immunostained with anti-CD34 antibody and Alexa-594 conjugated secondary antibody. (C) Co-immunostaining of CD31 (green) and CD34 (red) in heart tissue sections. Five fluorescent Images from each slide were taken using a Leica Confocal microscope. The percentage of CD31 or CD34 positive area was analyzed using FiJi ImageJ software. Scale bar is 25 µm.

Figure 8

E-cigarette increase CD31 and CD34 expression in kidney tissue. Kidney tissue from air control (Air) and e-cigarette (E-cig) were co-immunostained with CD31 (green) and CD34 (red) with anti-CD31 and anti-CD34 antibodies. Five fluorescent images from each slide were taken using a Leica confocal microscope. The percentage of CD31 or CD34 positive area was analyzed using FiJi ImageJ software. Scale bar is 25 μm.

Figure 9

Effect of e-cigarettes on CD31 expression in cardiac tissue. Heart tissue homogenates from female or male mice were separated on a SDS-PAGE gel and the expression of CD31 was analyzed using Western blot with anti-CD31.

Figure 10

E-cigarette vaping on plasma VEGF levels. Blood samples were collected at the last day of experiment immediately after e-cigarette or air exposure. Plasma was prepared by centrifugation and plasma levels of VEGF were measured using a commercial ELISA kit.