Atherosclerosis is the main cause of many complications of cardiovascular and cerebrovascular disease. Many factors, such as smoking can lead to initiation of atherosclerosis.1 Epidemiological studies have shown that both active and passive cigarette smoking increase the risk of atherosclerosis.2 Although evidence has shown that active cigarette smoking can stimulate atherogenesis, very little is known about the biological processes induced by passive cigarette smoking that contribute to atherosclerosis. Although it has been reported that second-hand smoke could induce atherogenesis in a mouse model,3 the research about the effect of second-hand smoke on the injury in the human vascular wall is seldom considered.
As we know, atherosclerosis is an inflammatory disease of arterial walls.4 In the early stage of atherosclerosis, dysfunction of the vascular endothelium is currently thought to represent the most important event, lots of inflammatory cells are recruited to the endothelial layer from the circulation. The factors that mediate the process include many cell adhesive molecules such as vascular cell adhesion molecule-1 (VCAM-1) and inflammatory chemokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-8.5 They attract inflammatory cells to migrate into the endothelial layers and accelerate the inflammation of arterial walls. The study aimed to investigate the expression in human arterial walls of a few of biological markers (platelet endothelial cell adhesion molecule-1 (PECAM-1); α-smooth muscle actin (α-SMA); collagen IV (Col IV)) and inflammatory markers, VCAM-1, MCP-1, IL-8, by immunostaining and reverse transcription-polymerase chain reaction (RT-PCR). Measurements were following in vitro exposure to a second-hand smoke solution (sidestream whole, SSW) for 48 hours. We discussed a possible mechanism of inflammatory injury of arterial walls induced by passive smoking.
Second-hand smoke solutions (preparation and quantification6) primary antibodies and secondary antibodies were provided by Prof. Manuela Martins-Green's Lab at Department of Neurology & Cell Biology, California University, Riverside, USA. Human aortal wall was derived from a brain dead donor who was male, 34 years old, having been healthy without history of smoking or any chronic disease in his life-time. We drew the arterial wall from his body 4 minutes after he died (warm ischemia time). The donation is authorized by Ethic Committee of Shanghai First People's Hospital.
The human aortal tissue was cut into pieces of about 0.5 cm × 0.8 cm by longitudinal dissociation. Samples were put into a 24-well culture dish, filled in with 1.0 ml of three different mixtures of SSW and DMEM culture media (GIBCO, USA). The 24-well culture dish was placed in incubator for 48 hours. The medium was replaced with fresh media once after 24 hours. According to different type of media, the samples were assigned into three groups: Group A: 1:40 dilution SSW + DMEM; group B: 1:20 SSW + DMEM; group C: DMEM as the control group. Parts of the sample were embedded in Tissue-Tek Optimal Cutting Temperature (OCT, Sakura Finetek, USA) and prepared for frozen sections, and the rest of the sample was stored in liquid nitrogen for RT-PCR.
Cross frozen sections of the human aortal walls (5 μm thick) were used for immunostaining. In brief, after the sections were fixed, incubated and blocked, mouse anti-human PECAM-1 (1:200), Col IV (1:200), α-SMA (1:150), VCAM-1 (1:100), IL-8 (1:150), rat anti-human-MCP-1 (1:150) were used as primary antibodies overnight 4°C. Goat anti-mouse-IgG-FITC PECAM-1 (1:200), Col IV (1:200), α-SMA (1:200), VCAM-1 (1:200), IL-8 (1:50), and goat anti-rat-IgG-FITC MCP-1 (1:50) were used as secondary antibodies the following day. Finally, sections were mounted with antifading mounting medium (Beyotime Institute of Biotechnology, China). All images were captured by using an Olympus microscope equipped with a video camera (ZEISS CO., Germany).
