Discovery of Transcription Factors Involved in the Maintenance of Resident Vascular Endothelial Stem Cell Properties.

A resident vascular endothelial stem cell (VESC) population expressing CD157 has been identified recently in mice. Herein, we identified transcription factors (TFs) regulating CD157 expression in endothelial cells (ECs) that were associated with drug resistance, angiogenesis, and EC proliferation. In the first screening, we detected 20 candidate TFs through the CD157 promoter and gene expression analyses. We found that 10 of the 20 TFs induced CD157 expression in ECs. We previously reported that 70% of CD157 VESCs were side population (SP) ECs that abundantly expressed ATP-binding cassette (ABC) transporters. Here, we found that the 10 TFs increased the expression of several ABC transporters in ECs and increased the proportion of SP ECs. Of these 10 TFs, we found that six (Atf3, Bhlhe40, Egr1, Egr2, Elf3, and Klf4) were involved in the manifestation of the SP phenotype. Furthermore, the six TFs enhanced tube formation and proliferation in ECs. Single-cell RNA sequence data in liver ECs suggested that Atf3 and Klf4 contributed to the production of CD157+ VESCs in the postnatal period. We concluded that Klf4 might be important for the development and maintenance of liver VESCs. Our work suggests that a TF network is involved in the differentiation hierarchy of VESCs.

Single-cell sequencing reveals the existence of fetal vascular endothelial stem cell-like cells in mouse liver.

BACKGROUND: A resident vascular endothelial stem cell (VESC) population expressing CD157 and CD200 has been identified recently in the adult mouse. However, the origin of this population and how it develops has not been characterized, nor has it been determined whether VESC-like cells are present during the perinatal period. Here, we investigated the presence of perinatal VESC-like cells and their relationship with the adult VESC-like cell population. METHODS: We applied single-cell RNA sequencing of endothelial cells (ECs) from embryonic day (E) 14, E18, postnatal day (P) 7, P14, and week (W) 8 liver and investigated transcriptomic changes during liver EC development. We performed flow cytometry, immunofluorescence, colony formation assays, and transplantation assays to validate the presence of and to assess the function of CD157+ and CD200+ ECs in the perinatal period. RESULTS: We identified CD200- expressing VESC-like cells in the perinatal period. These cells formed colonies in vitro and had high proliferative ability. The RNA velocity tool and transplantation assay results indicated that the projected fate of this population was toward adult VESC-like cells expressing CD157 and CD200 1 week after birth. CONCLUSION: Our study provides a comprehensive atlas of liver EC development and documents VESC-like cell lineage commitment at single-cell resolution.

Aging impairs the ability of vascular endothelial stem cells to generate endothelial cells in mice.

Tissue-resident vascular endothelial stem cells (VESCs), marked by expression of CD157, possess long-term repopulating potential and contribute to vascular regeneration and homeostasis in mice. Stem cell exhaustion is regarded as one of the hallmarks of aging and is being extensively studied in several types of tissue-resident stem cells; however, how aging affects VESCs has not been clarified yet. In the present study, we isolated VESCs from young and aged mice to compare their potential to differentiate into endothelial cells in vitro and in vivo. Here, we report that the number of liver endothelial cells (ECs) including VESCs was lower in aged (27-28 month-old) than young (2-3 month-old) mice. In vitro culture of primary VESCs revealed that the potential to generate ECs is impaired in aged VESCs isolated from liver and lung relative to young VESCs. Orthotopic transplantation of VESCs showed that aged VESCs and their progeny expand less efficiently than their young counterparts when transplanted into aged mice, but they are equally functional in young recipients. Gene expression analysis indicated that inflammatory signaling was more activated in aged ECs including VESCs. Using single-cell RNA sequencing data from the Tabula Muris Consortium, we show that T cells and monocyte/macrophage lineage cells including Kupffer cells are enriched in the aged liver. These immune cells produce IL-1ß and several chemokines, suggesting the possible involvement of age-associated inflammation in the functional decline of VESCs with age.

Cancer apelin receptor suppresses vascular mimicry in malignant melanoma.

Several reports indicate that apelin is often over-expressed in tumors, and therefore it has been suggested that the apelin-apelin receptor (APJ) system may induce tumor progression. In contrast, our previous research revealed high expression of the apelin-APJ system in tumor blood vessels, suggesting its involvement in the regulation of tumor vessel formation and normalization, resulting in the suppression of tumor growth by promoting the infiltration of T cells. Thus, the effect of the apelin-APJ system on tumors remains controversial. In this report, to clarify the effect of apelin in tumor cells, we analyzed the function of APJ in tumor cells using APJ knock out (KO) mice. In APJ-KO mice, Apelin overexpression in B16/BL6 (B16) melanoma cells induced greater tumor growth than controls. In an APJ-KO melanoma inoculation model, although angiogenesis is suppressed compared to wild type, no difference is evident in tumor growth. We found that APJ deficiency promoted vascular mimicry in tumors. In vitro, cultured APJ-KO B16 cells demonstrated a spindle-like shape. This phenotypic change was thought to be induced by epithelial-mesenchymal transition (EMT) based on evidence that APJ-KO B16 cells show persistently high levels of the mesenchymal maker, Zeb1; however, we found that EMT did not correlate with the transforming growth factor-ß/smad signaling pathway in our model. We propose that apelin-APJ system in cancer cells induces tumor growth but negatively regulates EMT and tumor malignancy.

