N-myc Downstream Regulated Gene 1 Promotes Adipocyte Differentiation in Endothelial Cells of Infantile Hemangioma

Dear Editor:

Infantile hemangioma (IH) is characterized by the initial phase of rapid proliferation followed by slow spontaneous involution and a fibrofatty residuum¹. The accumulation of fibrofatty residuum in IH suggests the adipogenesis in the involution. The CD90, CD133, VEGFR, and CD146 are hemangioma-derived stem cell markers that confer multilineage differentiation to endothelial cells, adipocytes, fibroblasts, and neuronal cells. Stem cell reprogramming factors OCT4, SOX2, and MYC are also highly expressed in IH².

N-myc downstream regulated gene 1 (NDRG1) is a distal effector of mTORC2 activation in the cells³. SGK and PKC are the two key phosphorylation substrates of mTORC2⁴. SGK substrates include NDRG1 and FOXO, which regulate cell survival under oxygen or nutrient depletion conditions⁴. In hypoxia, the upregulation of NDRG1 leads to cellular differentiation, proliferation, and redistribution of the cell cycle⁵. Previously, we have reported that NDRG1 expression is increased in the proliferating IHs⁶. A recent research showed that NDRG1 expression and its SGK1-dependent phosphorylation are induced during adipogenesis, and also NDRG1 promotes adipocyte differentiation by inducing PPARγ expression⁶. However, the impact of NDRG1 on adipocyte differentiation in IH involution has not yet been defined.

In accordance, we hypothesized that NDRG1 is involved in the involution phase of IH and regulates adipogenesis of hemangioma-derived endothelial cells (HemECs). To analyze gene expression of HemECs and identify molecular signatures, only CD31 expressing HemECs were isolated from specimens of 4 patients with proliferating IHs. The study was approved by the Institutional Review Board of the Inha University Hospital (IRB no. 2016-10-018). Written informed consent was obtained from all participants. The HemECs were cultured on endothelial cell growth medium MV2 (PromoCell), characterized by flow cytometry (Supplementary Fig. 1A). Normal endothelial cells such as human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HDMECs) were used as controls and cultured in the same medium. In order to construct cDNA libraries with the TruSeq Stranded mRNA LT Sample Prep Kit, total RNA was used. The protocol consisted of polyA-selected RNA extraction, RNA fragmentation, random hexamer primed reverse transcription and 100nt paired-end sequencing by Illumina NovaSeq 6000. The libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide and qualified using an Agilent Technologies 2100 Bioanalyzer. Statistical significance of the differential expression data was determined using independent t-test and fold change in which the null hypothesis was that no difference exists among groups. False discovery rate was controlled by adjusting p-value using Benjamini-Hochberg algorithm. Gene set enrichment analysis for module genes was performed using GSEA Tool⁷.

During the involuting phase, the proliferating capillaries are replaced spontaneously with fibrofatty tissue and capillary-sized channels (Fig. 1A, B). We evaluated the expression of human stem/progenitor cell marker CD133 and induced pluripotent stem cell factors such as SOX2 and c-MYC in HemECs. Immunohistochemical staining for CD133 was detected on both nucleus and membranes in some HemECs (Fig. 1C). Western blot analysis and RNA seq revealed that expression levels of SOX2 and c-MYC were increased in HemECs compared with HUVEC and HDMEC (Fig. 1D, E). This results demonstrated that some CD31+HemECs display stemness marker. In addition, consistent with our previous results⁵, the mTORC2-SGK1-NDRG1-cMYC pathway is activated in HemECs (Supplementary Fig. 1B, C, 2). Using RNA sequencing to compare HDMEC and HemECs, we identified the top 20 gene sets with the most differences including pathways of HIF-1 and FOXO signaling. The heat map showed hypoxia-inducible genes, including NDRG1 were detected in HemECs (Fig. 2A).

