Tumor endothelial cell-derived Sfrp1 supports the maintenance of cancer stem cells via Wnt signaling.

Cancer stem cells (CSCs), which are critical targets for cancer therapy as they are involved in drug resistance to anticancer drugs, and metastasis, are maintained by angiocrine factors produced by particular niches that form within tumor tissue. Secreted frizzled-related protein 1 (Sfrp1) is an extracellular protein that modulates Wnt signaling. However, the cells that produce Sfrp1 in the tumor environment and its function remain unclear. We aimed to elucidate angiocrine factors related to CSC maintenance, focusing on Sfrp1. Although Sfrp1 is a Wnt pathway-related factor, its impact on tumor tissues remains unknown. We investigated the localization of Sfrp1 in tumors and found that it is expressed in some tumor vessels. Analysis of mice lacking Sfrp1 showed that tumor growth was suppressed in Sfrp1-deficient tumor tissues. Flow cytometry analysis indicated that CSCs were maintained in the early tumor growth phase in the Sfrp1 knockout (KO) mouse model of tumor-bearing cancer. However, tumor growth was inhibited in the late tumor growth phase because of the inability to maintain CSCs. Real-time PCR results from tumors of Sfrp1 KO mice showed that the expression of Wnt signaling target genes significantly decreased in the late stage of tumor growth. This suggests that Sfrp1, an angiocrine factor produced by the tumor vascular niche, is involved in Wnt signaling-mediated mechanisms in tumor tissues.

Hypoxia activates SREBP2 through Golgi disassembly in bone marrow-derived monocytes for enhanced tumor growth.

Bone marrow-derived cells (BMDCs) infiltrate hypoxic tumors at a pre-angiogenic state and differentiate into mature macrophages, thereby inducing pro-tumorigenic immunity. A critical factor regulating this differentiation is activation of SREBP2-a well-known transcription factor participating in tumorigenesis progression-through unknown cellular mechanisms. Here, we show that hypoxia-induced Golgi disassembly and Golgi-ER fusion in monocytic myeloid cells result in nuclear translocation and activation of SREBP2 in a SCAP-independent manner. Notably, hypoxia-induced SREBP2 activation was only observed in an immature lineage of bone marrow-derived cells. Single-cell RNA-seq analysis revealed that SREBP2-mediated cholesterol biosynthesis was upregulated in HSCs and monocytes but not in macrophages in the hypoxic bone marrow niche. Moreover, inhibition of cholesterol biosynthesis impaired tumor growth through suppression of pro-tumorigenic immunity and angiogenesis. Thus, our findings indicate that Golgi-ER fusion regulates SREBP2-mediated metabolic alteration in lineage-specific BMDCs under hypoxia for tumor progression.

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.

Upregulation of Robo4 expression by SMAD signaling suppresses vascular permeability and mortality in endotoxemia and COVID-19 models.

There is an urgent need to develop novel drugs to reduce the mortality from severe infectious diseases with the emergence of new pathogens, including Coronavirus disease 2019 (COVID-19). Although current drugs effectively suppress the proliferation of pathogens, immune cell activation, and inflammatory cytokine functions, they cannot completely reduce mortality from severe infections and sepsis. In this study, we focused on the endothelial cell-specific protein, Roundabout 4 (Robo4), which suppresses vascular permeability by stabilizing endothelial cells, and investigated whether enhanced Robo4 expression could be a novel therapeutic strategy against severe infectious diseases. Endothelial-specific overexpression of Robo4 suppresses vascular permeability and reduces mortality in lipopolysaccharide (LPS)-treated mice. Screening of small molecules that regulate Robo4 expression and subsequent analysis revealed that two competitive small mothers against decapentaplegic (SMAD) signaling pathways, activin receptor-like kinase 5 (ALK5)-SMAD2/3 and ALK1-SMAD1/5, positively and negatively regulate Robo4 expression, respectively. An ALK1 inhibitor was found to increase Robo4 expression in mouse lungs, suppress vascular permeability, prevent extravasation of melanoma cells, and decrease mortality in LPS-treated mice. The inhibitor suppressed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced endothelial barrier disruption and decreased mortality in mice infected with SARS-CoV-2. These results indicate that enhancing Robo4 expression is an efficient strategy to suppress vascular permeability and mortality in severe infectious diseases, including COVID-19, and that small molecules that upregulate Robo4 can be potential therapeutic agents against these diseases.

