VEGF-B prevents excessive angiogenesis by inhibiting FGF2/FGFR1 pathway.

Although VEGF-B was discovered as a VEGF-A homolog a long time ago, the angiogenic effect of VEGF-B remains poorly understood with limited and diverse findings from different groups. Notwithstanding, drugs that inhibit VEGF-B together with other VEGF family members are being used to treat patients with various neovascular diseases. It is therefore critical to have a better understanding of the angiogenic effect of VEGF-B and the underlying mechanisms. Using comprehensive in vitro and in vivo methods and models, we reveal here for the first time an unexpected and surprising function of VEGF-B as an endogenous inhibitor of angiogenesis by inhibiting the FGF2/FGFR1 pathway when the latter is abundantly expressed. Mechanistically, we unveil that VEGF-B binds to FGFR1, induces FGFR1/VEGFR1 complex formation, and suppresses FGF2-induced Erk activation, and inhibits FGF2-driven angiogenesis and tumor growth. Our work uncovers a previously unrecognized novel function of VEGF-B in tethering the FGF2/FGFR1 pathway. Given the anti-angiogenic nature of VEGF-B under conditions of high FGF2/FGFR1 levels, caution is warranted when modulating VEGF-B activity to treat neovascular diseases.

PDGFD switches on stem cell endothelial commitment.

The critical factors regulating stem cell endothelial commitment and renewal remain not well understood. Here, using loss- and gain-of-function assays together with bioinformatic analysis and multiple model systems, we show that PDGFD is an essential factor that switches on endothelial commitment of embryonic stem cells (ESCs). PDGFD genetic deletion or knockdown inhibits ESC differentiation into EC lineage and increases ESC self-renewal, and PDGFD overexpression activates ESC differentiation towards ECs. RNA sequencing reveals a critical requirement of PDGFD for the expression of vascular-differentiation related genes in ESCs. Importantly, PDGFD genetic deletion or knockdown increases ESC self-renewal and decreases blood vessel densities in both embryonic and neonatal mice and in teratomas. Mechanistically, we reveal that PDGFD fulfills this function via the MAPK/ERK pathway. Our findings provide new insight of PDGFD as a novel regulator of ESC fate determination, and suggest therapeutic implications of modulating PDGFD activity in stem cell therapy.

Expression and function of PDGF-C in development and stem cells.

Platelet-derived growth factor C (PDGF-C) is a relatively new member of the PDGF family, discovered nearly 20 years after the finding of platelet-derived growth factor A (PDGF-A) and platelet-derived growth factor B (PDGF-B). PDGF-C is generally expressed in most organs and cell types. Studies from the past 20 years have demonstrated critical roles of PDGF-C in numerous biological, physiological and pathological processes, such as development, angiogenesis, tumour growth, tissue remodelling, wound healing, atherosclerosis, fibrosis, stem/progenitor cell regulation and metabolism. Understanding PDGF-C expression and activities thus will be of great importance to various research disciplines. In this review, however, we mainly discuss the expression and functions of PDGF-C and its receptors in development and stem cells.

Platelet-derived growth factor C signaling is a potential therapeutic target for radiation proctopathy.

Radiation proctopathy (RP) is characterized by inflammation of colorectal tissue and is a common complication of radiation therapy for pelvic malignancies with high incidence but lacking effective treatment. Here, we found that platelet-derived growth factor C (PDGF-C) and fibrosis markers were up-regulated in tissue samples from patients with RP and in rectal tissues after irradiation in a mouse model of RP. Genetic deletion of Pdgf-c in mice ameliorated RP-induced injuries. Genome-wide gene expression profiling and in vitro assays revealed that the promotive effect of PDGF-C in RP development was mediated by activation of PDGF receptors (PDGFRs) and C-X-C motif chemokine receptor 4, a proinflammatory chemokine regulated by transcription factor ETS variant transcription factor 1. Treatment with crenolanib, a selective inhibitor of PDGFRs, prevented or reduced RP in mice after irradiation. These results reveal that inhibition of PDGF-C signaling may have therapeutic value for the treatment of RP.

Single-Cell RNA Sequencing Reveals Renal Endothelium Heterogeneity and Metabolic Adaptation to Water Deprivation.

BACKGROUND: Renal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown. METHODS: We inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo. RESULTS: We identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and-surprisingly-oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration. CONCLUSIONS: This study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation.

ILF3 is a substrate of SPOP for regulating serine biosynthesis in colorectal cancer.

