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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 16 h.The molecular gene targets, respective signaling pathways and molecule mediators associated with the biological responses was demonstrated in NF3-treated HUVEC at 16 h. Yellow molecule 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.g006: Hypothetical diagram of mechanistic actions of NF3 on HUVEC at 16 h.The molecular gene targets, respective signaling pathways and molecule mediators associated with the biological responses was demonstrated in NF3-treated HUVEC at 16 h. Yellow molecule 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: As demonstrated by the gene expression results of NF3-treated HUVEC versus control HUVEC at 16 h, genes (EPB41, LCN1, NR1H4, PRL, SCG2 and TFAP2A) were significantly up-regulated while genes (GRIN1, IL1RAPL2 and miR28) were significantly down-regulated. Among those up-regulated genes of NF3-treated HUVEC at 16 h, protein 4.1 encoded by EPB41 gene was required for the regulation of channel activation and ion permeation for signaling transduction in ECs [36, 37]. LCN1 gene encoded a member of the lipocalin family of small secretory proteins. LCN1 was devoted to the cellular defense against oxidative stress [38]. NR1H4 (protein farnesoid X receptor (FXR)) was a member of the nuclear receptor superfamily. FXR was confirmed to induce eNOS expression at transcriptional level since up-regulation and subsequent NO production [39]. FXR activation in turn down-regulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which were the pro-inflammatory mediators. FXR signaling pathway played a pivotal role in vascular anti-inflammation and remodeling [40]. Substantial reports demonstrated the advantageous role of PRL (prolactin) in angiogenesis [41–43]. SCG2 gene encoded the protein secretogranin, which was converted to chemotactic active secretoneurin. Secretoneurin was involved in inflammation and wound healing [44]. Secretoneurin significantly induced the chemotaxis ability of ECs, confirming the regulatory role of secretoneurin in vascular cells migration [45]. The protein named transcription factor activator protein-2A (AP-2A) was encoded by TFAP2A gene, which was involved in transcriptional regulation and cell proliferation [46, 47]. For those down-regulated genes, study revealed that GRIN1 (N-methyl-D-aspartate receptor (NMDAR)) activation in human cerebral ECs promoted intracellular oxidative stress [48]. IL1RAPL2 (interleukin-1 receptor accessory protein-like 2) was the member of interleukin-1 receptor (IL-1R) family for signaling transduction of pro-inflammatory cytokines within cells [49]. miR28 was a member of the small non-coding RNAs called microRNAs (miRNAs). Elevation of endothelial miR28 impaired angiogenesis in diabetic patients [50]. Nrf2 regulated the expression of detoxifying enzymes, protecting cells towards tumorogenesis. Up-regulation of miR28 inhibited Nrf2 mRNA and protein expression, leading to breast cancer progression [51]. Besides, miR28 inhibited the translation of thrombopoietin receptor (TPOR). Activation of TPOR in HUVEC demonstrated enhanced cell motility through synthesis of 1-alkyl-/1-acyl-platelet-activating factor (PAF), IL-8, and phosphorylation of STAT1 and STAT5B [52, 53]. From numerous literature support, those differentially expressed genes at 16 h were proposed to devote pivotal role in wound healing angiogenesis, governing the diverse signaling pathways of transcriptional and translational regulation, anti-tumor activity, anti-oxidant activity, angiogenesis and anti-inflammation in NF3-treated HUVEC (Fig 6).


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 16 h.The molecular gene targets, respective signaling pathways and molecule mediators associated with the biological responses was demonstrated in NF3-treated HUVEC at 16 h. Yellow molecule 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4591983&req=5

pone.0139342.g006: Hypothetical diagram of mechanistic actions of NF3 on HUVEC at 16 h.The molecular gene targets, respective signaling pathways and molecule mediators associated with the biological responses was demonstrated in NF3-treated HUVEC at 16 h. Yellow molecule 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: As demonstrated by the gene expression results of NF3-treated HUVEC versus control HUVEC at 16 h, genes (EPB41, LCN1, NR1H4, PRL, SCG2 and TFAP2A) were significantly up-regulated while genes (GRIN1, IL1RAPL2 and miR28) were significantly down-regulated. Among those up-regulated genes of NF3-treated HUVEC at 16 h, protein 4.1 encoded by EPB41 gene was required for the regulation of channel activation and ion permeation for signaling transduction in ECs [36, 37]. LCN1 gene encoded a member of the lipocalin family of small secretory proteins. LCN1 was devoted to the cellular defense against oxidative stress [38]. NR1H4 (protein farnesoid X receptor (FXR)) was a member of the nuclear receptor superfamily. FXR was confirmed to induce eNOS expression at transcriptional level since up-regulation and subsequent NO production [39]. FXR activation in turn down-regulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which were the pro-inflammatory mediators. FXR signaling pathway played a pivotal role in vascular anti-inflammation and remodeling [40]. Substantial reports demonstrated the advantageous role of PRL (prolactin) in angiogenesis [41–43]. SCG2 gene encoded the protein secretogranin, which was converted to chemotactic active secretoneurin. Secretoneurin was involved in inflammation and wound healing [44]. Secretoneurin significantly induced the chemotaxis ability of ECs, confirming the regulatory role of secretoneurin in vascular cells migration [45]. The protein named transcription factor activator protein-2A (AP-2A) was encoded by TFAP2A gene, which was involved in transcriptional regulation and cell proliferation [46, 47]. For those down-regulated genes, study revealed that GRIN1 (N-methyl-D-aspartate receptor (NMDAR)) activation in human cerebral ECs promoted intracellular oxidative stress [48]. IL1RAPL2 (interleukin-1 receptor accessory protein-like 2) was the member of interleukin-1 receptor (IL-1R) family for signaling transduction of pro-inflammatory cytokines within cells [49]. miR28 was a member of the small non-coding RNAs called microRNAs (miRNAs). Elevation of endothelial miR28 impaired angiogenesis in diabetic patients [50]. Nrf2 regulated the expression of detoxifying enzymes, protecting cells towards tumorogenesis. Up-regulation of miR28 inhibited Nrf2 mRNA and protein expression, leading to breast cancer progression [51]. Besides, miR28 inhibited the translation of thrombopoietin receptor (TPOR). Activation of TPOR in HUVEC demonstrated enhanced cell motility through synthesis of 1-alkyl-/1-acyl-platelet-activating factor (PAF), IL-8, and phosphorylation of STAT1 and STAT5B [52, 53]. From numerous literature support, those differentially expressed genes at 16 h were proposed to devote pivotal role in wound healing angiogenesis, governing the diverse signaling pathways of transcriptional and translational regulation, anti-tumor activity, anti-oxidant activity, angiogenesis and anti-inflammation in NF3-treated HUVEC (Fig 6).

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