Apelin Resistance Contributes to Muscle Loss during Cancer Cachexia in Mice.

Cancer cachexia consists of dramatic body weight loss with rapid muscle depletion due to imbalanced protein homeostasis. We found that the mRNA levels of apelin decrease in muscles from cachectic hepatoma-bearing rats and three mouse models of cachexia. Furthermore, apelin expression inversely correlates with MuRF1 in muscle biopsies from cancer patients. To shed light on the possible role of apelin in cachexia in vivo, we generated apelin 13 carrying all the last 13 amino acids of apelin in D isomers, ultimately extending plasma stability. Notably, apelin D-peptides alter cAMP-based signaling in vitro as the L-peptides, supporting receptor binding. In vitro apelin 13 protects myotube diameter from dexamethasone-induced atrophy, restrains rates of degradation of long-lived proteins and MuRF1 expression, but fails to protect mice from atrophy. D-apelin 13 given intraperitoneally for 13 days in colon adenocarcinoma C26-bearing mice does not reduce catabolic pathways in muscles, as it does in vitro. Puzzlingly, the levels of circulating apelin seemingly deriving from cachexia-inducing tumors, increase in murine plasma during cachexia. Muscle electroporation of a plasmid expressing its receptor APJ, unlike apelin, preserves myofiber area from C26-induced atrophy, supporting apelin resistance in vivo. Altogether, we believe that during cachexia apelin resistance occurs, contributing to muscle wasting and nullifying any possible peptide-based treatment.

Neurovascular Interactions in the Development of the Vasculature.

Vertebrates have developed a network of blood vessels and nerves throughout the body that enables them to perform complex higher-order functions and maintain homeostasis. The 16th-century anatomical text 'De humani corporis fabrica' describes the networks of blood vessels and nerves as having a branching pattern in which they are closely aligned and run parallel one to another. This close interaction between adjacent blood vessels and nerves is essential not only for organogenesis during development and repair at the time of tissue damage but also for homeostasis and functional expression of blood vessels and nerves. Furthermore, it is now evident that disruptions in neurovascular interactions contribute to the progression of various diseases including cancer. Therefore, we highlight recent advances in vascular biology research, with a particular emphasis on neurovascular interactions.

Blood vessels sense dermal stiffness via a novel mechanotransducer, APJ.

Microvascular dysfunction accompanied by a dramatic alteration of stable capillary structure is a major hallmark of numerous age-related diseases. In skin, although the role of angiogenesis during dermal reconstitution is well documented, the functional relevance of the extracellular matrix (ECM) stiffness to vascular remodeling and its molecular mechanisms was poorly understood. Here, we developed an ex vivo 3-dimensional angiogenic model using human fat, revealing that "appropriate" stiffness induces vascular maturation associated with upregulated APJ expression, whereas the overexpression of APJ promotes the formation of large vessels even in the absence of the "appropriate" stiffness. Taken together, APJ could be a novel mechanotransducer that accelerates the maturation of cutaneous blood vessels, leading to the prevention of human skin aging.

Endothelial cell-derived Apelin inhibits tumor growth by altering immune cell localization.

