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Published in final edited form as: Microvasc Res. 2016 May 31;107:76–82. doi: 10.1016/j.mvr.2016.05.005

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

MARGARET MARKIEWICZ 1,*, KAVIN PANNEERSELVAM 1, NATALIA MARKS 2
PMCID: PMC4938740  NIHMSID: NIHMS793941  PMID: 27260080

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 48 hours. 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: Klotho, Angiogenesis, dermal microvascular endothelial cells, dermal fibroblasts

Introduction

Angiogenesis is a process through which new vessels are formed from pre-existing capillaries or venules. The whole process is divided into two phases: the activation phase and the resolution phase. During activation phase there is increased vascular permeability, detachment of periendothelial cells from endothelium, degradation and remodeling of basement membrane followed by migration and increased cell division of endothelial cells. 1–5 In the resolution phase, proliferation and migration of endothelial cells is decreased and the basement membrane is rebuilt leading to vessel maturation.3 The molecular and cellular mechanisms that regulate these processes are under investigation. Cell migration is essential to angiogenesis. This process is directionally regulated by chemotactic, haptotactic, and mechanotactic stimuli and further involves degradation of the extracellular matrix to enable progression of the migrating cells.6

The human Klotho gene encodes the α-Kl protein. Three α-Kl protein types with possibly different functions have been identified: a full-length transmembrane α-Kl, a secreted α-Kl and a truncated soluble α-Kl (sKl). sKl is a protein released from the cell membrane and after entering the urine and/or the blood, sKl functions as a hormone.7 The transmembrane Kl protein, homologous to β-glucuronidase was shown to be required for FGF23 (fibroblast growth factor 23) -mediated receptor activation. Kl binds to multiple FGFRs and increases their affinity for FGF23. Klotho-FGFR co-expression delineates the tissue specificity of FGF23 effects.8,9 Klotho which is an anti-aging gene plays an important role in angiogenesis. It has been shown in hindlimb ischemia heterozygously K1 gene deficient mice model that capillary density is decreased and Kl plays a role in restoration of blood flow in these mice.10 Additionally, in mice which lack the Kl gene, aortic-ring culture assay demonstrated reduced angiogenesis accompanied by reduced endothelium derived nitric oxide release.11 Studies published by Kusaba at al., reported that vascular endothelium in Klotho deficient mice is hyperpermeable because of increased apoptosis and decreased expression of VE-cadherin (vascular endothelial).12 Interestingly, Kl suppresses tumor necrosis factor-α (TNF-α) induced expression of adhesion molecules such as intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells.13 These adhesion molecules are essential for formation of new vessels.14 Although previous studies have shown that Kl has anti-apoptotic and anti-senescent effects on endothelial cells,15 Klotho’s role in migration and proliferation of endothelial cells is not well understood. Expression of Kl in human umbilical vein endothelial cells (HUVECs) decreases with cellular senescence suggesting the role of this gene in aging as well as in age related vascular diseases.16

It is known that angiogenesis requires an interaction between cells and extracellular matrix (ECM). However, the contribution of the cellular and fibrillar microenvironment in angiogenesis still remains unresolved. Fibroblasts and extracellular matrix deposited by these cells are the major players involved in this process. Matrix metalloproteinases (MMPs) are proteinases which take part in ECM degradation. Moreover, these proteinases play a significant role in many biological processes, such as embryogenesis, normal tissue remodeling, wound healing, and angiogenesis.17 MMP’s especially MMP-2 and MMP-9 play a key role in angiogenesis by degrading basement membrane and other ECM components, allowing endothelial cells to detach and migrate into new tissue.18,19 They are also involved in the release of ECM bound proangiogenic factors (bFGF, VEGF and TGF β).19

This is the first report showing expression of Kl in HDMECs and HFb. In this study, we will investigate the possible role of Kl in migration and proliferation of these cells. Furthermore, we will explore the role of Kl in regulation of genes involved in the activation of endothelial cells and ECM remodeling and organization of connective tissue.