The Serine-Glycine-One-Carbon (SGOC) pathway is pivotal in multiple anabolic processes. Expression levels of SGOC genes are deregulated under tumorigenic conditions, suggesting participation of oncogenes in deregulating the SGOC biosynthetic pathway. However, the underlying mechanism remains elusive. Here, we identified that Interleukin enhancer-binding factor 3 (ILF3) is overexpressed in primary CRC patient specimens and correlates with poor prognosis. ILF3 is critical in regulating the SGOC pathway by directly regulating the mRNA stability of SGOC genes, thereby increasing SGOC genes expression and facilitating tumor growth. Mechanistic studies showed that the EGF-MEK-ERK pathway mediates ILF3 phosphorylation, which hinders E3 ligase speckle-type POZ protein (SPOP)-mediated poly-ubiquitination and degradation of ILF3. Significantly, combination of SGOC inhibitor and the anti-EGFR monoclonal antibody cetuximab can hinder the growth of patient-derived xenografts that sustain high ERK-ILF3 levels. Taken together, deregulation of ILF3 via the EGF-ERK signaling plays an important role in systemic serine metabolic reprogramming and confers a predilection toward CRC development. Our findings indicate that clinical evaluation of SGOC inhibitor is warranted for CRC patients with ILF3 overexpression.

The long non-coding RNA Snhg3 is essential for mouse embryonic stem cell self-renewal and pluripotency.

BACKGROUND: Small nucleolar RNA host gene 3 (Snhg3) is a long non-coding RNA (lncRNA) that was shown to participate in the tumorigenesis of certain cancers. However, little is known about its role in embryonic stem cells (ESCs). METHODS: Here, we investigated the role of Snhg3 in mouse ESCs (mESCs) through both loss-of-function (knockdown) and gain-of-function (overexpression) approaches. Alkaline phosphatase staining, secondary colony formation, propidium iodide staining, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to access self-renewal capacity, whereas immunofluorescence, qRT-PCR, and embryoid body formation were performed to examine pluripotency. In addition, the effect of Snhg3 on mouse embryonic development was determined based on the morphological changes, blastocyst rate, and altered pluripotency marker (Nanog, Oct4) expression. Moreover, the relationship between Snhg3 and key pluripotency factors was evaluated by chromatin immunoprecipitation qPCR, qRT-PCR, subcellular fractionation, and RNA immunoprecipitation. Finally, RNA pull-down and mass spectrometry were applied to explore the potential interacting proteins of Snhg3 in mESCs. RESULTS: We demonstrated that Snhg3 is essential for self-renewal and pluripotency maintenance in mESCs. In addition, Snhg3 knockdown disrupted mouse early embryo development. Mechanistically, Snhg3 formed a positive feedback network with Nanog and Oct4, and 126 Snhg3-interacting proteins were identified in mESCs. CONCLUSIONS: Snhg3 is essential for mESC self-renewal and pluripotency, as well as mouse early embryo development.

Novel multi-targeted inhibitors suppress ocular neovascularization by regulating unique gene sets.

Neovascular diseases, such as many cancers and ocular disorders, are life threatening and devastating. Although anti-vascular endothelial growth factor A (VEGF-A) therapy is available, many patients are not responsive and drug resistance can develop. To try to overcome these problems, combination therapy targeting VEGF-A and platelet-derived growth factor B (PDGF-B) was tested. However, one obvious drawback was that the other VEGF and PDGF family members were not inhibited and therefore could compensate. Indeed, this was, at least to some extent, demonstrated by the disappointing outcomes. To this end, we designed novel multi-targeted inhibitors that can block most of the VEGF and PDGF family members simultaneously by making a fusion protein containing the ligand-binding domains of vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2) and platelet-derived growth factor receptor beta (PDGFRß), which can therefore act as a decoy blocker for most of the VEGF and PDGF family members. Indeed, in cultured cells, the novel inhibitors suppressed the migration and proliferation of both vascular endothelial cells and smooth muscle cells, and abolished VEGFR2 and PDGFRß activation. Importantly, in a choroidal neovascularization model in vivo, the novel inhibitor inhibited ocular neovascularization more efficiently than the mono-inhibitors against VEGFR or PDGFR alone respectively. Mechanistically, a genome-wide microarray analysis unveiled that the novel inhibitor regulated unique sets of genes that were not regulated by the mono-inhibitors, further demonstrating the functional uniqueness and superiority of the novel inhibitor. Together, we show that the multi-targeted inhibitors that can block VEGFR1, VEGFR2 and PDGFRß simultaneously suppress pathological angiogenesis more efficiently than monotherapy, and may therefore have promising therapeutic value for the treatment of neovascular diseases.

