Cellular Heterogeneity and Stem Cells of Vascular Endothelial Cells in Blood Vessel Formation and Homeostasis: Insights from single-cell RNA Sequencing

Vascular endothelial cells (ECs) that constitute the inner surface of blood vessels are essential for new vessel formation and organ homeostasis. ECs display remarkable phenotypic heterogeneity across different organs and the vascular tree during angiogenesis and homeostasis. Recent advances in single cell RNA sequencing (scRNA-seq) technologies have allowed a new understanding of EC heterogeneity in both mice and humans. In particular, scRNA-seq has identified new molecular signatures for arterial, venous and capillary ECs in different organs, as well as previously unrecognized specialized EC subtypes, such as the aerocytes localized in the alveolar capillaries of the lung. scRNA-seq has also revealed the gene expression profiles of specialized tissue-resident EC subtypes that are capable of clonal expansion and contribute to adult angiogenesis, a process of new vessel formation from the pre-existing vasculature. These specialized tissue-resident ECs have been identified in various different mouse tissues, including aortic endothelium, liver, heart, lung, skin, skeletal muscle, retina, choroid, and brain. Transcription factors and signaling pathways have also been identified in the specialized tissue-resident ECs that control angiogenesis. Furthermore, scRNA-seq has also documented responses of ECs in diseases such as cancer, age-related macular degeneration, Alzheimer\u27s disease, atherosclerosis, and myocardial infarction. These new findings revealed by scRNA-seq have the potential to provide new therapeutic targets for different diseases associated with blood vessels. In this article, we summarize recent advances in the understanding of the vascular endothelial cell heterogeneity and endothelial stem cells associated with angiogenesis and homeostasis in mice and humans, and we discuss future prospects for the application of scRNA-seq technology

腫瘍血管における血管内皮幹細胞システムの破綻

金沢大学医薬保健研究域医学系 / 大阪大学血管内皮細胞には多様性が存在し、一部には幹細胞様の性質を持つ内皮細胞が存在することが分かっている。今回腫瘍血管内皮細胞中の内皮幹細胞をより詳細に検討し、腫瘍血管内皮細胞が通常の内皮細胞とどのように異なるか解析を行った。腫瘍血管では内皮幹細胞が非常に高い割合で存在することが明らかとなった。また移植実験から、腫瘍血管内皮細胞も階層性を持つ内皮細胞集団であることが分かった。さらに遺伝子解析で薬剤耐性に関与する蛋白の上昇を特に幹細胞分画で認めた。薬剤耐性能に関わる蛋白の阻害剤にて内皮幹細胞の増殖阻害を認め、さらには腫瘍縮小効果も認めた。今後、血管内皮幹細胞システムを標的とした治療が期待できる。We have been studying the endothelial stem cell system in the vasculature. Here we identified this endothelial stem cell system in the tumor vasculature. In the tumor vasculature, the percentage of endothelial stem cells was significantly increased compared to normal tissue. Moreover, we showed endothelial hierarchy by transplantation model. By gene expression analysis, we found that endothelial stem cell in the tumor highly express drug resistance genes. Addition of an inhibitor which blocks the function of drug resistance protein inhibited the colony formation of endothelial stem cells. Furthermore, administration of this inhibitor to the tumor bearing mice induced regression of tumor size of these mice. Our results suggest the existence of endothelial hierarchy system in the tumor vasculature. Targeting this system may be a promising therapeutic target in the future.研究課題/領域番号:15K18409, 研究期間(年度):2015 - 201

Cellular heterogeneity and stem cells of vascular endothelial cells in blood vessel formation and homeostasis: Insights from single-cell RNA sequencing