Total RNA was extracted using Trizol reagent (Shanghai Sangon, China) from three groups of arterial wall tissues. RT-PCR was performed using β-actin specific primers as the internal standard, and the synthesis of First Strand cDNA was followed by the recommended protocol of RevertAid First Strand cDNA Synthesis Kit (Fermentas Life Sciences, Canada). It was followed by PCR amplification with 2×Taq PCR MasterMix (Tiangen Biotech, Beijing, China). Cycling conditions were (1) 94°C 3 minutes; (2) 94°C 30 seconds, 55°C 30 seconds, 72°C 1 minute for extension at 30 cycles; (3) 72°C 5 minutes to extend the strands for IL-8, and (1) 94°C 5 minutes; (2) 94°C 60 seconds, 60°C 60 seconds, 72°C 1 minute for extension at 35 cycles; (3) 72°C 5 minutes to extend the strands for MCP-1. Primers used for the amplification were:7,8 IL-8 (404 bp): 5′-GGGTCTGTT-GTAGGGTTGCC-3′, 5′-TGTGGATCCTGGCTAGCAG-A-3′; MCP-1 (171 bp): 5′-CCCCAGTCACCTGCTGT-TAT-3′, 5′-TGGAATCCTGAACCCACTTC-3′; β-actin (712 bp): 5′-CAAAGGGTCGTGCGTGACAT-3′, 5′-GA-ACTTTGGGGGATGCTCGC-3′. RT-PCR was analyzed by electrophoresis in 1.5% agarose.
Results were analyzed by one-way analysis of variance (ANOVA) using SPSS 11.0. Data were expressed as mean ± standard deviation (SD) from six to nine independent experiments. A P <0.05 was considered statistically significant.
We identified endothelial cells by staining for PECAM-1, smooth muscle cells by staining for α-SMA, and subendothelial layer by staining for Col IV. No distinct difference was observed between 1:20 SSW and the control group with these biological markers (Figure 1).
Immunostaining of VCAM-1 (Figure 2A) shows a deposit of VCAM-1 in the subendothelial layer and smooth muscle cell layers, which are near the endothelium of the arterial wall, is increased in the SSW group (Figure 2Ab, Figure 2Ac) compared to the control group (Figure 2Ad) which has no positive staining. By semi-quantitative analysis of immunofluorescence the intensity of VCAM-1 in the 1:40 SSW group (0.35±0.04) and 1:20 SSW group (0.40±0.04) are significantly higher than that in the control group (0.12±0.04) (P <0.001) (Figure 2B).
Immunostaining of MCP-1 (Figure 3A) and IL-8 (Figure 4A) shows that the deposit of these factors in the subendothelial and smooth muscle cell layers, which are near the endothelium of arterial wall, are strongly stained in the SSW group as compared with the control group. The fluorescence intensities of MCP-1 (Figure 3B) in the 1:40 SSW group (0.34±0.05) and 1:20 SSW group (0.52±0.09) are significantly stronger than in the control group (MCP-1: 0.06±0.02), and the fluorescence intensities of IL-8 (Figure 4B) in the 1:40 SSW group (0.37±0.05) and 1:20 SSW group (0.51±0.07) are also significantly stronger than in the control group (0.24±0.03) (P <0.001). MCP-1 mRNA (Figure 3C) expression in the 1:40 SSW (0.15±0.04) and 1:20 SSW (0.19 ±0.06) group is significantly higher than in the control group (0.09±0.03, P 0.001). IL-8 mRNA (Figure 4C) expression in the 1:40 SSW (0.64±0.12) and 1:20 SSW (0.72±0.13) groups is also significantly higher than in the control group (0.49±0.13) (P <0.05, P <0.01).
Vascular inflammatory injury is the basis of the pathogeneses of atherosclerosis:9 the early events in atherosclerosis are stimulated by some inflammatory components, such as cytokines with proinflammatory activity (IL-1β; tumor necrosis factor-alpha, TNF-α; IL-6),10 inflammatory chemokines (MCP-1, IL-8) and adhesive molecules (VCAM-1).11 They interact with each other, promoting inflammatory cells such as neutrophil, monocytes, macrophages to adhere and migrate to the local place of inflammatory lesion and aggravate inflammation.12
Human aortal wall tissue was, for the first time, used in our study of passive smoking by SSW. We carried out the preliminary study on cultured tissue for 24 hours, but we did not find any expression of these inflammatory cytokines (data not shown). Then we extended the culture time to 48 hours and found significant changes.