Interaction Between Sympk and Oct4 Promotes Mouse Embryonic Stem Cell Proliferation.

The scaffold protein Symplekin (Sympk) is involved in cytoplasmic RNA polyadenylation, transcriptional modulation, and the regulation of epithelial differentiation and proliferation via tight junctions. It is highly expressed in embryonic stem cells (ESCs), in which its role remains unknown. In this study, we found Sympk overexpression in mouse ESCs significantly increased colony formation, and Sympk deletion via CRISPR/Cas9 decreased colony formation. Sympk promoted ESC growth and its overexpression sustained ESC pluripotency, as assessed by teratoma and chimeric mouse formation. Genomic stability was preserved in these cells after long-term passage. The domain of unknown function 3453 (DUF3453) in Sympk was required for its interaction with the key pluripotent factor Oct4, and its depletion led to impaired colony formation. Sympk activated proliferation-related genes and suppressed differentiation-related genes. Our results indicate that Sympk interacts with Oct4 to promote self-renewal and pluripotency in ESCs and preserves genome integrity; accordingly, it has potential value for stem cell therapies. Stem Cells 2019;37:743-753.

VEGF-B is a potent antioxidant.

VEGF-B was discovered a long time ago. However, unlike VEGF-A, whose function has been extensively studied, the function of VEGF-B and the mechanisms involved still remain poorly understood. Notwithstanding, drugs that inhibit VEGF-B and other VEGF family members have been used to treat patients with neovascular diseases. It is therefore critical to have a better understanding of VEGF-B function and the underlying mechanisms. Here, using comprehensive methods and models, we have identified VEGF-B as a potent antioxidant. Loss of Vegf-b by gene deletion leads to retinal degeneration in mice, and treatment with VEGF-B rescues retinal cells from death in a retinitis pigmentosa model. Mechanistically, we demonstrate that VEGF-B up-regulates numerous key antioxidative genes, particularly, Gpx1 Loss of Gpx1 activity largely diminished the antioxidative effect of VEGF-B, demonstrating that Gpx1 is at least one of the critical downstream effectors of VEGF-B. In addition, we found that the antioxidant function of VEGF-B is mediated mainly by VEGFR1. Given that oxidative stress is a crucial factor in numerous human diseases, VEGF-B may have therapeutic value for the treatment of such diseases.

Vascular stem/progenitor cells: functions and signaling pathways.

Vascular stem/progenitor cells (VSCs) are an important source of all types of vascular cells needed to build, maintain, repair, and remodel blood vessels. VSCs, therefore, play critical roles in the development, normal physiology, and pathophysiology of numerous diseases. There are four major types of VSCs, including endothelial progenitor cells (EPCs), smooth muscle progenitor cells (SMPCs), pericytes, and mesenchymal stem cells (MSCs). VSCs can be found in bone marrow, circulating blood, vessel walls, and other extravascular tissues. During the past two decades, considerable progress has been achieved in the understanding of the derivation, surface markers, and differentiation of VSCs. Yet, the mechanisms regulating their functions and maintenance under normal and pathological conditions, such as in eye diseases, remain to be further elucidated. Owing to the essential roles of blood vessels in human tissues and organs, understanding the functional properties and the underlying molecular basis of VSCs is of critical importance for both basic and translational research.

PDGFs and their receptors in vascular stem/progenitor cells: Functions and therapeutic potential in retinal vasculopathy.

Vascular stem/progenitor cells (VSCs) include endothelial progenitor cells, smooth muscle progenitor cells, pericytes, and mesenchymal stem cells. VSCs can produce functional and mature vascular cells required to build blood vessels. VSCs therefore play critical roles in vascular repair and regeneration, particularly, in various retinal vasculopathies, in which vascular defects are a devastating pathology. The platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) are important regulators of numerous physiological events and diseases, and they play key roles in regulating the formation and function of blood vessels. A better understanding of the effects of PDGFs/PDGFRs on VSCs and a thorough elucidation of their therapeutic potential in the treatment of retinal vasculopathies are critical for both basic and translational research and may lead to better therapies for human vascular diseases.

Inhibitory effect of caveolin-1 in vascular endothelial cells, pericytes and smooth muscle cells.