Vascular endothelial cells (ECs) that constitute the inner surface of blood vessels are essential for new vessel formation and organ homeostasis. ECs display remarkable phenotypic heterogeneity across different organs and the vascular tree during angiogenesis and homeostasis. Recent advances in single cell RNA sequencing (scRNA-seq) technologies have allowed a new understanding of EC heterogeneity in both mice and humans. In particular, scRNA-seq has identified new molecular signatures for arterial, venous and capillary ECs in different organs, as well as previously unrecognized specialized EC subtypes, such as the aerocytes localized in the alveolar capillaries of the lung. scRNA-seq has also revealed the gene expression profiles of specialized tissue-resident EC subtypes that are capable of clonal expansion and contribute to adult angiogenesis, a process of new vessel formation from the pre-existing vasculature. These specialized tissue-resident ECs have been identified in various different mouse tissues, including aortic endothelium, liver, heart, lung, skin, skeletal muscle, retina, choroid, and brain. Transcription factors and signaling pathways have also been identified in the specialized tissue-resident ECs that control angiogenesis. Furthermore, scRNA-seq has also documented responses of ECs in diseases such as cancer, age-related macular degeneration, Alzheimer’s disease, atherosclerosis, and myocardial infarction. These new findings revealed by scRNA-seq have the potential to provide new therapeutic targets for different diseases associated with blood vessels. In this article, we summarize recent advances in the understanding of the vascular endothelial cell heterogeneity and endothelial stem cells associated with angiogenesis and homeostasis in mice and humans, and we discuss future prospects for the application of scRNA-seq technology

L-carnitine prevents lenvatinib-induced muscle toxicity without impairment of the anti-angiogenic efficacy

Lenvatinib is an oral tyrosine kinase inhibitor that acts on multiple receptors involved in angiogenesis. Lenvatinib is a standard agent for the treatment of several types of advanced cancers; however, it frequently causes muscle-related adverse reactions. Our previous study revealed that lenvatinib treatment reduced carnitine content and the expression of carnitine-related and oxidative phosphorylation (OXPHOS) proteins in the skeletal muscle of rats. Therefore, this study aimed to evaluate the effects of L-carnitine on myotoxic and anti-angiogenic actions of lenvatinib. Co-administration of L-carnitine in rats treated with lenvatinib for 2 weeks completely prevented the decrease in carnitine content and expression levels of carnitine-related and OXPHOS proteins, including carnitine/organic cation transporter 2, in the skeletal muscle. Moreover, L-carnitine counteracted lenvatinib-induced protein synthesis inhibition, mitochondrial dysfunction, and cell toxicity in C2C12 myocytes. In contrast, L-carnitine had no influence on either lenvatinib-induced inhibition of vascular endothelial growth factor receptor 2 phosphorylation in human umbilical vein endothelial cells or angiogenesis in endothelial tube formation and mouse aortic ring assays. These results suggest that L-carnitine supplementation could prevent lenvatinib-induced muscle toxicity without diminishing its antineoplastic activity, although further clinical studies are needed to validate these findings

Fluorescence and Bioluminescence Imaging of Angiogenesis in Flk1-Nano-lantern Transgenic Mice

Angiogenesis is important for normal development as well as for tumour growth. However, the molecular and cellular mechanisms underlying angiogenesis are not fully understood, partly because of the lack of a good animal model for imaging. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a bioluminescent reporter protein, Nano-lantern, under the control of Fetal liver kinase 1 (Flk1). Flk1-Nano-lantern BAC Tg mice recapitulated endogenous Flk1 expression in endothelial cells and lymphatic endothelial cells during development and tumour growth. Importantly, bioluminescence imaging of endothelial cells from the aortic rings of Flk1-Nano-lantern BAC Tg mice enabled us to observe endothelial sprouting for 18 hr without any detectable phototoxicity. Furthermore, Flk1-Nano-lantern BAC Tg mice achieved time-lapse luminescence imaging of tumour angiogenesis in freely moving mice with implanted tumours. Thus, this transgenic mouse line contributes a unique model to study angiogenesis within both physiological and pathological contexts

TAK1 safeguards endothelial cells from gut microbes and inflammation

How endothelial cells (ECs) survive in inflammatory environments remains unclear. We recently showed that TAK1 protects ECs against apoptosis induced by TNFα under physiological conditions in the intestine and liver, and under inflammatory conditions in other organs. Our results document that a single gene can affect cell fate decisions in mature ECs