The Apelin/APJ signalling pathway, involved in multiple physiological and pathological processes, has been attracting increasing interest recently. In our previous study, Apelin overexpression in colon26 tumor cells suppressed tumor growth by inducing vascular maturation. Here, we found that MC38 and LLC tumor growth were greater in the absence of Apelin than in wild-type (WT) mice, suggesting that Apelin acts as a tumor suppressor. Consistent with this, treating WT mice with [Pyr1]Apelin-13 inhibited tumor growth. In MC38 tumors, only endothelial cells (ECs) strongly express APJ, a cognate receptor for Apelin, indicating that EC-derived Apelin might regulate tumor formation in an autocrine manner. Comparing with WT mice, larger numbers of vessels with narrower diameters were observed in tumors of Apelin knockout mice and lack of Apelin enhanced tumor hypoxia. Investigating immune cells in the tumor revealed that [Pyr1]Apelin-13 infusion induced the accumulation of CD8+ and CD4+ T cells in central areas. Moreover, RNA-sequencing analysis showed that Apelin induces chemokine CCL8 expression in ECs. Thus, enhancing anti-tumor immunity might be one of the mechanisms by which Apelin is involved in tumor growth. Our result indicated that increased CCL8 expression might induce CD8 + T cells infiltration into tumor and tumor inhibition.

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.

Vasculature-driven stem cell population coordinates tissue scaling in dynamic organs.

Stem cell (SC) proliferation and differentiation organize tissue homeostasis. However, how SCs regulate coordinate tissue scaling in dynamic organs remain unknown. Here, we delineate SC regulations in dynamic skin. We found that interfollicular epidermal SCs (IFESCs) shape basal epidermal proliferating clusters (EPCs) in expanding abdominal epidermis of pregnant mice and proliferating plantar epidermis. EPCs consist of IFESC-derived Tbx3+-basal cells (Tbx3+-BCs) and their neighboring cells where Adam8-extracellular signal-regulated kinase signaling is activated. Clonal lineage tracing revealed that Tbx3+-BC clones emerge in the abdominal epidermis during pregnancy, followed by differentiation after parturition. In the plantar epidermis, Tbx3+-BCs are sustained as long-lived SCs to maintain EPCs invariably. We showed that Tbx3+-BCs are vasculature-dependent IFESCs and identified mechanical stretch as an external cue for the vasculature-driven EPC formation. Our results uncover vasculature-mediated IFESC regulations, which explain how the epidermis adjusts its size in orchestration with dermal constituents in dynamic skin.

An in vivo model allowing continuous observation of human vascular formation in the same animal over time.

Angiogenesis contributes to numerous pathological conditions. Understanding the molecular mechanisms of angiogenesis will offer new therapeutic opportunities. Several experimental in vivo models that better represent the pathological conditions have been generated for this purpose in mice, but it is difficult to translate results from mouse to human blood vessels. To understand human vascular biology and translate findings into human research, we need human blood vessel models to replicate human vascular physiology. Here, we show that human tumor tissue transplantation into a cranial window enables engraftment of human blood vessels in mice. An in vivo imaging technique using two-photon microscopy allows continuous observation of human blood vessels until at least 49 days after tumor transplantation. These human blood vessels make connections with mouse blood vessels as shown by the finding that lectin injected into the mouse tail vein reaches the human blood vessels. Finally, this model revealed that formation and/or maintenance of human blood vessels depends on VEGFR2 signaling. This approach represents a useful tool to study molecular mechanisms of human blood vessel formation and to test effects of drugs that target human blood vessels in vivo to show proof of concept in a preclinical model.

Age-dependent decline in remyelination capacity is mediated by apelin-APJ signaling.

Age-related regeneration failure in the central nervous system can occur as a result of a decline in remyelination efficacy. The responsiveness of myelin-forming cells to signals for remyelination is affected by aging-related epigenetic modification; however, the molecular mechanism is not fully clarified. In the present study, we report that the apelin receptor (APJ) mediates remyelination efficiency with age. APJ expression in myelin-forming cells is correlated with age-associated changes in remyelination efficiency, and the activation of APJ promotes remyelination through the translocation of myelin regulatory factor. APJ signaling activation promoted remyelination in both aged mice with toxin-induced demyelination and mice with experimental autoimmune encephalomyelitis. In human cells, APJ activation enhanced the expression of remyelination markers. Impaired oligodendrocyte function in aged animals can be reversibly reactivated; thus, the results demonstrate that dysfunction of the apelin-APJ system mediates remyelination failure in aged animals, and that their myelinating function can be reactivated by APJ activation.