Fig. 1. HemECs containing CD133+ stem cell property spontaneously involute and change to adipocytes. (A) Proliferative phase infantile hemangioma (IH). (Left) Clinical picture of proliferating IH. (Right) Well-circumscribed lobules of downregulation packed capillaries composed of plump endothelial cells and pericytes, separated by fibrous septae or by normal intervening tissue (H&E original magnification ×100). (B) Involutive phase IH. (Left) Clinical picture of involulting IH. (Right) Lesional capillaries are replaced with loose fibroadipose tissue. Note residual endothelial cells lined by the thickened and hyalinized basement membranes, reflective of the involutive process (H&E original magnification ×100). (C) HemEC displays stemness markers. Immunostaining for CD133 (green) and CD31 (red), and nuclei staining for 4’,6-diamidino-2-phenylindole (blue), in HemECs. Scale bar=50 mm. HemECs of proliferative-phase stained for the human stem/progenitor cell marker CD133 (green stain, CD133+ cells; blue, nuclei). CD133+ cells are sparse and double-stained for the endothelial marker CD31 (red) (Confocal microscopy, 400×). (D) Protein levels of iPSC factors (c-MYC and SOX2) were upregulated in HemEC. (E) Heat maps for MYC target genes in HemECs compared with HDMECs. We received the patient’s consent form about publishing all photographic materials. Hem-ECs: hemangioma-derived endothelial cells, HUVEC: human umbilical vein endothelial cell, HDMEC: human dermal microvascular endothelial cell.

Fig. 2. N-myc downstream regulated gene 1 (NDRG1) expression is critical for adipocyte differentiation in HemEC. (A) Heat map of the RNA-seq data for the top 20 upregulated pathways in HemECs compared with HDMEC. Shown are heat maps for hypoxia genes including NDRG1. (B) To examine whether NDRG1 regulates IH involution to adipocyte differentiation, HemECs were transfected with NDRG1 and control siRNA (p=0.0029). (C) The knockdown of NDRG1 reduced the level of iPSC factor, c-Myc, but it did not affect the expression of SOX2. Protein levels and mRNA expression of c-Myc were downregulated in siNDRG1-transfected HemEC. mRNA expression measured by RT-PCR was graphed as the relative expression of siNDRG1-transfected HemEC, compared with those transfected with control siRNA. Heat map (right) of MYC genes for siNDRG1 transfected HemECs and siControl transfected HemECs. (D) Neutral lipid staining (Oil Red O) of siControl transfected HemECs and siNDRG1 transfected HemECs. Oil Red O stain in siRNA NDRG1-expressing cells compared to control cells. Scale bar=100 µm and 20 µm. (E) Relative mRNA levels for indicated genes in differentiated control and siRNA NDRG1-expressing cells. Induction of C/Ebpα, FABP4, and leptin transcriptional factors are required for proper adipocyte differentiation. TGF-β signaling pathway functions as a key suppressor of the commitment phase of adipocyte differentiation. The knockdown of NDRG1 in HemECs has increased the mRNA expression of TGF-β. (F) Heat map of adipogenesis genes for siNDRG1 transfected HemECs and siControl transfected HemECs. One representative of 3 experiments is shown. The data represent the mean±standard error of mean. HemECs: hemangioma-derived endothelial cells, HDMEC: human dermal microvascular endothelial cell, siControl: small interfering control, siNDRG1: small interfering N-myc downstream regulated gene 1. Statistical analysis by a student t-test (^*p<0.05, ^**p<0.001).

To investigate the effect of NDRG1 on adipocyte differentiation, the inhibition of NDRG1 by RNA interference was performed using ON-TARGETplus small interfering RNA (siRNA) targeting NDRG1 and Non-targeting siRNA (Dhamacon) according to the manufacturer’s instruction. First, HemECs, HUVECs and HDMECs were knockdown using siRNA NDRG1 transfection for 48 hours before adipogenic differentiation. As shown in Fig. 2C, gene and protein expressions of c-MYC were significantly reduced in HemECs after transfecting NDRG1-siRNA compared to HUVECs and HDMECs, but there was no significant difference in SOX2. Next, we evaluated whether NRDG1 is essential for differentiation into adipocyte in HemECs of IH. Primary HemECs, HUVECs and HDMECs were cultured under differentiation condition into adipocytes for 7 days and the presence of adipocytes was confirmed by using neutral lipid Oil Red O (ORO) staining. NDRG1 knockdown HemECs resulted in reduced adipocytes differentiation and lipid accumulation compared with the control cells. Under control condition, HemECs differentiated into adipocytes (Fig. 2D). The level of ORO stain in NDRG1 knockdown HemECs was profoundly reduced compared with control cells (p=0.008; Fig. 2E). However, HDMEC did not differentiate into adipocytes under the same differentiation condition (Supplementary Fig. 3). We also tested the expression level of the key transcription factors regulating adipocyte differentiation. mRNA levels of PPARγ were markedly reduced (p=0.010). The knockdown of NDRG1 also downregulated the mRNA expression of C/Ebpα, FABP4, and leptin and increased that of TGF-β (p=0.007, p=0.002, and p=0.002, respectively) (Fig. 2E). The heat map showed the differences of adipogenesis genes in the control group and the NDRG1 knockdown group (Fig. 2F). This result demonstrated that expression of NDRG1 is important for differentiation into adipocytes in HemECs.

