Yonsei Med J.  2010 May;51(3):438-447. 10.3349/ymj.2010.51.3.438.

Knockdown of Moesin Expression Accelerates Cellular Senescence of Human Dermal Microvascular Endothelial Cells

• Lee JH
• Yoo JH
• Oh SH
• Lee KY
• Lee KH

• KMID: 1074998
• DOI: http://doi.org/10.3349/ymj.2010.51.3.438

Abstract

PURPOSE
Endothelial cells maintain the homeostasis of blood, which consists of plasma and cellular components, and regulate the interaction between blood and the surrounding tissues. They also have essential roles in vascular permeability, the circulation, coagulation, inflammation, wound healing, and tissue growth. The senescence of endothelial cells is closely related to the aging of the adjacent tissues and to age-related vascular disease. Recently, the expression of moesin was found to be decreased in elderly human dermal microvascular endothelial cells (HDMECs), and an association between moesin and senescence has been suggested. This study examined the functional role of moesin in cellular senescence.
MATERIALS AND METHODS
To study the effects of decreased moesin expression on cellular senescence and metabolism, HDMECs were transfected with short hairpin-RNA (shRNA) lentivirus to silence moesin gene expression. In addition, specimens from young and old human skin were stained with anti-moesin and anti-p16 antibodies as an in vivo study.
RESULTS
Using shRNAl-entivirus, moesin knock-down HDMECs developed characteristics associated with aging and expressed senescence associated-beta-galactosidase during early passages. They also showed increased p16 expression, decreased metabolic activity, and cell growth retardation. Human skin tissue from elderly persons showed decreased moesin expression and increased p16 expression.
CONCLUSION
These findings suggest that there is a functional association between moesin expression and cellular senescence. Further study of the functional mechanism of moesin in the cytoskeleton and cellular senescence is needed. In addition, this study provides a useful model for developing anti-aging treatments.

Keyword

Aging; endothelial cell; shRNA lentivirus; moesin; p16

MeSH Terms

Aged, 80 and over
Antigens, CD31/metabolism
Blotting, Western
Cell Aging/genetics/*physiology
Cell Line
Child
Endothelial Cells/*cytology/*metabolism
Humans
Immunohistochemistry
Microfilament Proteins/genetics/*physiology
Microscopy, Phase-Contrast
Microvessels/*cytology
RNA, Small Interfering/genetics/physiology
Reverse Transcriptase Polymerase Chain Reaction
Skin/*blood supply

Figure

• 

  Fig. 1 Confirming the infection of HDMEC by shRNA-lentivirus. Control vector-treated HDMECs and shRNA-lentivirus-treated HDMECs showed RFP expression at p6, p10, and p20. HDMEC, human dermal microvasular endothelial cell; shRNA, short hairpin-RNA; CTR, control; CTR-V, control-virus; RFP, red fluorescent protein.

• 

  Fig. 2 Comparison of senescent level in HDMEC transfected with shRNA-lentivirus during subculture. Control vector-treated HDMECs without moesin knock-down (CTR-V) from passage 12, moesin knock-down HDMECs (Moesin-V) from passage 6, and HDMEC at over passage 25 (CTR-Aged) were stained as blue with SA-β-gal staining. HDMEC, human dermal microvasular endothelial cell; SA-β-gal, senescence-associated-β-galactosidase.

• 

  Fig. 3 Doubling time and doubling level of cumulative cell population. (A) The doubling time of Moesin-V group was longer than those of CTR-V group at passage 6 to 22. (B) Doubling level of Moesin-V group was much less than that of CTR or CTR-V group. Error bars indicates standard deviations. These experiments have been performed in a pair three times.

• 

  Fig. 4 Expression of moesin/p16 RNA and protein. (A) Moesin RNA expression in Moesin-V group and CTR-Aged group was lower than that in CTR-V group. (B) In comparison with CTR-V group, CTR-Aged and Moesin-V groups showed higher RNA expression of p16. (C) Applying Western blotting, reduced expression of moesin and higher expression of p16 were observed in Moesin-V group, compared to CTR-V group at passage 14. Left panels, autoradiographs of mRNA or protein levels; right panels, relative amounts of mRNA or protein as estimated by an imaging analyzer. The values obtained were normalized by β-actin mRNA or protein content. Error bars indicates standard deviations. These experiments have been performed in a pair three times.

• 

  Fig. 5 Comparison of cellular metabolic activity. Cellular metabolic activity was measured by MTT assay (48 hours). Cellular metabolic activity significantly decreased in CTR-Aged group (passage 39) and Moesin V group compared to CTR-V group (*p = 0.016, **p = 0.012). Mann-Whitney U-test was performed for statistical analysis. Error bars indicates standard deviations. These experiments have been performed in a pair three times.

• 

  Fig. 6 Immunohistochemical staining of moesin and p16. (A) Immunohistochemical staining of moesin and p16 was performed on the neonatal prepuce (Age 1) and the skin of the 10-year-old child (Age 10) and the 86-year-old adult (Age 86). Moesin was stained well as pink or brown in Age 1 and Age 10, on the other hand, moesin was not detected well in Age 86. In contrast, p16 stained cells were not detected well in Age 1 and Age 10 and numerous p16 stained cells were observed in Age 86. (B) Comparable CD31 positive cells and tissues were observed at double staining of moesin/CD31 and p16/CD31 between Age 10 and Age 86. Double stained cells were observed as purple.

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