Centriole Translational Planar Polarity in Monociliated Epithelia.

Ciliated epithelia are widespread in animals and play crucial roles in many developmental and physiological processes. Epithelia composed of multi-ciliated cells allow for directional fluid flow in the trachea, oviduct and brain cavities. Monociliated epithelia play crucial roles in vertebrate embryos, from the establishment of left-right asymmetry to the control of axis curvature via cerebrospinal flow motility in zebrafish. Cilia also have a central role in the motility and feeding of free-swimming larvae in a variety of marine organisms. These diverse functions rely on the coordinated orientation (rotational polarity) and asymmetric localization (translational polarity) of cilia and of their centriole-derived basal bodies across the epithelium, both being forms of planar cell polarity (PCP). Here, we review our current knowledge on the mechanisms of the translational polarity of basal bodies in vertebrate monociliated epithelia from the molecule to the whole organism. We highlight the importance of live imaging for understanding the dynamics of centriole polarization. We review the roles of core PCP pathways and of apicobasal polarity proteins, such as Par3, whose central function in this process has been recently uncovered. Finally, we emphasize the importance of the coordination between polarity proteins, the cytoskeleton and the basal body itself in this highly dynamic process.

Vangl2 regulates intercellular junctions by remodeling actin-based cytoskeleton through the Rock signaling pathway during spermatogenesis in Eriocheir sinensis.

As a key planar cell polarity protein, Van Gogh-like 2 (Vangl2) is essential for mammalian spermatogenesis. As a decapod crustacean, Eriocheir sinensis exhibits distinct spermatogenic processes due to its unique seminiferous tubule morphology and hemolymph-testis barrier (HTB). To determine whether Vangl2 performs analogous functions in E. sinensis, we identified the Es-Vangl2. Es-Vangl2 exhibited high expression and wide distribution in the testes, indicating its crucial involvement in spermatogenesis. Following targeted knockdown of Es-Vangl2in vivo, the structure of seminiferous tubules was disrupted, characterized by vacuolization of the germinal zone and obstruction of spermatozoon release. Concurrently, the integrity of the HTB was compromised, accompanied by reduced expression and aberrant localization of junction proteins. More importantly, the regulatory influence of Es-Vangl2 was manifested through modulating the organization of microfilaments, a process mediated by epidermal growth factor receptor pathway substrate 8 (Eps8). Further studies demonstrated that these phenotypes resulting from Es-Vangl2 knockdown were attributed to the inhibition of Rock signaling pathway activity, which was verified by the Es-Rock interference and Y27632 inhibition assays. In summary, the findings highlight the pivotal role of Es-Vangl2 in stabilizing HTB integrity by regulating Eps8-mediated actin remodeling through the Rock signaling pathway in the spermatogenesis of E. sinensis.

Vangl2 suppresses NF-κB signaling and ameliorates sepsis by targeting p65 for NDP52-mediated autophagic degradation.

Van Gogh-like 2 (Vangl2), a core planar cell polarity component, plays an important role in polarized cellular and tissue morphology induction, growth development, and cancer. However, its role in regulating inflammatory responses remains elusive. Here, we report that Vangl2 is upregulated in patients with sepsis and identify Vangl2 as a negative regulator of The nuclear factor-kappaB (NF-κB) signaling by regulating the protein stability and activation of the core transcription component p65. Mice with myeloid-specific deletion of Vangl2 (Vangl2ΔM) are hypersusceptible to lipopolysaccharide (LPS)-induced septic shock. Vangl2-deficient myeloid cells exhibit enhanced phosphorylation and expression of p65, therefore, promoting the secretion of proinflammatory cytokines after LPS stimulation. Mechanistically, NF-κB signaling-induced-Vangl2 recruits E3 ubiquitin ligase PDLIM2 to catalyze K63-linked ubiquitination on p65, which serves as a recognition signal for cargo receptor NDP52-mediated selective autophagic degradation. Taken together, these findings demonstrate Vangl2 as a suppressor of NF-κB-mediated inflammation and provide insights into the crosstalk between autophagy and inflammatory diseases.

Stearoylation cycle regulates the cell surface distribution of the PCP protein Vangl2.