腫瘍血管における新しい内皮細胞の探索と新規血管阻害療法の開発

金沢大学医薬保健研究域医学系 / 大阪大学本研究ではマウス腫瘍モデルを用いて、腫瘍血管を構築する全血管内皮細胞を１細胞レベルで遺伝子発現解析を行い、腫瘍血管内皮細胞の多様性を解明することを目的とした。これまで血管内皮細胞は全て均一であると考えられてきたが、本研究で実施した１細胞遺伝子発現解析の結果、血管内皮細胞の遺伝子発現は多様性に富むことが明らかになった。また腫瘍血管内皮細胞には正常組織の血管内皮細胞とは明らかに遺伝子発現が異なる細胞集団が存在することが明らかになった。その腫瘍特異的血管内皮細胞集団で高発現している遺伝子と発現低下している遺伝子を同定した。今後これらの遺伝子を標的として、新たな血管新生阻害剤が開発できる可能性がある。In this project, we aimed to identify tumor endothelial cell (EC) heterogeneity by single cell analysis of whole ECs isolated from mouse tumor model. It is believed that all ECs are equal and possess high plasticity and proliferative potential. However, the result of single analysis suggests that they are heterogenous in terms of gene expression and can be clustered to several distinct subpopulations. Interestingly, we found tumor specific EC subpopulation which is not detected in normal tissue. This subpopulation clearly shows different gene expression profile compared to that of any EC subpopulation in normal tissue. We identified several genes which are highly and lowly expressed in this specific EC subpopulation. These genes may be a new target for developing novel anti-angiogenic drugs in the future.出典：「腫瘍血管における新しい内皮細胞の探索と新規血管阻害療法の開発」研究成果報告書　課題番号19K22562（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-19K22562/19K22562seika/）を加工して作

腫瘍血管内皮幹細胞制御による新規血管新生阻害療法の開発

金沢大学医薬保健研究域医学系 / 大阪大学腫瘍の増大には血管新生が必須である。これまでに増殖能が非常に高い幹・前駆細胞様の性質を持った血管内皮細胞が全身の血管に存在する事を明らかにした。血管新生時にはこの特殊な内皮細胞が血管を構築する内皮細胞を大量に産生する。本研究では腫瘍血管で、このような幹・前駆細胞様の内皮細胞の解析を行った。腫瘍血管においては増殖能の高い内皮細胞は正常組織よりも高頻度で認めること、さらに正常組織とは異なる表面マーカーを持つことが明らかになった。また、この内皮細胞と腫瘍をマウス皮下に共移植するとこの内皮細胞由来の腫瘍血管が構築された。この細胞を特異的に阻害することにより、効果的な血管新生阻害剤が開発できる。Angiogenesis is regarded as a hallmark in cancer development.In the previous work, we identified stem/progenitor like endothelial cell(EC) in the peripheral blood vessel. These endothelial stem-like cell possess EC colony-forming potential in vitro and contribute to angiogenesis by generating functional mature blood vessels in vivo. In this study, we characterized endothelial stem-like cells in the tumor vasculature and found that in the tumor vasclature, the percentage of stem-like cells were higher than normal tissues. We further examined contribution of these cells to the tumor vasculature by transplantation model and found that they produces numbers of ECs and contribute to the tumor blood vessels as functional vessels. Moreover, administration of anti-angiogenic drugs revealed their potential role for resistance to anti-angiogenic therapy.研究課題/領域番号:25830080, 研究期間(年度):2013 - 201

Tumor endothelial cell-specific drug delivery system using apelin-conjugated liposomes.

BACKGROUND: A drug delivery system specifically targeting endothelial cells (ECs) in tumors is required to prevent normal blood vessels from being damaged by angiogenesis inhibitors. The purpose of this study was to investigate whether apelin, a ligand for APJ expressed in ECs when angiogenesis is taking place, can be used for targeting drug delivery to ECs in tumors. METHODS AND RESULTS: Uptake of apelin via APJ stably expressed in NIH-3T3 cells was investigated using TAMRA (fluorescent probe)-conjugated apelin. Both long and short forms of apelin (apelin 36 and apelin 13) were taken up, the latter more effectively. To improve efficacy of apelin- liposome conjugates, we introduced cysteine, with its sulfhydryl group, to the C terminus of apelin 13, resulting in the generation of apelin 14. In turn, apelin 14 was conjugated to rhodamine-encapsulating liposomes and administered to tumor-bearing mice. In the tumor microenvironment, we confirmed that liposomes were incorporated into the cytoplasm of ECs. In contrast, apelin non-conjugated liposomes were rarely found in the cytoplasm of ECs. Moreover, non-specific uptake of apelin-conjugated liposomes was rarely detected in other normal organs. CONCLUSIONS: ECs in normal organs express little APJ; however, upon hypoxic stimulation, such as in tumors, ECs start to express APJ. The present study suggests that apelin could represent a suitable tool to effectively deliver drugs specifically to ECs within tumors