Role of Klotho in migration and proliferation of human dermal microvascular endothelial cells

Abstract

Purpose: To examine the possible role of Klotho (Kl) in human microvasculature.

Methods: The expression level of Kl in primary human dermal microvascular endothelial cells (HDMECs) and primary human dermal fibroblasts (HFb) was detected by real-time polymerase chain reaction amplification (qRT-PCR), Western blot analyses and immunohistochemistry. Migration of HDMECs and HFb was examined in monolayer wound healing "scratch assay" and Transwell assay. Proliferation of these cells was examined using Cell Proliferation BrdU incorporation assay.

Results: Our results have shown that downregulation of Kl abrogated HDMECs migration after 48h. On the other hand, migration of HFb significantly increased after blocking Kl. Lack of Kl decreased expression of genes involved in the activation of endothelial cells and enhanced expression of genes involved in extracellular matrix remodeling and organization of connective tissue.

Conclusions: This study for the first time provides the evidence that Kl is expressed in HDMECs and HFb. Additionally, we have demonstrated that Kl is implicated in the process of angiogenesis of human dermal microvasculature.

Keywords: Angiogenesis; Dermal fibroblasts; Dermal microvascular endothelial cells; Klotho.

Figure 1

Expression of Klotho in HDMECs (A,B,C), and HFb (A,B,D). (A) qRT-PCR analysis shows Kl mRNA in both HDMECs and HFb. Kl mRNA is normalized for B₂MG (beta-2 microglobulin) mRNA. (B) Klotho protein level was determined by Western blot analysis. The blot was probed O/N with primary antibody at 4°C.β-actin was used as a control for equal loading. (C,D) Cultured HDMECs and HFb were isolated from foreskins and subjected to IHC for Klotho. Immunoreactivity was detected by using DAB substrate kit. C(−) represents the negative control (no primary Ab) in HDMECs and HFb. Isolated HDMECs were labeled with Dil-Ac-LDL and visualized under fluorescence microscopy. α-SMA is the positive control for HFb. Bars represent mean ± SEM. Asterisk symbol indicate statistically significant values:* P<0.05.

Figure 2

Migration of HDMECs was significantly diminished by blocking Klotho. qRT-PCR analyses in HDMECs and HFb (A). Western blot analyses of Kl in HDMECS (B) and HFb (C). HDMECs or HFb were cultured to 70% confluence in full medium followed by addition of rhKl or transfection with ScrRNA or siKl. After 24hrs, cells were stained with SYTOgreen followed by cells scratches and floating cells were removed by washing with 1xPBS. Incubation was continued for 48 hours in 1% serum EBM2 (endothelial basal cell growth medium) with addition of Mitomycin C (10μg/ml) to prevent cell proliferation. Representative images are presented (D,E). The bar graphs represent migration rate expressed as percentage of cells that crossed into the “scratch” area in comparison with identical “nonscratch” area (F,G) Graphical presentation of the transwell co-culture migration assay (H). Briefly, dermal fibroblasts (C-ECs only) were untreated, transfected with indicated siRNA or rhKl was added. Next day, the medium was changed and HDMECs were plated on the cell culture inserts. 24 hours later the inserts were removed and migrated endothelial cells were counted as previously described. Bars represent mean ± SEM. Asterisk symbol indicate statistically significant values:* P<0.05.

Figure 3

Proliferation of endothelial cells decreased after blocking endogenous Klotho. (A,B) Cell proliferation of HDMECs or HFb was performed using Cell proliferation BrdU incorporation assay as described previously. Bars represent mean ± SEM. There were not statistically significant values: P>0.05

Figure 4

Interaction between KL and FGFR1 in HDMECs. No Co-IP complexes of KL and FGFR1 were formed in HDMECs. Kl and FGFR1 was detected by IB (Immunoblot) with KL and FGFR1 antibody respectively. Negative control-IgG-isotype control.

Figure 5

Changes in gene expression after blocking Klotho in HDMECs (A,B,C) and HFb (A,C,D). Samples were assayed by qRT-PCR to determine mRNA levels (B) or by Western blot analysis (C,D) to determine protein levels for the genes indicated. Fold change shown in the graph (A) is normalized to control B2MG (beta-2-microglobulin). The experiments were performed three times and representative blots are shown. Band intensities were quantified by densitometric analysis (D). Bars represent mean ± SEM.*P<0.05