Materials and methods

Cell Culture

Human dermal microvascular endothelial cells and human dermal fibroblasts were isolated from foreskins as previously described.20 The cells were cultured on collagen type I coated flasks in the presence of endothelial cell growth medium 5% EBM2- MV (endothelial basal cell growth medium) with supplements (Lonza, Inc.) and incubated at 37°C with humidified 95% air/5% CO2. Human dermal fibroblasts culture was established from foreskins of healthy newborns from the Medical University of South Carolina Hospital in compliance with the Institutional Review Board for Human Studies.

Immunohistochemistry

Klotho expression was identified in HDMECs and HFb. α-SMA (NeoMarkers Inc.) was detected in HFbs by IHC, according to the previously described protocol.21 Isolated HDMECs were labeled with Dil-Ac-LDL (Low Density Liproprotein acetylated and labeled with fluorescent probe Dil), according to manufacturer’s protocol (Biomedical Technology Inc.).

Quantitive Real time PCR (qRT-PCR)

Total RNA was isolated from HDMECs or HFb using RNeasy Mini Kit (Qiagen, Inc). Briefly, 5μg of total RNA was reverse transcribed with random hexamers using Transcriptor First Strand cDNA Synthesis Kit (Roche, Inc) according to manufacturer’s protocol.

B2MG (beta-2 microglobulin) was used as a control. Amplification of product was performed using the PTC-200 Peltier Termal Cycler at the reaction condition described by Tang et al.22.

Table 1.

Primers for real time RT-PCR

B2MG F 5′-GGCATTCCTGAAGCTGACAG -3′, R 5′-TGGATGACGTGAGTAAACCTG -3′
VCAM1 F 5′-TCTACGCTGACAATGAATCCTG -3′, R 5′-AGGGCCACTCAAATGAATCTC -3′
PECAM1 F 5′-GAGTCTGGAGAGGACATTGTG- 3′, R 5′-CTTCTGCTTGGTCCAAAATGC -3′
MMP9 F 5′-CAGTTTCCATTCATCTTCCAAGG-3′, R 5′-CATCACCGTCGAGTCAGC -3′
KLOTHO F 5′-GACCACCAAGAGAGATGATGC -3′, R 5′-CTGTAACCTCTGTGCCACTC -3′
LAMB3 F 5′-CTTCACTGGACTCACCTACG -3′, R 5′-GCACACTGGTCACATTTGG - 3′
MMP8 F 5′-GCAACCCTATCCAACCTACTG -3′, R 5′-CGACTCTTTGTAGCTGAGGATG -3′
MMP15 F 5′-CTTCTCCAGCACTGACCTG - 3′, R 5′-TTGTCAACGTCCTTCCACTG -3′
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Western Blot analyses

Confluent untreated HDMECs, HFb or transfected with scrRNA (control), Klotho siRNA (siKl) (Origene, Inc) or treated with recombinant human Klotho (rhKL) (0.5μg/ml) (Prospec, Inc.) were lysed in RIPA buffer. Protein concentration was quantified using the BCA Protein Assay kit (Pierce, Rockford, IL). Forty micrograms of protein was separated via SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA) which was then blocked with TBST (Tris buffered saline-Tween) at RT for 1 hour and probed ON with primary antibody directed against Klotho (Thermo Fisher Scientific Inc.) or MMP9, PECAM-1, VCAM-1, LAMB3, MMP8, MMP15 (Santa Cruz Biotechnology Inc). Following washes with TBST, blots were incubated with appropriate conjugated secondary antibody and developed with ECL kit (Pierce). As a control for equal protein loading, membranes were stripped and re-probed for β-actin using a monoclonal antibody to β-actin (Sigma).

Co-Immunoprecipitation assay

Co-IP assay was performed as described previously by Maltare et al.,23. Briefly, proteins were extracted (500μg) following the procedures described for Western blot analysis. The supernatant with primary antibody (FGFR1, Thermo Fisher Scientific Inc.) or IgG-isotype control (Abcam) was shaken ON/4°C and the next day incubated with 70 μl of agarose beads (Protein G sepharose, Amersham ) at 4 °C for 2 hrs. The samples were cleared with RIPA buffer and centrifuged twice at 3000 rpm for10 min. Washed beads were suspended in an equal volume of 5xSDS sample loading buffer, boiled, and the supernatant was loaded onto 10% polyacrylamide gel. Immunoblots were processed as above.