Defects in planar cell polarity (PCP) have been implicated in diverse human pathologies. Vangl2 is one of the core PCP components crucial for PCP signaling. Dysregulation of Vangl2 has been associated with severe neural tube defects and cancers. However, how Vangl2 protein is regulated at the posttranslational level has not been well understood. Using chemical reporters of fatty acylation and biochemical validation, here we present that Vangl2 subcellular localization is regulated by a reversible S-stearoylation cycle. The dynamic process is mainly regulated by acyltransferase ZDHHC9 and deacylase acyl-protein thioesterase 1 (APT1). The stearoylation-deficient mutant of Vangl2 shows decreased plasma membrane localization, resulting in disruption of PCP establishment during cell migration. Genetically or pharmacologically inhibiting ZDHHC9 phenocopies the effects of the stearoylation loss of Vangl2. In addition, loss of Vangl2 stearoylation enhances the activation of oncogenic Yes-associated protein 1 (YAP), serine-threonine kinase AKT, and extracellular signal-regulated protein kinase (ERK) signaling and promotes breast cancer cell growth and HRas G12V mutant (HRasV12)-induced oncogenic transformation. Our results reveal a regulation mechanism of Vangl2, and provide mechanistic insight into how fatty acid metabolism and protein fatty acylation regulate PCP signaling and tumorigenesis by core PCP protein lipidation.

Expression Profiles of ITGA8 and VANGL2 Are Altered in Congenital Anomalies of the Kidney and Urinary Tract (CAKUT).

Kidney failures in infants are mostly caused by congenital anomalies of the kidney and urinary tract (CAKUT), which are among the most common congenital birth disorders worldwide when paired with cardiac abnormalities. People with CAKUT often have severe kidney failure as a result of a wide range of abnormalities that can occur alone or in conjunction with other syndromic disorders. In this study, we aimed to investigate the expression pattern of CAKUT candidate genes alpha-8 integrin (ITGA8) and Van Gogh-like 2 (VANGL2) in fetal tissues of healthy and CAKUT-affected kidneys using immunohistochemistry and immunofluorescence. We found that under CAKUT circumstances, the expressions of ITGA8 and VANGL2 are changed. Additionally, we showed that VANGL2 expression is constant during fetal aging, but ITGA8 expression varies. Moreover, compared to normal healthy kidneys (CTRL), ITGA8 is poorly expressed in duplex kidneys (DKs) and dysplastic kidneys (DYS), whereas VANGL2 is substantially expressed in dysplastic kidneys (DYS) and poorly expressed in hypoplastic kidneys (HYP). These results point to VANGL2 and ITGA8 as potential prognostic indicators for CAKUT malformations. Further research is necessary to explore the molecular mechanisms underlying this differential expression of ITGA8 and VANGL2.

Non-canonical Wnt pathway expression in the developing mouse and human retina.

The non-canonical Wnt pathway is an evolutionarily conserved pathway essential for tissue patterning and development across species and tissues. In mammals, this pathway plays a role in neuronal migration, dendritogenesis, axon growth, and synapse formation. However, its role in development and synaptogenesis of the human retina remains less established. In order to address this knowledge gap, we analyzed publicly available single-cell RNA sequencing (scRNAseq) datasets for mouse retina, human retina, and human retinal organoids over multiple developmental time points during outer retinal maturation. We identified ligands, receptors, and mediator genes with a putative role in retinal development, including those with novel or species-specific expression, and validated this expression using fluorescence in situ hybridization (FISH). By quantifying outer nuclear layer (ONL) versus inner nuclear layer (INL) expression, we provide evidence for the differential expression of certain non-canonical Wnt signaling components in the developing mouse and human retina during outer plexiform layer (OPL) development. Importantly, we identified distinct expression patterns of mouse and human FZD3 and WNT10A, as well as previously undescribed expression, such as for mouse Wnt2b in Chat+ starburst amacrine cells. Human retinal organoids largely recapitulated the human non-canonical Wnt pathway expression. Together, this work provides the basis for further study of non-canonical Wnt signaling in mouse and human retinal development and synaptogenesis.

Vangl-dependent mesenchymal thinning shapes the distal lung during murine sacculation.