Loss of Down Syndrome Critical Region-1 Mediated-Hypercholesterolemia Accelerates Corneal Opacity Via Pathological Neovessel Formation.

OBJECTIVE: The calcineurin-NFAT (nuclear factor for activated T cells)-DSCR (Down syndrome critical region)-1 pathway plays a crucial role as the downstream effector of VEGF (vascular endothelial growth factor)-mediated tumor angiogenesis in endothelial cells. A role for DSCR-1 in different organ microenvironment such as the cornea and its role in ocular diseases is not well understood. Corneal changes can be indicators of various disease states and are easily detected through ocular examinations. Approach and Results: The presentation of a corneal arcus or a corneal opacity due to lipid deposition in the cornea often indicates hyperlipidemia and in most cases, hypercholesterolemia. Although the loss of Apo (apolipoprotein) E has been well characterized and is known to lead to elevated serum cholesterol levels, there are few corneal changes observed in ApoE-/- mice. In this study, we show that the combined loss of ApoE and DSCR-1 leads to a dramatic increase in serum cholesterol levels and severe corneal opacity with complete penetrance. The cornea is normally maintained in an avascular state; however, loss of Dscr-1 is sufficient to induce hyper-inflammatory and -oxidative condition, increased corneal neovascularization, and lymphangiogenesis. Furthermore, immunohistological analysis and genome-wide screening revealed that loss of Dscr-1 in mice triggers increased immune cell infiltration and upregulation of SDF (stromal derived factor)-1 and its receptor, CXCR4 (C-X-C motif chemokine ligand receptor-4), potentiating this signaling axis in the cornea, thereby contributing to pathological corneal angiogenesis and opacity. CONCLUSIONS: This study is the first demonstration of the critical role for the endogenous inhibitor of calcineurin, DSCR-1, and pathological corneal angiogenesis in hypercholesterolemia induced corneal opacity.

Zinc Finger Protein St18 Protects against Septic Death by Inhibiting VEGF-A from Macrophages.

Zinc finger protein St18 was initially reported as candidate tumor suppressor gene, and also suggested that fibroblast St18 positively regulates NF-κB activation. Despite the pleiotropic functions of St18, little is known about its roles in macrophages. Here, we report that myeloid St18 is a potent inhibitor of VEGF-A. Mice lacking St18 in myeloid lineages exhibit increased retinal vasculature with enhanced serum VEGF-A concentrations. Despite the normal activation of NF-κB target genes, these mice are highly susceptible to LPS-induced shock, polymicrobial sepsis, and experimental colitis, accompanied by enhanced vascular and intestinal leakage. Pharmacological inhibition of VEGF signaling rescued the high mortality rate of myeloid-specific St18-deficient mice in response to inflammation. Mechanistically, St18 directly binds to Sp1 and attenuates its activity, leading to the suppression of Sp1 target gene VEGF-A. Using mouse genetic and pharmacological models, we reveal myeloid St18 as a critical septic death protector.

The Bartonella autotransporter BafA activates the host VEGF pathway to drive angiogenesis.

Pathogenic bacteria of the genus Bartonella can induce vasoproliferative lesions during infection. The underlying mechanisms are unclear, but involve secretion of an unidentified mitogenic factor. Here, we use functional transposon-mutant screening in Bartonella henselae to identify such factor as a pro-angiogenic autotransporter, called BafA. The passenger domain of BafA induces cell proliferation, tube formation and sprouting of microvessels, and drives angiogenesis in mice. BafA interacts with vascular endothelial growth factor (VEGF) receptor-2 and activates the downstream signaling pathway, suggesting that BafA functions as a VEGF analog. A BafA homolog from a related pathogen, Bartonella quintana, is also functional. Our work unveils the mechanistic basis of vasoproliferative lesions observed in bartonellosis, and we propose BafA as a key pathogenic factor contributing to bacterial spread and host adaptation.