Cell migration wound healing (“scratch”) assay

HDMECs and HFb were plated on collagen type I coated or uncoated twelve well plates respectively. The cells were cultured to 80% confluence in 5% EBM-2 medium or 10% DMEM respectively followed by addition of rhKl (0.5μg/ml), transfection with scrRNA or siKl and proceed as previously described by Markiewicz et al.,1.

Transwell assay

24-multiwell plate with cell culture HTS FluoroBlok Inserts (BD Falcon, Inc) were used in this assay. At 90% confluence dermal fibroblasts seeded on the bottom wells (4×104 cells per well) were transduced with siKl, scrRNA or treated with rhKl in 1% DMEM. Next day, the medium was removed and replaced with 1% EBM2 medium and 2×104 HDMECs were plated on the cell culture inserts. Twenty four hours later, inserts were removed and the migrated cells attached to the bottom surface of the membrane were stained with DAPI and counted in five fields of view under the microscope.

Cell proliferation analysis

Cell proliferation assay was performed using Cell Proliferation BrdU incorporation assay (Millipore, INC) as described previously by Markiewicz et al.,1.

Microarrays

HDMECs or HFb were transfected with siKl or scrRNA. Obtained RNA samples were converted into cDNA using the RT2 First Strand Kit (Roche, Inc.). The cDNA was then mixed with RT2 SYBR Green Mastermix. This mixture was aliquoted into the wells of the RT2 Profiler PCR Array of Human Endothelial Cell Biology or Extracellular Matrix& Adhesion Molecules PCR Array (SaBiosciences, Inc). Each of the PCR Array profiled the expression of 84 genes related to human endothelial cell biology or for cell-cell and cell-matrix interactions. PCR was performed and finally the relative expression was determined using data from the real-time cycler and the ΔΔCT method.

Statistical analysis

Results were compared using Students’ unpaired t test. Asterisk symbol indicate statistically significant values:* P<0.05.

Results

The expression of Klotho in human dermal microvascular endothelial cells and human dermal fibroblasts

To investigate the role of Klotho in the HDMECs and HFb the mRNA and protein expression of Klotho was examined in these cells (Fig. 1A–D). Our data have shown that 60% of endothelial cells were Klotho positive as compared to ~ 80% of positive fibroblasts (A,D). These data show an abundant expression of this protein in the primary cells isolated from foreskins which may suggest that endogenous Klotho may have an effect on migration and proliferation of both cell types.

Figure 1.

Figure 1

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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.

Lack of Klotho abrogates migration of HDMECs

Furthermore, we sought to determine whether Kl is involved in migration of HDMECs and HFb using wound healing “scratch” assay. This assay measures cell migration toward the injured sites resulting in the closure of the wound/scratch. Confluent monolayers of endothelial cells or fibroblasts were transfected with siKl, scrRNA or treated with rhKl. Pictures were taken at 48 hours and representative images are shown in Fig. 2D,E. We observed that blockade of endogenous Klotho abrogated endothelial cell migration after 48 hours. On the other hand, addition of Klotho significantly enhanced migration of endothelial cells and fibroblasts. The migration rate of HDMECs and HFb at 48 hours reached 50% and 40% respectively (Fig. 2F,G). These results suggest that Klotho plays the promigratory role in both cells type. To check whether blockage of Kl or addition of rhKl to fibroblasts will influence HDMECs migration we used transwell assay as described in the Materials and method section. Relative number of migrated cells is presented in Fig. 2H. Migration of endothelial cells significantly increased in the wells with fibroblasts treated with rhKl.

Figure 2.

Figure 2

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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.1 Bars represent mean ± SEM. Asterisk symbol indicate statistically significant values:* P<0.05.