The planar cell polarity (PCP) complex is speculated to function in murine lung development, where branching morphogenesis generates an epithelial tree whose distal tips expand dramatically during sacculation. Here, we show that PCP is dispensable in the airway epithelium for sacculation. Rather, we find a Celsr1-independent role for the PCP component Vangl in the pulmonary mesenchyme: loss of Vangl1/2 inhibits mesenchymal thinning and expansion of the saccular epithelium. Further, loss of mesenchymal Wnt5a mimics sacculation defects observed in Vangl2-mutant lungs, implicating mesenchymal Wnt5a/Vangl signaling as a key regulator of late lung morphogenesis. A computational model predicts that sacculation requires a fluid mesenchymal compartment. Lineage-tracing and cell-shape analyses are consistent with the mesenchyme acting as a fluid tissue, suggesting that loss of Vangl1/2 impacts the ability of mesenchymal cells to exchange neighbors. Our data thus identify an explicit function for Vangl and the pulmonary mesenchyme in actively shaping the saccular epithelium.

A vertebrate Vangl2 translational variant required for planar cell polarity.

First described in the milkweed bug Oncopeltus fasciatus, planar cell polarity (PCP) is a developmental process essential for embryogenesis and development of polarized structures in Metazoans. This signaling pathway involves a set of evolutionarily conserved genes encoding transmembrane (Vangl, Frizzled, Celsr) and cytoplasmic (Prickle, Dishevelled) molecules. Vangl2 is of major importance in embryonic development as illustrated by its pivotal role during neural tube closure in human, mouse, Xenopus, and zebrafish embryos. Here, we report on the molecular and functional characterization of a Vangl2 isoform, Vangl2-Long, containing an N-terminal extension of about 50 aa, which arises from an alternative near-cognate AUA translation initiation site, lying upstream of the conventional start codon. While missing in Vangl1 paralogs and in all invertebrates, including Drosophila, this N-terminal extension is conserved in all vertebrate Vangl2 sequences. We show that Vangl2-Long belongs to a multimeric complex with Vangl1 and Vangl2. Using morpholino oligonucleotides to specifically knockdown Vangl2-Long in Xenopus, we found that this isoform is functional and required for embryo extension and neural tube closure. Furthermore, both Vangl2 and Vangl2-Long must be correctly expressed for the polarized distribution of the PCP molecules Pk2 and Dvl1 and for centriole rotational polarity in ciliated epidermal cells. Altogether, our study suggests that Vangl2-Long significantly contributes to the pool of Vangl2 molecules present at the plasma membrane to maintain PCP in vertebrate tissues.

Nuclear VANGL2 Inhibits Lactogenic Differentiation.

Planar cell polarity (PCP) proteins coordinate tissue morphogenesis by governing cell patterning and polarity. Asymmetrically localized on the plasma membrane of cells, transmembrane PCP proteins are trafficked by endocytosis, suggesting they may have intracellular functions that are dependent or independent of their extracellular role, but whether these functions extend to transcriptional control remains unknown. Here, we show the nuclear localization of transmembrane, PCP protein, VANGL2, in the HCC1569 breast cancer cell line, and in undifferentiated, but not differentiated, HC11 cells that serve as a model for mammary lactogenic differentiation. The loss of Vangl2 function results in upregulation of pathways related to STAT5 signaling. We identify DNA binding sites and a nuclear localization signal in VANGL2, and use CUT&RUN to demonstrate recruitment of VANGL2 to specific DNA binding motifs, including one in the Stat5a promoter. Knockdown (KD) of Vangl2 in HC11 cells and primary mammary organoids results in upregulation of Stat5a, Ccnd1 and Csn2, larger acini and organoids, and precocious differentiation; phenotypes are rescued by overexpression of Vangl2, but not Vangl2ΔNLS. Together, these results advance a paradigm whereby PCP proteins coordinate tissue morphogenesis by keeping transcriptional programs governing differentiation in check.

Folate regulation of planar cell polarity pathway and F-actin through folate receptor alpha.

