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Identification of Target Genes Involved in Wound Healing Angiogenesis of Endothelial Cells with the Treatment of a Chinese 2-Herb Formula.

Tam JC, Ko CH, Koon CM, Cheng Z, Lok WH, Lau CP, Leung PC, Fung KP, Chan WY, Lau CB - PLoS ONE (2015)

Bottom Line: We had previously demonstrated that a Chinese 2-herb formula (NF3) significantly stimulated angiogenesis of HUVEC in wound healing.The microarray results illustrated that different panels of differentially expressed genes were strictly governed in NF3-treated HUVEC in a time-regulated manner.This study provided concrete scientific evidence in support of the regulatory role of NF3 on endothelial cells involved in wound healing angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong; State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.

ABSTRACT
Angiogenesis is vitally important in diabetic wound healing. We had previously demonstrated that a Chinese 2-herb formula (NF3) significantly stimulated angiogenesis of HUVEC in wound healing. However, the molecular mechanism has not yet been elucidated. In line with this, global expression profiling of NF3-treated HUVEC was performed so as to assess the regulatory role of NF3 involved in the underlying signaling pathways in wound healing angiogenesis. The microarray results illustrated that different panels of differentially expressed genes were strictly governed in NF3-treated HUVEC in a time-regulated manner. The microarray analysis followed by qRT-PCR and western blotting verification of NF3-treated HUVEC at 6 h revealed the involvement of various genes in diverse biological process, e.g., MAP3K14 in anti-inflammation; SLC5A8 in anti-tumorogenesis; DNAJB7 in protein translation; BIRC5, EPCAM, INSL4, MMP8 and NPR3 in cell proliferation; CXCR7, EPCAM, HAND1 and MMP8 in migration; CXCR7, EPCAM and MMP8 in tubular formation; and BIRC5, CXCR7, EPCAM, HAND1, MMP8 and UBD in angiogenesis. After 16 h incubation of NF3, other sets of genes were shown with differential expression in HUVEC, e.g., IL1RAPL2 and NR1H4 in anti-inflammation; miR28 in anti-tumorogenesis; GRIN1 and LCN1 in anti-oxidation; EPB41 in intracellular signal transduction; PRL and TFAP2A in cell proliferation; miR28, PRL and SCG2 in cell migration; PRL in tubular formation; and miR28, NR1H4 and PRL in angiogenesis. This study provided concrete scientific evidence in support of the regulatory role of NF3 on endothelial cells involved in wound healing angiogenesis.

No MeSH data available.


Related in: MedlinePlus

Hypothetical diagram of mechanistic actions of NF3 on HUVEC at 6 h.The molecular gene targets, respective signaling pathways and molecule mediators associated with the biological responses was demonstrated in NF3-treated HUVEC at 6 h. Yellow molecules represented differentially expressed genes identified from microarray analysis. Blue box represented the suggested signaling pathway. Purple box represented the molecular mediators. Green box represented the biological responses. + indicated as positive regulation and–indicated negative regulation.
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pone.0139342.g005: Hypothetical diagram of mechanistic actions of NF3 on HUVEC at 6 h.The molecular gene targets, respective signaling pathways and molecule mediators associated with the biological responses was demonstrated in NF3-treated HUVEC at 6 h. Yellow molecules represented differentially expressed genes identified from microarray analysis. Blue box represented the suggested signaling pathway. Purple box represented the molecular mediators. Green box represented the biological responses. + indicated as positive regulation and–indicated negative regulation.