Lack of Klotho changed proliferation of HDMECs

Furthermore, we assessed endothelial cell and fibroblast proliferation after addition of rhKl and/or rhFGF23 (5ng/ml), or transfection with siRNAs (Fig. 3A,B). It was demonstrated that Kl protein is required for FGF23-mediated receptor activation. Treatment of HDMECs with Kl alone or in association with FGF23 increased cell proliferation ~30% within 36 hours (Fig. 3A). In the absence of Kl with addition of FGF23 ECs proliferation decreased indicating that Kl is required for FGF23 effect on ECs proliferation. On the other hand, we observed ~ 20% in decrease of fibroblast proliferation after addition of Kl and in association with FGF23. Taken together, this data suggests that lack of Klotho significantly diminishes proliferation of HDMECs and HFb.

Figure 3.

Figure 3

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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 previously1. Bars represent mean ± SEM. There were not statistically significant values: P>0.05

Lack of interaction between Kl and FGFR1 in HDMECs

It has already been shown that Kl is a co-receptor with FGFR in transduction of the FGF23 signaling pathway in the bone and kidney (7, 24–26). Using a Co-IP assay, we next examined the Kl-FGFR1 interaction in HDMECs. Our result showed that Kl was not able to bind to FGFR1 effectively (Fig. 4). This result indicates that Kl-FGFR1 interaction is not essential for Kl activation and proangiogenic function.

Figure 4.

Figure 4

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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.

Microarray analyses in HDMECs and HFb after blocking endogenous Klotho

Microarray analyses were used to investigate the effects of Klotho on gene expression in HDMECs and HFb (Fig. 5A). We employed two different commercially available PCR Arrays as described in the Materials and method section. We performed the analyses in HDMECs and HFb after suppression of Kl with siRNA oligos. We observed several genes upregulated or downregulated in both cell types after blocking an endogenous Klotho (Fig. 5A,B,C,D). This screen identified genes involved in the activation of endothelial cells (MMP9, PECAM-1, VCAM-1) suggests changes in activation phase of angiogenesis (Fig. 5A, B, C). An absence of Kl in dermal fibroblasts lead to changes in the expression of genes involved in ECM remodeling and organization of connective tissue (LAMB3, MMP8, MMP15). Messenger RNA levels of target genes were examined by qRT-PCR. As shown in Figure 4A, levels of mRNA for MMP9, PECAM-1 and VCAM-1 were diminished in HDMECs by approximately 20–40%, while in dermal fibroblasts, the expression level of LAMB3, MMP8, MMP15 were increased between 1.2–1.6 fold after suppression of Kl (Fig. 5A). Western blotting analysis confirmed differential expression of MMP9, PECAM-1, VCAM-1, LAMB3, MMP8 and MMP15 at the protein level (Fig. 5C,D). These data strongly suggest that Kl may play a role in regulating genes that promote vascular remodeling.

Figure 5.

Figure 5

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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