Folate deficiency contribute to neural tube defects (NTDs) which could be rescued by folate supplementation. However, the underlying mechanisms are still not fully understood. Besides, there is considerable controversy concerning the forms of folate used for supplementation. To address this controversy, we prepared culture medium with different forms of folate, folic acid (FA), and 5-methyltetrahydrofolate (5mTHF), at concentrations of 5 µM, 500 nM, 50 nM, and folate free, respectively. Mouse embryonic fibroblasts (MEFs) were treated with different folates continuously for three passages, and cell proliferation and F-actin were monitored. We determined that compared to 5mTHF, FA showed stronger effects on promoting cell proliferation and F-actin formation. We also found that FOLR1 protein level was positively regulated by folate concentration and the non-canonical Wnt/planar cell polarity (PCP) pathway signaling was significantly enriched among different folate conditions in RNA-sequencing analyses. We demonstrated for the first time that FOLR1 could promote the transcription of Vangl2, one of PCP core genes. The transcription of Vangl2 was down-regulated under folate-deficient condition, which resulted in a decrease in PCP activity and F-actin formation. In summary, we identified a distinct advantage of FA in cell proliferation and F-actin formation over 5mTHF, as well as demonstrating that FOLR1 could promote transcription of Vangl2 and provide a new mechanism by which folate deficiency can contribute to the etiology of NTDs.

PDZome-wide and structural characterization of the PDZ-binding motif of VANGL2.

VANGL2 is a core component of the non-canonical Wnt/Planar Cell Polarity signaling pathway that uses its highly conserved carboxy-terminal type 1 PDZ-binding motif (PBM) to bind a variety of PDZ proteins. In this study, we characterize and quantitatively assess the largest VANGL2 PDZome-binding profile documented so far, using orthogonal methods. The results of our holdup approach support VANGL2 interactions with a large panel of both long-recognized and unprecedented PDZ domains. Truncation and point mutation analyses of the VANGL2 PBM establish that, beyond the strict requirement of the P-0 / V521 and P-2 / T519 amino acids, upstream residues, including E518, Q516 and R514 at, respectively, P-3, P-5 and P-7 further contribute to the robustness of VANGL2 interactions with two distinct PDZ domains, SNX27 and SCRIBBLE-PDZ3. In agreement with these data, incremental amino-terminal deletions of the VANGL2 PBM causes its overall affinity to progressively decline. Moreover, the holdup data establish that the PDZome binding repertoire of VANGL2 starts to diverge significantly with the truncation of E518. A structural analysis of the SYNJ2BP-PDZ/VANGL2 interaction with truncated PBMs identifies a major conformational change in the binding direction of the PBM peptide after the P-2 position. Finally, we report that the PDZome binding profile of VANGL2 is dramatically rearranged upon phosphorylation of S517, T519 and S520. Our crystallographic approach illustrates how SYNJ2BP accommodates a S520-phosphorylated PBM peptide through the ideal positioning of two basic residues, K48 and R86. Altogether our data provides a comprehensive view of the VANGL2 PDZ network and how this network specifically responds to the post-translation modification of distinct PBM residues. These findings should prove useful in guiding future functional and molecular studies of the key PCP component VANGL2.

The loop-tail mouse model displays open and closed caudal neural tube defects.

Neural tube defects (NTDs) are the second most common cause of congenital malformations and are often studied in animal models. Loop-tail (Lp) mice carry a mutation in the Vangl2 gene, a member of the Wnt-planar cell polarity pathway. In Vangl2+/Lp embryos, the mutation induces a failure in the completion of caudal neural tube closure, but only a small percentage of embryos develop open spina bifida. Here, we show that the majority of Vangl2+/Lp embryos developed caudal closed NTDs and presented cellular aggregates that may facilitate the sealing of these defects. The cellular aggregates expressed neural crest cell markers and, using these as a readout, we describe a systematic method to assess the severity of the neural tube dorsal fusion failure. We observed that this defect worsened in combination with other NTD mutants, Daam1 and Grhl3. Besides, we found that in Vangl2+/Lp embryos, these NTDs were resistant to maternal folic acid and inositol supplementation. Loop-tail mice provide a useful model for research on the molecular interactions involved in the development of open and closed NTDs and for the design of prevention strategies for these diseases.

Inversin (NPHP2) and Vangl2 are required for normal zebrafish cloaca formation.