Mentions: In the present study, we investigated the effect of NF3 treatment on global gene expression of HUVEC governing angiogenesis in wound healing after 6 h and 16 h. From the microarray analysis associated with qRT-PCR and western blotting verification, target genes (BIRC5, CXCR7, DNAJB7, EPCAM, HAND1, MMP8, NPR3 and SLC5A8) were significantly up-regulated, while target genes (INSL4, MAP3K14 and UBD) were significantly down-regulated in NF3-treated HUVEC when compared to that of control HUVEC at 6 h. BIRC5 gene encoded the protein survivin, which regulated the apoptosis pathway. BIRC5 was crucial for optimal angiogenesis after brain injury in VEGF-treated HUVEC [14]. CXCR7, a member of CXC chemokine receptor family, possessed high binding affinity to chemokine stromal derived factor-1α (SDF-1α). Up-regulation of CXCR7 was vitally important in angiogenesis, stimulating the production of interleukin-8 (IL-8) and VEGF [15–17]. The protein of DNAJB7 gene belonged to the DNAJ/HSP40 family, which regulated molecular chaperone activity [18]. EPCAM mediated cell-cell adhesion, migration, proliferation and differentiation. EPCAM was asserted to possess oncogenic potential and up-regulation of EPCAM induced angiogenesis [19–21]. The protein encoded by HAND1 was classified into the basic helix-loop-helix family of transcription factors. Recent finding identified that thymosin β4 was an in vivo downstream target of HAND1, which was crucial for cell migration and angiogenesis as well as yolk sac vasculogenesis [22, 23]. The functional involvement of MMP8 (matrix metalloproteinase 8) in angiogenesis and tissue remodeling was extensively investigated [24, 25]. Up-regulation of NPR3 (natriuretic peptide receptor 3) activated ECs proliferation and meanwhile inhibited vascular smooth muscle cells (VSMCs) growth [26, 27]. SLC5A8 was identified as one of the tumor suppressor genes [28, 29]. For those down-regulated genes, INSL4 (insulin-like 4 protein) was the member of insulin superfamily. It was suggested that down-regulation of INSL4 might contribute to the cell survival and anti-apoptotic ability [30, 31]. MAP3K14 (mitogen-activated protein kinase kinase kinase 14) was a serine/threonine protein-kinase. Up-regulation of MAP3K14 had been linked to the persistent inflammatory response in diabetes [32, 33]. UBD was one of the components in the ubiquitin-proteasome system (UPS). It had been found that inhibition of UPS up-regulated endothelial nitric oxide synthase (eNOS) expression and activity, thus improving the endothelial function [34, 35]. By summarizing the biological function among the differentially expressed genes in NF3-treated HUVEC at 6 h, their respective roles were postulated, suggesting their beneficial effects on diverse mechanisms related to migration, tube formation, proliferation, angiogenesis as well as anti-inflammation in HUVEC (Fig 5).


Identification of Target Genes Involved in Wound Healing Angiogenesis of Endothelial Cells with the Treatment of a Chinese 2-Herb Formula.

Tam JC, Ko CH, Koon CM, Cheng Z, Lok WH, Lau CP, Leung PC, Fung KP, Chan WY, Lau CB - PLoS ONE (2015)

Hypothetical diagram of mechanistic actions of NF3 on HUVEC at 6 h.The molecular gene targets, respective signaling pathways and molecule mediators associated with the biological responses was demonstrated in NF3-treated HUVEC at 6 h. Yellow molecules represented differentially expressed genes identified from microarray analysis. Blue box represented the suggested signaling pathway. Purple box represented the molecular mediators. Green box represented the biological responses. + indicated as positive regulation and–indicated negative regulation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4591983&req=5