The dilution of SSW was taken from the previous study of Bernhard et al.13 They used a dilution of about 2% and 4% smoke solution to expose human umbilical vein endothelial cell in vitro. Therefore, we chose the dose of SSW of 1:20 and 1:40 dilution (smoke solution: media, about 2.5%-5.0% concentration of SSW solution) to ensure that the endothelial cell will not be killed and the dilution of SSW is similar to the exposure of tissue of passive smokers in vivo.
Effects of SSW on biological markers in human aortal walls
When we observed the structure of the arterial walls under a light microscope, we found no distinct changes of endothelial cells, smooth muscle cells or of the subendothelial layer of the arterial wall when stimulated by 1:20 SSW for 48 hours as compared to without SSW The previous studies of endothelium lesions, when stimulated by SSW, are focused on the structural changes seen under the electronic microscope. These included microtubule collapse and breakdown, cytoskeletal and intermediate filament breakdown, leading to detachment of endothelial cells.13 This indicated that it will be helpful to investigate lesions of the endothelium simulated by second-hand smoke, by doing research under the electronic microscope in future studies.
Effects of SSW on inflammatory markers in human aortal walls
We found that SSW stimulated the increasing expression of VCAM-1 as detected by immunostaining. VCAM-1 expression in the subendothelial layer and smooth muscle cell layers, which are near the endothelium of the arterial wall, is stronger in the 1:20 SSW than in the 1:40 SSW group. The control group has no positive staining in these layers. VCAM-1 is a cytokine which could potentially contribute to the promotion of leukocyte adhere to the endothelium, and migration into the deep endothelium.14 It activates signals within endothelial cells resulting in the opening of an “endothelial cell gate” through which leukocytes migrate.15 Additionally, VCAM-1 is also expressed vascular smooth muscle cells and influences its proliferation and differentiation.16 A report has shown that the serum concentration of VCAM-1 was elevated significantly in patients with acute coronary syndrome.17 So our study also indicated that second-hand smoke could stimulate the inflammatory lesion in the arterial wall by release this adhesion molecule and could be related to atherosclerosis.
We also found that the expression of the inflammatory attractant factors IL-8 and MCP-1 are significantly increased following stimulation by SSW solutions; especially high dose SSW (1:20). IL-8 is an example of a CXC chemokine, which possess cysteine residues with an intervening amino acid.18 IL-8 bears principal responsibility for recruitment of neutrophils and monocytes, and directs inflammatory cells to migrate to inflammatory sites. In addition to recruitment, IL-8 also serves to stimulate the neutrophil to a higher activation state.19 Additionally, it may be produced early in the inflammatory response and has also been linked to the development of atherosclerosis.7 So IL-8 is a strong inflammatory factor, and the increase of its secretion suggested that inflammatory injury occurred locally in the arterial wall. MCP-1 is a CC chemokine, which does not have the intervening amino acid between the cysteines. MCP-1, which is secreted from endothelial cells, attracts monocytes to adhere to endothelial cells and to migrate into the atherosclerotic lesion.20 Plasma levels of MCP-1 showed significant increases in cases of unstable angina and acute myocardial infarction.21 Our results implied that second-hand smoke solution induces the inflammatory reaction in the arterial wall by expression and release of these inflammatory chemokines, indicating that passive smoking is closely related with inflammatory injury in the human arterial wall.
It is implied that second-hand smoke solutions induce the inflammatory reaction of the arterial wall by release of inflammatory factors even though there is no distinct structural change of the arterial walls viable under the light microscope. This indicates that passive cigarette smoking is related to inflammatory injury in the human arterial wall and highlights a novel potential mechanism by which passive smoking induces or accelerates the early inflammatory stage of atherosclerosis. As a result, we should not only control the expansion of smokers, but also initiate limits to the harm of passive smoking to nonsmokers. We should build more awareness to decrease second-hand smoke in public in order to reduce the threat of atherosclerosis to human's health.
We thank Manuela Martins-Green Lab Neorology & Cell Biology for presenting the second-hand smoke solution and all the mono-antibodies for immunostaining. We also acknowledge the donors for the aorta arterial wall and the help of Department of Surgery of Shanghai First People's Hospital.
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