Nephronophthisis (NPH), an autosomal recessive ciliopathy, results from mutations in more than 20 different genes (NPHPs). These gene products form protein complexes that regulate trafficking within the cilium, a microtubular structure that plays a crucial role in developmental processes. Several NPHPs, including NPHP2/Inversin, have been linked to extraciliary functions. In addition to defining a specific segment of primary cilia (Inversin compartment), NPHP2 participates in planar cell polarity (PCP) signaling along with Dishevelled and Vangl family members. We used the mutant zebrafish line invssa36157, containing a stop codon at amino acid 314, to characterize tissue-specific functions of zebrafish Nphp2. The invssa36157 line exhibits mild ciliopathy phenotypes and increased glomerular and cloaca cyst formation. These mutants showed enhanced susceptibility to the simultaneous depletion of the nphp1/nphp2/nphp8 module, known to be involved in the cytoskeletal organization of epithelial cells. Notably, simultaneous depletion of zebrafish nphp1 and vangl2 led to a pronounced increase in cloaca malformations in the invssa36157 mutant embryos. Time-lapse imaging showed that the pronephric cells correctly migrated towards the ectodermal cells in these embryos, but failed to form the cloaca opening. Despite these abnormal developments, cellular fate does not seem to be affected in nphp1 and vangl2 MO-depleted invssa36157 mutants, as shown by in situ hybridizations for markers of pronephros and ectodermal cell development. However, significantly reduced apoptotic activity was observed in this double knockdown model, signifying the role of apoptosis in cloacal morphogenesis. Our findings underscore the critical interplay of nphp1, nphp2/Inversin, and vangl2 in orchestrating normal cloaca formation in zebrafish, shedding light on the complex molecular mechanisms underlying ciliopathy-associated phenotypes.

An unanticipated discourse of HB-EGF with VANGL2 signaling during embryo implantation.

Implantation is the first direct encounter between the embryo and uterus during pregnancy, and Hbegf is the earliest known molecular signaling for embryo-uterine crosstalk during implantation. The downstream effectors of heparin-binding EGF (HB-EGF) in implantation remain elusive due to the complexity of EGF receptor family. This study shows that the formation of implantation chamber (crypt) triggered by HB-EGF is disrupted by uterine deletion of Vangl2, a key planar cell polarity component (PCP). We found that HB-EGF binds to ERBB2 and ERBB3 to recruit VANGL2 for tyrosine phosphorylation. Using in vivo models, we show that uterine VAGL2 tyrosine phosphorylation is suppressed in Erbb2/Erbb3 double conditional knockout mice. In this context, severe implantation defects in these mice lend support to the critical role of HB-EGF-ERBB2/3-VANGL2 in establishing a two-way dialogue between the blastocyst and uterus. In addition, the result addresses an outstanding question how VANGL2 is activated during implantation. Taken together, these observations reveal that HB-EGF regulates the implantation process by influencing uterine epithelial cell polarity comprising VANGL2.

Emerging roles of planar cell polarity proteins in glutamatergic synapse formation, maintenance and function in health and disease.

The local signaling mechanism which directly assembles and maintains glutamatergic synapses has not been well understood. Glutamatergic synapses are made of presynaptic and postsynaptic compartments with distinct sets of proteins. The planar cell polarity (PCP) pathway is highly conserved and responsible for establishing and maintaining the cell and tissue polarity along the tissue plane. The six core PCP proteins form antagonizing complexes within the cells and asymmetric intercellular complexes across neighboring cells which regulate cell-cell interactions during planar polarity signaling. Accumulating evidence suggests that the PCP proteins play essential roles in glutamatergic synapse assembly, maintenance and function in the brain. This review summarizes the key evidence that PCP proteins may be responsible for the formation and stability of the vast majority of the glutamatergic synapses in hippocampus and medial prefrontal cortex, the progress in understanding the mechanisms of how PCP proteins assemble and maintain glutamatergic synapses and initial insights on how disruption of the function of the PCP proteins can lead to neurodegenerative, neurodevelopmental and neuropsychiatric disorders. The PCP proteins may be the missing pieces of a long-standing puzzle and filling this gap of knowledge may provide the basis for understanding many unsolved questions in synapse biology.

MicroRNA-27a-3p targeting Vangl1 and Vangl2 inhibits cell proliferation in mouse granulosa cells.

