Department of Dermatology, The Second Hospital of Jilin University, Changchun, P.R. China.
Department of Nephrology, The Second Hospital of Jilin University, Changchun, P.R. China.
Lasers Surg Med. 2022 Oct;54(8):1157-1166. doi: 10.1002/lsm.23586. Epub 2022 Aug 2.
Pulsed dye laser (PDL) is the main treatment for port wine stain (PWS), but a considerable number of patients show low clearances. The reason for the poor efficacy is related to PDL-induced angiogenesis. Vascular endothelial growth factor (VEGF) plays an important role in PDL-induced angiogenesis and can activate the tyrosine kinase activity of VEGF receptor (VEGFR) in endothelial cells. It triggers a full range of responses, and then participates in the regulation of angiogenesis. Tivozanib is an inhibitor of VEGFR tyrosine kinase activity, which can block the pro-angiogenic effect of VEGF and reduce vascular permeability.
Different energy densities of PDL were used to irradiate the abdominal skin of rats. According to the general and pathological changes of the irradiated area, the energy density of 8 J/cm with smaller scab and stronger vascular effect was selected for follow-up experiments. Divided the rat abdomen skin into four areas, irradiated three of them uniformly with an energy density of 8 J/cm , and applied different concentrations of Tivozanib coating agent to the laser irradiation area, and grouped them as follows: (1) vacant group, (2) control group, (3) 0.5% Tivozanib group, (4) 1% Tivozanib group. Camera and dermoscopy were used to observe skin changes. Hematoxylin-eosin staining, immunohistochemical staining, and blood vessels were counted to detect dermal vascular regeneration. Transcriptome sequencing and real-time polymerase chain reaction (PCR) were conducted to elucidate the mechanism and validate the reliability.
The number of blood vessels in the 0.5% Tivozanib group and 1% Tivozanib group was significantly reduced on the 7, 10, and 14 days compared with the control group. The number of blood vessels in the 1% Tivozanib group was significantly reduced compared with the 0.5% Tivozanib group, indicating that Tivozanib successfully inhibited PDL-induced angiogenesis, and the inhibitory effect of 1% Tivozanib was more significant than that of 0.5% Tivozanib. Transcriptome sequencing results showed a total of 588 significantly differentially expressed genes, including 90 upregulated genes and 498 downregulated genes. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that the significantly differentially expressed genes were mainly enriched in the metabolic pathways which were closely related to angiogenesis. Finally, real-time PCR was used to verify the genes with higher expression differences, the top ranking and closely related to angiogenesis, namely, Cxcl1, Cxcl2, Cxcl3, Cxcl6, Ccl3, Csf3, IL1β, iNOS, Mmp9, Mmp13, Plau, Ets1, Spp1, Nr4a1. The results were consistent with the trend of transcriptome sequencing results, which proved the reliability of this study.
This study explored the inhibitory effect of Tivozanib on PDL-induced angiogenesis, and provided a new idea for the treatment of clinical PWS. Transcriptome sequencing explored the mechanism and provided reliable clues for later in-depth research.
脉冲染料激光（PDL）是治疗葡萄酒色斑（PWS）的主要方法，但相当一部分患者的清除率较低。疗效不佳的原因与 PDL 诱导的血管生成有关。血管内皮生长因子（VEGF）在 PDL 诱导的血管生成中起着重要作用，可激活血管内皮细胞中 VEGF 受体（VEGFR）的酪氨酸激酶活性。它引发了一系列全面的反应，然后参与了血管生成的调节。替沃扎尼布是一种 VEGFR 酪氨酸激酶活性抑制剂，可阻断 VEGF 的促血管生成作用，并降低血管通透性。
用不同能量密度的 PDL 辐照大鼠腹部皮肤。根据辐照区域的一般和病理变化，选择 8 J/cm 的能量密度进行后续实验，该能量密度产生的结痂较小且血管生成效果较强。将大鼠腹部皮肤分为四个区域，用 8 J/cm 的能量密度均匀照射其中三个区域，并在激光照射区域涂抹不同浓度的替沃扎尼布涂层剂，将其分组如下：（1）空白组，（2）对照组，（3）0.5%替沃扎尼布组，（4）1%替沃扎尼布组。使用相机和皮肤镜观察皮肤变化。进行苏木精-伊红染色、免疫组织化学染色和血管计数，以检测真皮血管再生。进行转录组测序和实时聚合酶链反应（PCR）以阐明机制并验证可靠性。
与对照组相比，0.5%替沃扎尼布组和 1%替沃扎尼布组在第 7、10 和 14 天的血管数量明显减少。与 0.5%替沃扎尼布组相比，1%替沃扎尼布组的血管数量明显减少，表明替沃扎尼布成功抑制了 PDL 诱导的血管生成，且 1%替沃扎尼布的抑制作用比 0.5%替沃扎尼布更显著。转录组测序结果共显示 588 个显著差异表达基因，包括 90 个上调基因和 498 个下调基因。基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析显示，差异表达基因主要富集在与血管生成密切相关的代谢途径中。最后，使用实时 PCR 验证了具有更高表达差异的基因，这些基因排名靠前且与血管生成密切相关，即 Cxcl1、Cxcl2、Cxcl3、Cxcl6、Ccl3、Csf3、IL1β、iNOS、Mmp9、Mmp13、Plau、Ets1、Spp1、Nr4a1。结果与转录组测序结果的趋势一致，证明了本研究的可靠性。
本研究探讨了替沃扎尼布对 PDL 诱导的血管生成的抑制作用，为临床 PWS 的治疗提供了新的思路。转录组测序探索了其机制，并为后续的深入研究提供了可靠的线索。