Discussion

This is the first report showing a different expression level of Klotho gene in HDMECs and human dermal fibroblasts. Studies done by Carracedo et al.,16 have demonstrated that Klotho protein is expressed in cytoplasm of human umbilical vein endothelial cell (HUVEC) and is reduced in these cells with cell senescence. Klotho is an anti-aging gene playing an important role in angiogenesis. Angiogenesis is a formation of new blood vessels including two phases: activation and resolution phase. Activation phase contains endothelial cell activation, proliferation, adhesion and migration. 2,15,16 We have shown that lack of Kl abrogates the migration of endothelial cells (Fig. 2C,E) which may influence microvasculature formation and proper functioning in human skin. Furthermore, our microarray analyses have shown that lack of Kl decreased expression of VCAM-1 and PECAM-1 in HDMECs. Decreased expression of these proteins, which are known to be important for ECs migration may explain our results in Figure 2C,E. On the other hand, addition of recombinant human Kl significantly increased migration of HDMECs demonstrating a pro-migratory role of Kl. Data published by Park at al., demonstrated that lack of PECAM-1 had a significant influence on endothelial cell-cell and cell-matrix interactions, resulting in reduction of cell migration and capillary morphogenesis.27,28 These changes were associated with decreased expression of endothelial nitric oxide and nitric oxide bioavailability in PECAM-1 deficient retinal ECs. Furthermore, we have shown decreased expression of MMP9 in HDMECs (Fig. 4A,B,D). Endothelial expression of MMP9 is implicated as an essential for angiogenesis. 29,30 MMP9 degrades native collagen type IV, constituent of basement membranes which lead to vascular cell migration and invasion.31,32 New capillary formation is dependent on the neighboring extracellular architecture. It is known that angiogenesis requires an interaction between cells and ECM. Fibroblasts and extracellular matrix deposited by these cells are the major players involved in this process. Fibroblasts secrete MMPs that participate in remodeling of basement membrane and ECM.19 We demonstrated that absence of Kl in dermal fibroblasts leads to an increase in the expression of genes involved in ECM remodeling and organization of connective tissue. Specifically, lack of Kl increases MMP8 and MMP15 expression level in dermal fibroblasts (Fig. 4A,C,D) which may lead to decreased ECM deposition by fibroblasts needed to support the endothelial cell tubulogenesis. Cell proliferation is a part of activation phase of angiogenesis. We found that absence of Kl decreases proliferation of HDMECs and HFb (Fig. 3A,B). On the other hand, addition of Kl increased proliferation of ECs ~30% and diminished fibroblast proliferation ~20%. It has been shown that Klotho-FGF complex is necessary for eliciting the FGF23 mediated signaling events in bone. 25, 26 We have shown that Klotho is a necessary cofactor for FGF23 effects on proliferation of human ECs and human fibroblasts (Fig. 3A,B). To examine whether Kl can cooperate with FGFR1 in HDMECs we performed a Co-IP experiment (Fig. 4A). Our result demonstrated that Klotho did not bind to FGFR1. Because of the complex nature of Kl protein, it is possible that, in addition to FGFR1, other regulatory proteins/kinases might be involved in Kl activation and proangiogenic function in primary HDMECs. It should also be noted that, primary HDMECs, which were obtained from different donors are non-homogenous endothelial cells. Additionally, the endothelial cells obtained from different tissues may exhibit different tissue-specific gene expression profile 33. It has been suggested by other authors that the pathway by which Kl influences angiogenesis is comparable to action of known growth factor such as VEGF.10 Even though Kl gene is mainly expressed in the kidney; its anti-aging phenotypes are multi-systemic. Klotho has been shown to inhibit fibrosis associated with chronic airway diseases by suppressing TGFβ-1/Smad3 expression 34 and to suppresses renal fibrosis by partially inhibiting FGF2 signaling.35 On the other hand, Kl depletion can increase inflammatory or fibrotic mediators which may exacerbate inflammation, fibrosis, and cancer progression.36–38 The pathways involved in which Kl mediates angiogenesis should be intensively studied.

Conclusions

In conclusion, this study shows for the first time that Klotho is implicated in the process of angiogenesis of human dermal microvasculature. Specifically, lack of Kl may affect the migration of HDMECs by changing the expression of genes which play an important role in this process. Additionally, present studies suggest that Kl may influence gene expression in dermal fibroblasts leading to changes in ECM. Further investigations are required to explore the precise mechanism of these changes and investigate whether Kl has a role in resolution phase of angiogenesis.

Highlights.

  • Klotho is expressed in microvascular endothelial cells and dermal fibroblasts

  • Lack of Kl abrogates migration of human dermal microvascular endothelial cells

  • Lack of Kl enhances migration of human dermal fibroblasts

  • Decrease in gene expression involved in the activation of endothelial cells

Acknowledgments

The study was supported by grants from the National Institute of Health, K01AG031909 (MM) and the South Carolina Clinical & Translational Research (SCTR) Institute, with an academic home at the MUSC, through NIH - NCATS Grant Number UL1 TR001450.

Footnotes

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