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.

Spatiotemporally Dependent Vascularization Is Differently Utilized among Neural Progenitor Subtypes during Neocortical Development.

To facilitate efficient oxygen and nutrient delivery, blood vessels in the brain form three-dimensional patterns. However, little is known about how blood vessels develop stereographically in the neocortex and how they control the expansion and differentiation of neural progenitors during neocortical development. We show that highly vascularized and avascular regions are strictly controlled in a spatially and temporally restricted manner and are associated with distinct cell populations. Dividing basal progenitors and oligodendrocyte precursors preferentially contact honeycomb vessels, but dividing apical progenitors are localized in avascular regions without Flt1-positive endothelial cells but directly contact with sprouting neovascular tip cells. Therefore, not all blood vessels are associated equally with neural progenitors. Furthermore, a disruption of normal vascular patterning can induce abnormalities in neural development, whereas the impaired features of neural progenitors influenced angiogenesis patterning. These results indicate that close association between the nervous and vascular systems is essential for neocortex assembly.

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.

Endothelial Cells Regulate Physiological Cardiomyocyte Growth via VEGFR2-Mediated Paracrine Signaling.

BACKGROUND: Heart failure, which is a major global health problem, is often preceded by pathological cardiac hypertrophy. The expansion of the cardiac vasculature, to maintain adequate supply of oxygen and nutrients, is a key determinant of whether the heart grows in a physiological compensated manner or a pathological decompensated manner. Bidirectional endothelial cell (EC)-cardiomyocyte (CMC) cross talk via cardiokine and angiocrine signaling plays an essential role in the regulation of cardiac growth and homeostasis. Currently, the mechanisms involved in the EC-CMC interaction are not fully understood, and very little is known about the EC-derived signals involved. Understanding how an excess of angiogenesis induces cardiac hypertrophy and how ECs regulate CMC homeostasis could provide novel therapeutic targets for heart failure. METHODS: Genetic mouse models were used to delete vascular endothelial growth factor (VEGF) receptors, adeno-associated viral vectors to transduce the myocardium, and pharmacological inhibitors to block VEGF and ErbB signaling in vivo. Cell culture experiments were used for mechanistic studies, and quantitative polymerase chain reaction, microarrays, ELISA, and immunohistochemistry were used to analyze the cardiac phenotypes. RESULTS: Both EC deletion of VEGF receptor (VEGFR)-1 and adeno-associated viral vector-mediated delivery of the VEGFR1-specific ligands VEGF-B or placental growth factor into the myocardium increased the coronary vasculature and induced CMC hypertrophy in adult mice. The resulting cardiac hypertrophy was physiological, as indicated by preserved cardiac function and exercise capacity and lack of pathological gene activation. These changes were mediated by increased VEGF signaling via endothelial VEGFR2, because the effects of VEGF-B and placental growth factor on both angiogenesis and CMC growth were fully inhibited by treatment with antibodies blocking VEGFR2 or by endothelial deletion of VEGFR2. To identify activated pathways downstream of VEGFR2, whole-genome transcriptomics and secretome analyses were performed, and the Notch and ErbB pathways were shown to be involved in transducing signals for EC-CMC cross talk in response to angiogenesis. Pharmacological or genetic blocking of ErbB signaling also inhibited part of the VEGF-B-induced effects in the heart. CONCLUSIONS: This study reveals that cross talk between the EC VEGFR2 and CMC ErbB signaling pathways coordinates CMC hypertrophy with angiogenesis, contributing to physiological cardiac growth.