Indispensable role of Galectin-3 in promoting quiescence of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) in adult bone marrow (BM) are usually maintained in a state of quiescence. The cellular mechanism coordinating the balance between HSC quiescence and differentiation is not fully understood. Here, we report that galactose-binding lectin-3 (galectin-3; Gal-3) is upregulated by Tie2 or Mpl activation to maintain quiescence. Conditional overexpression of Gal-3 in mouse HSCs under the transcriptional control of Tie2 or Vav1 promoters (Gal-3 Tg) causes cell cycle retardation via induction of p21. Conversely, the cell cycle of long-term repopulating HSCs (LT-HSCs) in Gal-3-deficient (Gal-3-/-) mice is accelerated, resulting in their exhaustion. Mechanistically, Gal-3 regulates p21 transcription by forming a complex with Sp1, thus blocking cell cycle entry. These results demonstrate that Gal-3 is a negative regulator of cell-cycling in HSCs and plays a crucial role in adult hematopoiesis to prevent HSC exhaustion.

High expression of PSF1 promotes drug resistance and cell cycle transit in leukemia cells.

Escape of cancer cells from chemotherapy is a problem in the management of cancer patients. Research on chemotherapy resistance has mainly focused on the heterogeneity of cancer cells, multiple gene mutations, and quiescence of malignant cancer cells. However, some studies have indicated that interactions between cancer cells and vascular cells promote resistance to chemotherapy. Here, we established mouse leukemia models using the cell lines THP-1 or MEG-1. These were derived from acute and chronic myeloid leukemias, respectively, and highly expressed DNA replication factor PSF1, a member of the GINS complex. We found that, after anti-cancer drug administration, surviving GFP-positive leukemia cells in the bone marrow were located adjacent to blood vessels, as previously reported in a subcutaneous solid tumor transplantation model. Treating THP-1 and MEG-1 cells with anti-cancer drugs in vitro revealed that those most strongly expressing PSF1 were most chemoresistant, suggesting that PSF1 induces not only cell cycle progression but also facilitates cell survival. Indeed, when PSF1 expression was suppressed by shRNA, the growth rate was reduced and cell death was enhanced in both cell lines. Furthermore, PSF1 knockdown in leukemia cells led to a change in their location at a distance from the blood vessels in a bone marrow transplantation model. These findings potentially reflect a mechanism of escape of leukemic cells from chemotherapy and suggest that PSF1 may be a possible therapeutic target to enhance the effect of chemotherapy.

Embryonic expression of GINS members in the development of the mammalian nervous system.

The GINS (Go, Ichi, Nii, and San) complex contains four protein subunits (PSF1, PSF2, PSF3, and SLD5) and has been identified as a factor essential for the initiation and elongation stages of the DNA replication process. A previous study indicated that PSF2 participated in the developing central nervous system (CNS) of Xenopus laevis. However, the expression and function of GINS members in the mammalian developing nervous system remains unclear. Here, we examined the expression of GINS members in mice during nervous system development via immunofluorescence staining. At the beginning of neural development, PSF1 and SLD5 were highly expressed in neuroepithelial stem cells (NSCs) of the inner surface of neural tube (NT) and overlapped with proliferation marker Ki67. After entering the mid- and late-phase of neural development, PSF1 and SLD5 changed their regions of expression. These genes were highly expressed in dorsal root ganglion (DRG) progenitors, but they showed no overlap with Ki67 positive cells. Instead, a reduction of SLD5 expression promoted neuronal differentiation and maturation in the late-phase. PSF2 and PSF3 showed no tissue-specificity. PSF2 was constitutively and highly expressed whereas PSF3 was expressed at very low levels during neural development. In this study, we demonstrated variations in proteins and expression regions of the GINS members during mammalian CNS development and revealed a correlation between GINS expression and cell proliferation. Furthermore, we have suggested a novel function of GINS member SLD5, which regulates the differentiation of neural stem/progenitors.

Regnase-1-mediated post-transcriptional regulation is essential for hematopoietic stem and progenitor cell homeostasis.

