Yonsei Med J.  2019 Nov;60(11):1036-1044. 10.3349/ymj.2019.60.11.1036.

Adipose-Derived Stem Cell Transplantation Inhibits Vascular Inflammatory Responses and Endothelial Dysfunction in Rats with Atherosclerosis

• Fan M
• Bai J
• Ding T
• Yang X
• Si Q
• Nie D

• KMID: 2460220
• DOI: http://doi.org/10.3349/ymj.2019.60.11.1036

Abstract

PURPOSE
This study aimed to investigate the effect of adipose-derived stem cell (ADSC) transplantation on atherosclerosis (AS) and its underlying mechanisms.
MATERIALS AND METHODS
In our study, rat AS model was established, and ADSCs were isolated and cultured. Atherosclerotic plaque and pathological symptoms of thoracic aorta were measured by Oil Red O staining and Hematoxylin-Eosin staining, respectively. Total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were measured by an automatic biochemical analyzer. Expressions of vascular endothelial growth factor (VEGF), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), aortic endothelin-1 (ET-1), interleukin-6 (IL-6), c-reactive protein (CRP), and tumor necrosis factor α (TNF-α) were measured by enzyme linked immunosorbent assay, VEGF, VCAM-1, ICAM-1, ET-1, respectively, and NF-κB p65 mRNA expressions were detected by quantitative real-time polymerase chain reaction. Protein expressions of VEGF, VCAM-1, ICAM-1, ET-1, NF-κB p65, p-NF-κB p65, and IκBα were measured by western blot. Moreover, NF-κB p65 expression was measured by immunofluorescence staining.
RESULTS
ADSC transplantation alleviated the pathological symptoms of aortic AS. ADSC transplantation decreased the levels of TC, TG, and LDL-C and increased serum HDL-C level. Meanwhile, ADSC transplantation decreased the levels of IL-6, CRP, and TNF-α in AS rats. Moreover, the expressions of VEGF, ET-1, VCAM-1, and ICAM-1 were decreased by ADSC transplantation. ADSC transplantation inhibited phosphorylation of NF-κB p65 and promoted IκBα expression in AS rats.
CONCLUSION
Our study demonstrated that ADSC transplantation could inhibit vascular inflammatory responses and endothelial dysfunction by suppressing NF-κB pathway in AS rats.

Keyword

ADSC; atherosclerosis; endothelial dysfunction; inflammatory responses; NF-κB pathway

MeSH Terms

Animals
Aorta, Thoracic
Atherosclerosis*
Blotting, Western
C-Reactive Protein
Cholesterol
Endothelin-1
Enzyme-Linked Immunosorbent Assay
Fluorescent Antibody Technique
Intercellular Adhesion Molecule-1
Interleukin-6
Lipoproteins
Phosphorylation
Plaque, Atherosclerotic
Rats*
Real-Time Polymerase Chain Reaction
RNA, Messenger
Stem Cell Transplantation*
Stem Cells*
Triglycerides
Tumor Necrosis Factor-alpha
Vascular Cell Adhesion Molecule-1
Vascular Endothelial Growth Factor A
C-Reactive Protein
Cholesterol
Endothelin-1
Intercellular Adhesion Molecule-1
Interleukin-6
Lipoproteins
RNA, Messenger
Tumor Necrosis Factor-alpha
Vascular Cell Adhesion Molecule-1
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1 Successful construction of rat atherosclerosis (AS) model (n=8 in each group). (A) Hematoxylin-Eosin (HE) staining of aortic cross-sections. Arrows indicate atheromatous plaque. Magnification: ×200. (B) Oil Red O staining of aortic sections. Magnification: ×400. (C) Ratio of I/(I+M) (I=thickness of aortic intima, M=thickness of aortic media). (D) The levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured by automatic biochemical analyzer. Data are presented as mean±standard deviation, with repetition of three times. Bar=100 µm. *p<0.05, †p<0.01, ‡p<0.001, vs. Normal group.

• 

  Fig. 2 Identification of adipose-derived stem cells (ADSCs). (A) Morphological characteristics of ADSCs at three passages. Magnification: ×200. Bar=100 µm. (B) Expressions of ADSC surface antigens (CD34, CD44, CD45, and CD90) were detected by flow cytometry.

• 

  Fig. 3 Adipose-derived stem cells (ADSC) transplantation alleviated the pathological symptoms of aortic atherosclerosis (AS) (n=8 in each group). (A) Expression of PKH26-labeled ADSCs in aortic tissues of rats. Bar=100 µm. (B) Hematoxylin-Eosin (HE) staining of aortic sections. Magnification: ×200. (C) Ratio of I/(I+M) (I=thickness of aortic intima, M=thickness of aortic media). Data are presented as mean±standard deviation, with repetition of three times. ‡p<0.001, vs. Normal group and Normal+ADSCs group; ∥p<0.01, vs. AS group.

• 

  Fig. 4 Adipose-derived stem cell (ADSC) transplantation decreased the levels of lood lipids and inflammatory cytokines in atherosclerosis (AS) rats (n=8 in each group). (A) The levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured by automatic biochemical analyzer. (B–D) Expression of interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and c-reactive protein (CRP) was detected by Enzyme linked immunosorbent assay. Data are presented as mean±standard deviation, with repetition of three times. †p<0.01, ‡p<0.001, vs. Normal group and Normal+ADSCs group; §p<0.05, ∥p<0.01, ¶p<0.001, vs. AS group.

• 

  Fig. 5 Adipose-derived stem cell (ADSC) transplantation inhibited endothelial dysfunction in atherosclerosis (AS) rats (n=8 in each group). (A–D) The levels of vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) were detected by Enzyme linked immunosorbent assay. (E) The mRNA expressions of VEGF, ET-1, VCAM-1, and ICAM-1 were measured by quantitative real-time polymerase chain reaction (qRT-PCR). (F) Protein expressions of VEGF, ET-1, VCAM-1, and ICAM-1 were measured by western blot. Data are presented as mean±standard deviation, with repetition of three times. †p<0.01, ‡p<0.001, vs. Normal group and Normal+ADSCs group; §p<0.05, ∥p<0.01, ¶p<0.001, vs. AS group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 6 Adipose-derived stem cell (ADSC) transplantation inhibited NF-κB signaling pathway in atherosclerosis (AS) rats (n=8 in each group). (A) Expression of NF-κB p65 was measured by immunofluorescence staining. Scale bar=20 µm. (B) Expression of NF-κB p65 was detected by quantitative real-time polymerase chain reaction (qRT-PCR). (C) Expressions of NF-κB p65, p-NF-κB p65, and IκBα were measured by western blot. Data are presented as mean±standard deviation, with repetition of three times. †p<0.01, ‡p<0.001, vs. Normal group and Normal+ADSCs group; §p<0.05, ∥p<0.01, vs. AS group. DAPI, 4′, 6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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