As a main phosphorylation target in mTORC2 pathway, NDRG1 was implicated in regulation of HemEC proliferation⁶. In adipogenesis of IH, PPAR-γ signal pathway plays important role in differentiation into adipocytes⁸. Recently, NDRG1 is induced during adipocytes differentiation and promotes the expression of PPARγ and phosphorylation of C/Ebpα in 3t311 preadipocytes⁶. In this study, we found that NDRG1 is upregulated in the IH tissue and is involved in adipocyte differentiation. NDRG1 plays a role not only in proliferating HemECs but also in mediators essential for the differentiation of these cells into adipocytes regulating the expression of c-MYC, thus playing an important role in the differentiation of HemECs into adipocytes. NDRG1 knockdown decreased the levels of adipogenic transcription factors (C/EBPα and PPARγ) and late marker of adipogenesis (FABP-4). We found that TGF-β, a key suppressor of the commitment phase to a preadipocyte, was induced in NDRG1-knockdown HemECs.

Thus, our findings demonstrate that NDRG1 is overexpressed in IH endothelial cells and regulates proliferation and involution to adipocytes. Indeed, additional in vivo studies are needed to identify the impact of NDRG1 on the involution of IH.

SUPPLEMENTARY MATERIALS

Supplementary data can be found via http://anndermatol.org/src/sm/ad-20-262-s001.pdf.

Supplementary Fig. 1

(A) Flow cytometry analysis of HemECs from primary tumor (case #1 and #2) and HDMECs. After selecting CD31+ endothelial cells by magnetic-activated cell sorting, cells from the early passage (3~4) were incubated with antibodies for flow cytometry. To characterize the isolated cells, cells from the early passage (3~4) of HemECs were immune-stained with antibodies against endothelial marker CD31 conjugated to fluorescein isothiocyanate. HemECs were suspended in 1×D-PBS with 2% FBS, and then incubated with antibodies conjugated to fluorescein isothiocyanate, phycoerythrin, or allophycocyanin at room temperature for 30 minutes. The antibodies used for flow cytometric analysis were as follows: CD31, CD34, CD45, CD105, CD146, CD44 (BD Biosciences), CD90 (Bio-Rad). The labeled cells were analyzed using a FACSCalibur system and CellQuest™ (BD Biosciences). (B) Western blot analysis of RICTOR-NDRG1-FOXO1 pathway in HUVEC, HDMEC, and HemECs. During proliferative phase infantile hemangioma, NDRG1 is highly expressed in primary HemEC. Western blot analysis showed that NDRG1 protein levels were increased and FOXO1 protein levels were decreased in HemEC. (C) Comparison of relative mRNA levels of NDRG1 and FOXO1 in HUVEC, HDMEC, and HemECs. The mRNA levels of NDRG1 were enhanced in HemEC compared with HUVEC and HDMEC. The mRNA levels of FOXO1 were downregulated in HemEC compared with HUVEC and HDMEC. Error bars: ±standard error of mean. NDRG1: N-myc downstream regulated gene 1, HemECs: hemangioma-derived endothelial cells, HDMEC: human dermal microvascular endothelial cell, HUVEC: human umbilical vein endothelial cell. ^***p<0.001.

Supplementary Fig. 2

N-myc downstream regulated gene 1 (NDRG1) positively regulated the proliferation of HemECs. (A) HemECs were transfected with NDRG1 and control siRNA. Knockdown of NDRG1 negatively regulated the proliferation of HemECs. (B) Protein levels of Histone H3 were downregulated in siNDRG1-transfected HemEC. One representative of 3 experiments is shown. The data represent the mean±standard error of mean. HemECs: hemangioma-derived endothelial cells, siControl: small interfering control, siNDRG1: small interfering N-myc downstream regulated gene 1. Statistical analysis by a student t-test (^*p<0.05, ^***p<0.001).

Supplementary Fig. 3

To examine whether NDRG1 regulates infantile hemangioma involution to adipocyte differentiation, HemECs were transfected with NDRG1 and control siRNA. Normal human endothelial cells, HDMEC, were used for negative control. After incubation under condition of adipocyte differentiation, HDMEC does not show any adipogenic potential (ORO staining). ORO: Oil Red O, HemECs: hemangioma-derived endothelial cells, HDMEC: human dermal