The balance between self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) maintains hematopoietic homeostasis, failure of which can lead to hematopoietic disorder. HSPC fate is controlled by signals from the bone marrow niche resulting in alteration of the stem cell transcription network. Regnase-1, a member of the CCCH zinc finger protein family possessing RNAse activity, mediates post-transcriptional regulatory activity through degradation of target mRNAs. The precise function of Regnase-1 has been explored in inflammation-related cytokine expression but its function in hematopoiesis has not been elucidated. Here, we show that Regnase-1 regulates self-renewal of HSPCs through modulating the stability of Gata2 and Tal1 mRNA. In addition, we found that dysfunction of Regnase-1 leads to the rapid onset of abnormal hematopoiesis. Thus, our data reveal that Regnase-1-mediated post-transcriptional regulation is required for HSPC maintenance and suggest that it represents a leukemia tumor suppressor.

TAK1 Prevents Endothelial Apoptosis and Maintains Vascular Integrity.

TNF-α is a pleiotropic cytokine that has the potential to induce apoptosis under inflammation. How endothelial cells (ECs) are spared from this fate in inflammatory environments where TNF-α is present is not known. Here, we show that TGF-ß-activated kinase 1 (TAK1) ensures EC survival and maintains vascular integrity upon TNF-α stimulation. Endothelial-specific TAK1 knockout mice exhibit intestinal and liver hemorrhage due to EC apoptosis, leading to vascular destruction and rapid death. This EC apoptosis was induced by TNF-α from myeloid cells responding to intestinal microbiota. TNF-α secretion associated with inflammation also induced vascular defects in inflamed organs. Additionally, we determined that TAK1 deletion in tumor ECs resulted in blood vessel and hence tumor regression. Our results illuminate mechanisms ensuring survival of intestinal and liver ECs under physiological conditions and ECs of other organs under inflammatory conditions that could be exploited for anti-angiogenic therapy to treat cancer.

Galectin-3 Inhibits Cancer Metastasis by Negatively Regulating Integrin ß3 Expression.

Galectin-3 (Gal-3; gene LGALS3) is a member of the ß-galactose-binding lectin family. Previous studies showed that Gal-3 is expressed in several tissues across species and functions as a regulator of cell proliferation, apoptosis, adhesion, and migration, thus affecting many aspects of events, such as angiogenesis and tumorigenesis. Although several reports have suggested that the level of Gal-3 expression correlates positively with tumor progression, herein we show that highly metastatic mouse melanoma B16/BL6 cells express less Gal-3 than B16 cells with a lower metastatic potential. It was found that overexpression of Gal-3 in melanoma cells in fact suppresses metastasis. In contrast, knocking out Gal-3 expression in cancer cells promoted cell aggregation mediated through interactions with platelets and fibrinogen in vitro and increased the number of metastatic foci in vivo. Thus, reduced Gal-3 expression results in the up-regulation of ß3 integrin expression, and this contributes to metastatic potential. These findings indicate that changes of Gal-3 expression in cancer cells during tumor progression influence the characteristics of metastatic cells.

Visualization of Proliferative Vascular Endothelial Cells in Tumors in Vivo by Imaging Their Partner of Sld5-1 Promoter Activity.

Vascular endothelial cells (ECs) isolated from tumors characteristically express certain genes. It has recently been suggested that tumor vessel normalization facilitates effective drug delivery into tumors; however, how tumor vessel normalization can be recognized on the basis of the molecules expressed by tumor ECs is not clearly defined. The degree of cell proliferation is an important indicator to characterize the condition of the ECs. Herein, we generated transgenic mice expressing enhanced green fluorescent protein (EGFP) under the transcriptional control of the DNA replication factor partner of Sld5-1 (PSF1; official name GINS1) promoter to assess whether active ECs can be distinguished from dormant ECs. Predictably, ECs in the adult skin exhibited no EGFP signals. However, after s.c. injection of tumor cells, some ECs shifted to EGFP positivity, enabling distinction of EGFP-positive from EGFP-negative cells. We found that only a fraction of the EGFP-negative ECs strongly expressed the glycosylphosphatidylinositol-anchor protein CD109 associated with the phosphatidylinositol 3-kinase pathway. Taken together, these data indicate that areas of vascular normalization in tumors can be detected by CD109 expression, and this provides a window of opportunity for timing chemotherapy.

APJ Regulates Parallel Alignment of Arteries and Veins in the Skin.