Caveolin-1 (Cav1) is the principle structural protein of caveolae. It plays important roles in the vascular system under both physiological and pathological conditions. Although Cav1 has been shown to inhibit microvascular permeability and has been considered as a tumor-suppressor for years, the underlying cellular mechanism has yet to be discovered. Here, we systematically investigated Cav1 functions in the main types of vascular cells, including endothelial cells (ECs), pericytes (PCs) and smooth muscle cells (SMCs). We synthesized a cell-permeable peptide called cavtratin that is derived from the Cav1 scaffolding domain. We found that cavtratin inhibited ECs in all assays, including survival, proliferation, migration and permeability assays. It also inhibited the proliferation of PCs and SMCs but had no effect on their survival or migration. The inhibitory effect of cavtratin on the proliferation of all vascular cells suggests that Cav1 plays important roles in vascular development and angiogenesis. Under physiological condition, the main function of Cav1 is to inhibit EC permeability.

Rho GDP-dissociation inhibitor α is a potential prognostic biomarker and controls telomere regulation in colorectal cancer.

Rho GDP-dissociation inhibitor α (RhoGDIα) is an essential regulator for Rho GTPases. Although RhoGDIα may serve as an oncogene in colorectal cancer (CRC), the underlying mechanism is still unclear. We investigated the function, mechanism, and clinical significance of RhoGDIα in CRC progression. We founded that downregulation of RhoGDIα repressed CRC cell proliferation, motility, and invasion. Overexpression of RhoGDIα increased DNA damage response signals at telomeres, and led to telomere shortening in CRC cells, also being validated in 26 pairs of CRC tissues. Mechanistic studies revealed that RhoGDIα could promote telomeric repeat factor 1 (TRF1) expression through the phosphatidylinositol 3-kinase-protein kinase B signal pathway. Moreover, RhoGDIα protein levels were strongly correlated with TRF1 in CRC tissues. A cohort of 297 CRC samples validated the positive relationship between RhoGDIα and TRF1, and revealed that RhoGDIα and TRF1 levels were negatively associated with CRC patients' survival. Taken together, our results suggest that RhoGDIα regulate TRF1 and telomere length and may be novel prognostic biomarkers in colorectal cancer.

PDGF-CC underlies resistance to VEGF-A inhibition and combinatorial targeting of both suppresses pathological angiogenesis more efficiently.

Anti-VEGF-A therapy has proven to be effective for many neovascular diseases. However, drug resistance to anti-VEGF-A treatment can develop. Also, not all patients with neovascular diseases are responsive to anti-VEGF-A treatment. The mechanisms underlying these important issues remain unclear. In this study, using different model systems, we found that inhibition of VEGF-A directly upregulated PDGF-CC and its receptors in multiple cell types in pathological angiogenesis in vitro and in vivo. Importantly, we further revealed that combinatorial targeting of VEGF-A and PDGF-CC suppressed pathological angiogenesis more efficiently than monotherapy. Given the potent angiogenic activity of PDGF-CC, our findings suggest that the development of resistance to anti-VEGF-A treatment may be caused by the compensatory upregulation of PDGF-CC, and combined inhibition of VEGF-A and PDGF-CC may have therapeutic advantages in treating neovascular diseases.

The Daxx/Atrx Complex Protects Tandem Repetitive Elements during DNA Hypomethylation by Promoting H3K9 Trimethylation.

In mammals, DNA methylation is essential for protecting repetitive sequences from aberrant transcription and recombination. In some developmental contexts (e.g., preimplantation embryos) DNA is hypomethylated but repetitive elements are not dysregulated, suggesting that alternative protection mechanisms exist. Here we explore the processes involved by investigating the role of the chromatin factors Daxx and Atrx. Using genome-wide binding and transcriptome analysis, we found that Daxx and Atrx have distinct chromatin-binding profiles and are co-enriched at tandem repetitive elements in wild-type mouse ESCs. Global DNA hypomethylation further promoted recruitment of the Daxx/Atrx complex to tandem repeat sequences, including retrotransposons and telomeres. Knockdown of Daxx/Atrx in cells with hypomethylated genomes exacerbated aberrant transcriptional de-repression of repeat elements and telomere dysfunction. Mechanistically, Daxx/Atrx-mediated repression seems to involve Suv39h recruitment and H3K9 trimethylation. Our data therefore suggest that Daxx and Atrx safeguard the genome by silencing repetitive elements when DNA methylation levels are low.

ERK2-Dependent Phosphorylation of CSN6 Is Critical in Colorectal Cancer Development.

