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Integrated Transcriptome Analysis Reveals the Impact of Photodynamic Therapy on Cerebrovascular Endothelial Cells.

ABSTRACT: Blood vessels in the brain tissue form a compact vessel structure and play an essential role in maintaining the homeostasis of the neurovascular system. The low dosage of photodynamic intervention (PDT) significantly affects the expression of cellular biomarkers. To understand the impact of photodynamic interventions on cerebrovascular endothelial cells, we evaluated the dosage-dependent impact of porfimer sodium-mediated PDT on B.END3 cells using flow cytometer, comet assay, RNA sequencing, and bioinformatics analysis. To examine whether PDT can induce disorder of intracellular organelles, we did not observe any significance damage of DNA and cellular skeleton. Moreover, expression levels of cellular transporters-related genes were significantly altered, implying the drawbacks of PDT on cerebrovascular functions. To address the potential molecular mechanisms of these phenotypes, RNA sequencing and bioinformatics analysis were employed to identify critical genes and pathways among these processes. The gene ontology (GO) analysis and protein-protein interaction (PPI) identified 15 hub genes, highly associated with cellular mitosis process (CDK1, CDC20, MCM5, MCM7, MCM4, CCNA2, AURKB, KIF2C, ESPL1, BUB1B) and DNA replication (POLE2, PLOE, CDC45, CDC6). Gene set enrichment analysis (GSEA) reveals that TNF-α/NF-κB and KRAS pathways may play a critical role in regulating expression levels of transporter-related genes. To further perform qRT-PCR assays, we find that TNF-α/NF-κB and KRAS pathways were substantially up-regulated, consistent with GSEA analysis. The current findings suggested that a low dosage of PDT intervention may be detrimental to the homeostasis of blood-brain barrier (BBB) by inducing the inflammatory response and affecting the expression of surface biomarkers.

SUBMITTER: He Y

PROVIDER: S-EPMC8645902 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe immune microenvironment plays a critical role in maintaining skin homeostasis, which is closely related to the dysfunction in photoaged skin such as autoimmunity and tumorigenesis. Several recent studies have demonstrated the efficacy of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) in alleviating photoaging and skin cancer. However, the underlying immune mechanisms and the immune microenvironment change by ALA-PDT remain largely unknown.MethodsTo illustrate the effects of ALA-PDT on immune microenvironment in photoaged skin, single cell RNA sequencing (scRNA-seq) analysis of photoaged skin on the extensor side of the human forearm before and after ALA-PDT was performed. R-packages of Seurat, clusterProfiler, Monocle, CellChat were used for cell clustering, differentially expressed genes analysis, functional annotation, pseudotime analysis and cell-cell communication analysis. The gene sets related to specific functions were extracted from the MSigDB database, which were used to score the functions of immune cells in different states. We also compared our result with published scRNA-seq data of photoaged skin of the eyelids.ResultsThe increase score of cellular senescence, hypoxia and reactive oxygen species pathway in immune cells and the decrease of immune receptor activity function and proportion of naive T cells were found in skin photoaging. Moreover, the function of T cell ribosomal synthesis was also impaired or down regulated and function of G2M checkpoint was up regulated. However, ALA-PDT showed promising results in reversing these effects, as it improved the above functions of T cells. The ratio of M1/M2 and percentage of Langerhans cells also decreased with photoaging and increased after ALA-PDT. Additionally, ALA-PDT restored the antigen presentation and migration function of dendritic cells and enhanced cell-cell communication among immune cells. These effects were observed to last for 6 months.ConclusionALA-PDT has potential to rejuvenate immune cells, partially reversed immunosenescence and improved the immunosuppressive state, ultimately remodelling the immune microenvironment in photoaged skin. These results provide an important immunological basis for further exploring strategies to reverse skin photoaging, chronological aging and potentially systemic aging.

Project description:BackgroundInfantile hemangiomas are benign vascular tumors primarily found on the skin in 10% of the pediatric population. The etiology of this disease is largely unknown and while large scale genomic studies have examined the transcriptomes of infantile hemangioma tumors as a whole, no study to date has compared the global gene expression profiles of pure infantile hemangioma endothelial cells (HEMECs) to that of normal human dermal microvascular endothelial cells (HDMVECs).MethodsTo shed light on the molecular differences between these normal and aberrant dermal endothelial cell types, we performed whole genome microarray analysis on purified cultures of HEMECs and HDMVECs. We then utilized qPCR and immunohistochemistry to confirm our microarray results.ResultsOur array analysis identified 125 genes whose expression was upregulated and 104 genes whose expression was downregulated by greater than two fold in HEMECs compared to HDMVECs. Bioinformatics analysis revealed three major classifications of gene functions that were altered in HEMECs including cell adhesion, cell cycle, and arachidonic acid production. Several of these genes have been reported to be critical regulators and/or mutated in cancer, vascular tumors, and vascular malformations. We confirmed the expression of a subset of these differentially expressed genes (ANGPT2, ANTXR1, SMARCE1, RGS5, CTAG2, LTBP2, CLDN11, and KISS1) using qPCR and utilized immunohistochemistry on a panel of paraffin embedded infantile hemangioma tumor tissues to demonstrate that the cancer/testis antigen CTAG2 is highly abundant in vessel-dense proliferating infantile hemangiomas and with significantly reduced levels during tumor involution as vascular density decreases.ConclusionOur data reveal that the transcriptome of HEMECs is reflective of a pro-proliferative cell type with altered adhesive characteristics. Moveover, HEMECs show altered expression of many genes that are important in the progression and prognosis of metastatic cancers.

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Integrated Transcriptome Analysis Reveals the Impact of Photodynamic Therapy on Cerebrovascular Endothelial Cells.

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