Molecular pathways regulating the development of arterial and venous endothelial cells (ECs) are now well established, but control of parallel arterial-venous alignment is unclear. Here we report that arterial-venous alignment in the skin is determined by apelin receptor (APJ) expression in venous ECs. One of the activators of APJ is apelin. We found that apelin is produced by arterial ECs during embryogenesis, induces chemotaxis of venous ECs, and promotes the production of secreted Frizzled-related protein 1 (sFRP1) by APJ(+) ECs. sFRP1 stimulates matrix metalloproteinase production by Ly6B.2(+) neutrophil-like cells located between the arteries and veins, resulting in remodeling of extracellular matrices to support venous displacement. Moreover, using apelin- or APJ-deficient mice, which exhibit arterial-venous disorganization, we found that arterial-venous alignment is involved in thermoregulation, possibly by regulating countercurrent heat exchange. We hypothesize that the evolution of parallel juxtapositional arterial-venous alignment was an adaptation to reduce body heat loss.

Ligand-independent Tie2 dimers mediate kinase activity stimulated by high dose angiopoietin-1.

Tie2 is a receptor tyrosine kinase expressed on vascular endothelial cells (ECs). It has dual roles in promoting angiogenesis and stabilizing blood vessels, and it has been suggested that Tie2 forms dimers and/or oligomers in the absence of angiopoietin-1 (Ang1); however, the mechanism of ligand-independent dimerization of Tie2 and its biological significance have not been clarified. Using a bimolecular fluorescence complementation assay and a kinase-inactive Tie2 mutant, we show here that ligand-independent Tie2 dimerization is induced without Tie2 phosphorylation. Moreover, based on the fact that Tie1 never forms heterodimers with Tie2 in the absence of Ang1 despite having high amino acid sequence homology with Tie2, we searched for ligand-independent dimerization domains of Tie2 by reference to the difference with Tie1. We found that the YIA sequence of the intracellular domain of Tie2 corresponding to the LAS sequence in Tie1 is essential for this dimerization. When the YIA sequence was replaced by LAS in Tie2 (Tie2YIA/LAS), ligand-independent dimer was not formed in the absence of Ang1. When activation of Tie2YIA/LAS was induced by a high dose of Ang1, phosphorylation of Tie2 was limited compared with wild-type Tie2, resulting in retardation of activation of Erk downstream of Tie2. Therefore, these data suggest that ligand-independent dimerization of Tie2 is essential for a strong response upon stimulation with high dose Ang1.

Galectin-3 accelerates M2 macrophage infiltration and angiogenesis in tumors.

It is widely accepted that robust invasion of tumor-associated macrophages resembling M2 macrophage correlates with disease aggressiveness by affecting cancer cell invasion, metastasis, and angiogenesis. Many chemokines that induce migration of macrophages have been identified during inflammatory responses; however, further precise analysis of macrophage migration in the tumor microenvironment is required. Here, we analyzed the function of galectin-3 (Gal-3; gene LGALS3, alias Gal3) for macrophage chemotaxis using Gal3(-/-) mice as hosts, and a tumor allograft model. We engineered a concentration gradient of Gal-3 produced by the tumor. In this model, we found that macrophage infiltration was enhanced in tumors developing in these Gal3(-/-) mice relative to the Gal3(+/+) animals. This was accompanied by enhanced tumor angiogenesis and tumor growth in Gal3(-/-) mice. We found that macrophages of the M2 phenotype were dominant in infiltrates in the Gal3(-/-) mice and that they expressed only low levels of Gal-3. Gal3 knockdown by siRNA in macrophages resulted in enhanced chemotaxis. These data suggest that M2-like macrophages migrate into the tumor along a Gal-3 gradient and that high-level Gal-3 expression in the tumor results in acceleration of angiogenesis and tumor growth. Therefore, Gal-3 could be a potential target for the development of new treatments to inhibit tumor growth.

2-Methoxycinnamaldehyde inhibits tumor angiogenesis by suppressing Tie2 activation.

Blood vessels are mainly composed of intraluminal endothelial cells (ECs) and mural cells adhering to the ECs on their basal side. Immature blood vessels lacking mural cells are leaky; thus, the process of mural cell adhesion to ECs is indispensable for stability of the vessels during physiological angiogenesis. However, in the tumor microenvironment, although some blood vessels are well-matured, the majority is immature. Because mural cell adhesion to ECs also has a marked anti-apoptotic effect, angiogenesis inhibitors that destroy immature blood vessels may not affect mature vessels showing more resistance to apoptosis. Activation of Tie2 receptor tyrosine kinase expressed in ECs mediates pro-angiogenic effects via the induction of EC migration but also facilitates vessel maturation via the promotion of cell adhesion between mural cells and ECs. Therefore, inhibition of Tie2 has the advantage of completely inhibiting angiogenesis. Here, we isolated a novel small molecule Tie2 kinase inhibitor, identified as 2-methoxycinnamaldehyde (2-MCA). We found that 2-MCA inhibits both sprouting angiogenesis and maturation of blood vessels, resulting in inhibition of tumor growth. Our results suggest a potent clinical benefit of disrupting these two using Tie2 inhibitors.