pone.0139342.g005: Hypothetical diagram of mechanistic actions of NF3 on HUVEC at 6 h.The molecular gene targets, respective signaling pathways and molecule mediators associated with the biological responses was demonstrated in NF3-treated HUVEC at 6 h. Yellow molecules represented differentially expressed genes identified from microarray analysis. Blue box represented the suggested signaling pathway. Purple box represented the molecular mediators. Green box represented the biological responses. + indicated as positive regulation and–indicated negative regulation.
Mentions: In the present study, we investigated the effect of NF3 treatment on global gene expression of HUVEC governing angiogenesis in wound healing after 6 h and 16 h. From the microarray analysis associated with qRT-PCR and western blotting verification, target genes (BIRC5, CXCR7, DNAJB7, EPCAM, HAND1, MMP8, NPR3 and SLC5A8) were significantly up-regulated, while target genes (INSL4, MAP3K14 and UBD) were significantly down-regulated in NF3-treated HUVEC when compared to that of control HUVEC at 6 h. BIRC5 gene encoded the protein survivin, which regulated the apoptosis pathway. BIRC5 was crucial for optimal angiogenesis after brain injury in VEGF-treated HUVEC [14]. CXCR7, a member of CXC chemokine receptor family, possessed high binding affinity to chemokine stromal derived factor-1α (SDF-1α). Up-regulation of CXCR7 was vitally important in angiogenesis, stimulating the production of interleukin-8 (IL-8) and VEGF [15–17]. The protein of DNAJB7 gene belonged to the DNAJ/HSP40 family, which regulated molecular chaperone activity [18]. EPCAM mediated cell-cell adhesion, migration, proliferation and differentiation. EPCAM was asserted to possess oncogenic potential and up-regulation of EPCAM induced angiogenesis [19–21]. The protein encoded by HAND1 was classified into the basic helix-loop-helix family of transcription factors. Recent finding identified that thymosin β4 was an in vivo downstream target of HAND1, which was crucial for cell migration and angiogenesis as well as yolk sac vasculogenesis [22, 23]. The functional involvement of MMP8 (matrix metalloproteinase 8) in angiogenesis and tissue remodeling was extensively investigated [24, 25]. Up-regulation of NPR3 (natriuretic peptide receptor 3) activated ECs proliferation and meanwhile inhibited vascular smooth muscle cells (VSMCs) growth [26, 27]. SLC5A8 was identified as one of the tumor suppressor genes [28, 29]. For those down-regulated genes, INSL4 (insulin-like 4 protein) was the member of insulin superfamily. It was suggested that down-regulation of INSL4 might contribute to the cell survival and anti-apoptotic ability [30, 31]. MAP3K14 (mitogen-activated protein kinase kinase kinase 14) was a serine/threonine protein-kinase. Up-regulation of MAP3K14 had been linked to the persistent inflammatory response in diabetes [32, 33]. UBD was one of the components in the ubiquitin-proteasome system (UPS). It had been found that inhibition of UPS up-regulated endothelial nitric oxide synthase (eNOS) expression and activity, thus improving the endothelial function [34, 35]. By summarizing the biological function among the differentially expressed genes in NF3-treated HUVEC at 6 h, their respective roles were postulated, suggesting their beneficial effects on diverse mechanisms related to migration, tube formation, proliferation, angiogenesis as well as anti-inflammation in HUVEC (Fig 5).

Bottom Line: We had previously demonstrated that a Chinese 2-herb formula (NF3) significantly stimulated angiogenesis of HUVEC in wound healing.The microarray results illustrated that different panels of differentially expressed genes were strictly governed in NF3-treated HUVEC in a time-regulated manner.This study provided concrete scientific evidence in support of the regulatory role of NF3 on endothelial cells involved in wound healing angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong; State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.

ABSTRACT
Angiogenesis is vitally important in diabetic wound healing. We had previously demonstrated that a Chinese 2-herb formula (NF3) significantly stimulated angiogenesis of HUVEC in wound healing. However, the molecular mechanism has not yet been elucidated. In line with this, global expression profiling of NF3-treated HUVEC was performed so as to assess the regulatory role of NF3 involved in the underlying signaling pathways in wound healing angiogenesis. The microarray results illustrated that different panels of differentially expressed genes were strictly governed in NF3-treated HUVEC in a time-regulated manner. The microarray analysis followed by qRT-PCR and western blotting verification of NF3-treated HUVEC at 6 h revealed the involvement of various genes in diverse biological process, e.g., MAP3K14 in anti-inflammation; SLC5A8 in anti-tumorogenesis; DNAJB7 in protein translation; BIRC5, EPCAM, INSL4, MMP8 and NPR3 in cell proliferation; CXCR7, EPCAM, HAND1 and MMP8 in migration; CXCR7, EPCAM and MMP8 in tubular formation; and BIRC5, CXCR7, EPCAM, HAND1, MMP8 and UBD in angiogenesis. After 16 h incubation of NF3, other sets of genes were shown with differential expression in HUVEC, e.g., IL1RAPL2 and NR1H4 in anti-inflammation; miR28 in anti-tumorogenesis; GRIN1 and LCN1 in anti-oxidation; EPB41 in intracellular signal transduction; PRL and TFAP2A in cell proliferation; miR28, PRL and SCG2 in cell migration; PRL in tubular formation; and miR28, NR1H4 and PRL in angiogenesis. This study provided concrete scientific evidence in support of the regulatory role of NF3 on endothelial cells involved in wound healing angiogenesis.

No MeSH data available.


Related in: MedlinePlus