Biomarkers for predicting prognosis are critical to treating colorectal cancer (CRC) patients. We found that CSN6, a subunit of COP9 signalosome, is overexpressed in CRC samples and that CSN6 overexpression is correlated with poor patient survival. Mechanistic studies revealed that CSN6 is deregulated by epidermal growth factor receptor (EGFR) signaling, in which ERK2 binds directly to CSN6 Leu163/Val165 and phosphorylates CSN6 at Ser148. Furthermore, CSN6 regulated ß-Trcp and stabilized ß-catenin expression by blocking the ubiquitin-proteasome pathway, thereby promoting CRC development. High CSN6 expression was positively correlated with ERK2 activation and ß-catenin overexpression in CRC samples, and inhibiting CSN6 stability with cetuximab reduced colon cancer growth. Taken together, our study's findings indicate that the deregulation of ß-catenin by ERK2-activated CSN6 is important for CRC development.

Actl6a protects embryonic stem cells from differentiating into primitive endoderm.

Actl6a (actin-like protein 6A, also known as Baf53a or Arp4) is a subunit shared by multiple complexes including esBAF, INO80, and Tip60-p400, whose main components (Brg1, Ino80, and p400, respectively) are crucial for the maintenance of embryonic stem cells (ESCs). However, whether and how Actl6a functions in ESCs has not been investigated. ESCs originate from the epiblast (EPI) that is derived from the inner cell mass (ICM) in blastocysts, which also give rise to primitive endoderm (PrE). The molecular mechanisms for EPI/PrE specification remain unclear. In this study, we provide the first evidence that Actl6a can protect mouse ESCs (mESCs) from differentiating into PrE. While RNAi knockdown of Actl6a, which appeared highly expressed in mESCs and downregulated during differentiation, induced mESCs to differentiate towards the PrE lineage, ectopic expression of Actl6a was able to repress PrE differentiation. Our work also revealed that Actl6a could interact with Nanog and Sox2 and promote Nanog binding to pluripotency genes such as Oct4 and Sox2. Interestingly, cells depleted of p400, but not of Brg1 or Ino80, displayed similar PrE differentiation patterns. Mutant Actl6a with impaired ability to bind Tip60 and p400 failed to block PrE differentiation induced by Actl6a dysfunction. Finally, we showed that Actl6a could target to the promoters of key PrE regulators (e.g., Sall4 and Fgf4), repressing their expression and inhibiting PrE differentiation. Our findings uncover a novel function of Actl6a in mESCs, where it acts as a gatekeeper to prevent mESCs from entering into the PrE lineage through a Yin/Yang regulating pattern.

The death-inducer obliterator 1 (Dido1) gene regulates embryonic stem cell self-renewal.

The regulatory network of factors that center on master transcription factors such as Oct4, Nanog, and Sox2 help maintain embryonic stem (ES) cells and ensure their pluripotency. The target genes of these master transcription factors define the ES cell transcriptional landscape. In this study, we report our findings that Dido1, a target of canonical transcription factors such as Oct4, Sox2, and Nanog, plays an important role in regulating ES cell maintenance. We found that depletion of Dido1 in mouse ES cells led to differentiation, and ectopic expression of Dido1 inhibited differentiation induced by leukemia inhibitory factor withdrawal. We further demonstrated that whereas Nanog and Oct4 could occupy the Dido1 locus and promote its transcription, Dido1 could also target to the loci of pluripotency factors such as Nanog and Oct4 and positively regulate their expression. Through this feedback and feedforward loop, Dido1 is able to regulate self-renewal of mouse ES cells.

Akt regulates TPP1 homodimerization and telomere protection.

Telomeres are specialized structures at the ends of eukaryotic chromosomes that are important for maintaining genome stability and integrity. Telomere dysfunction has been linked to aging and cancer development. In mammalian cells, extensive studies have been carried out to illustrate how core telomeric proteins assemble on telomeres to recruit the telomerase and additional factors for telomere maintenance and protection. In comparison, how changes in growth signaling pathways impact telomeres and telomere-binding proteins remains largely unexplored. The phosphatidylinositol 3-kinase (PI3-K)/Akt (also known as PKB) pathway, one of the best characterized growth signaling cascades, regulates a variety of cellular function including cell proliferation, survival, metabolism, and DNA repair, and dysregulation of PI3-K/Akt signaling has been linked to aging and diseases such as cancer and diabetes. In this study, we provide evidence that the Akt signaling pathway plays an important role in telomere protection. Akt inhibition either by chemical inhibitors or small interfering RNAs induced telomere dysfunction. Furthermore, we found that TPP1 could homodimerize through its OB-fold, a process that was dependent on the Akt kinase. Telomere damage and reduced TPP1 dimerization as a result of Akt inhibition was also accompanied by diminished recruitment of TPP1 and POT1 to the telomeres. Our findings highlight a previously unknown link between Akt signaling and telomere protection.