BACKGROUND: Mammalian folliculogenesis is the complex process through which primordial follicles develop into preovulatory follicles. The chief function of ovarian follicle granulosa cells is to play a vital role in the growth, development and atresia of ovarian follicles via gap junctions. Increasing evidence suggests that microRNAs (miRNAs) are essential regulators of granulosa cell apoptosis or proliferation. METHODS: The expression level of miR-27a-3p, myogenic differentiation (MyoD), Vangl1 and Vangl2 was investigated by Real-time quantitative PCR (RT-qPCR) and Western blot. Luciferase reporter assay, bioinformatics analysis and ChIP-PCR was used to detect the binding sites between miR-27a-3p, transcription factor and target genes. KEGG pathway analyses were performed to reveal the predicted targets of miR-27a-3p. Ethynyl deoxyuridine (EdU) proliferation assay was used to measure cell proliferation. RESULTS: To explore the underlying mechanisms of the miR-27a-3p function in the development of mouse granulosa cells (mGCs), we screened for the target genes of miR-27a-3p, confirmed its interaction with Vangl1 and Vangl2 and elucidated their roles in mGCs. MiR-27a-3p inhibited the proliferation of mGCs, whereas target genes Vangl1 and Vangl2 had the opposite effect. In addition, the transcription factor MYOD bound to and activated the promoter of miR-27a-3p. MiR-27a-3p suppressed Vangl1 and Vangl2 expression by targeting their 3'-untranslated region (3'-UTR). Furthermore, Vangl1 and Vangl2 suppressed the Wnt pathway by reducing the expression of ß-catenin and B-cell lymphoma/leukemia-2 (Bcl-2). CONCLUSION: These findings indicate a pro-survival mechanism of the MyoD/miR-27a-3p/Vangl1/Vangl2 axis for granulosa cell proliferation and suggest a novel target for the improvement of female fertility.

Analysis of the Ubiquitination and Phosphorylation of Vangl Proteins.

The core planar cell polarity (PCP) protein Vang/Vangl, including Vangl1 and Vangl2 in vertebrates, is indispensable during development. Our previous studies showed that the activity of Vangl is tightly controlled by two important posttranslational modifications, ubiquitination and phosphorylation. Vangl is ubiquitinated through an endoplasmic reticulum-associated degradation (ERAD) pathway and is phosphorylated by casein kinase 1 (CK1) in response to Wnt. Here, we present step-by-step procedures to analyze Vangl ubiquitination and phosphorylation, including cell culture, transfection, sample preparation, and signal detection, as well as the use of newly available phospho-specific antibodies to detect Wnt-induced Vangl2 phosphorylation. The protocol described here can be applicable to the analysis of posttranslational modifications of other membrane proteins.

Wnt/planar cell polarity signaling controls morphogenetic movements of gastrulation and neural tube closure.

Gastrulation and neurulation are successive morphogenetic processes that play key roles in shaping the basic embryonic body plan. Importantly, they operate through common cellular and molecular mechanisms to set up the three spatially organized germ layers and to close the neural tube. During gastrulation and neurulation, convergent extension movements driven by cell intercalation and oriented cell division generate major forces to narrow the germ layers along the mediolateral axis and elongate the embryo in the anteroposterior direction. Apical constriction also makes an important contribution to promote the formation of the blastopore and the bending of the neural plate. Planar cell polarity proteins are major regulators of asymmetric cell behaviors and critically involved in a wide variety of developmental processes, from gastrulation and neurulation to organogenesis. Mutations of planar cell polarity genes can lead to general defects in the morphogenesis of different organs and the co-existence of distinct congenital diseases, such as spina bifida, hearing deficits, kidney diseases, and limb elongation defects. This review outlines our current understanding of non-canonical Wnt signaling, commonly known as Wnt/planar cell polarity signaling, in regulating morphogenetic movements of gastrulation and neural tube closure during development and disease. It also attempts to identify unanswered questions that deserve further investigations.

Dihydroartemisinin Attenuates Hypoxic Pulmonary Hypertension via the Downregulation of miR-335 Targeting Vangl2.

Dihydroartemisinin (DHA) is a traditional antimalarial drug. DHA plays a crucial role in preventing pulmonary hypertension (PH); however, its regulatory function on microRNAs (miRNAs) in PH remains unclear. This study aimed to investigate whether DHA exerts its protective functions by regulating miR-335 in PH. Hypoxia-induced PH models were induced both in vitro and in vivo. Mice were treated with various concentrations of DHA, and pulmonary arterial smooth muscle cells (PASMCs) were treated with DHA, miR-335 inhibitor, miR-335 mimic, or Van Gogh-like 2 (Vangl2) plasmid. The expression of miR-335 and Vangl2, pulmonary arterial remodeling index; right ventricular hypertrophy index; and proliferation and migration indexes were measured. DHA improved pulmonary vascular remodeling and alleviated PH in vivo. miRNA sequencing and real-time PCR results further show that the increase in hypoxia-induced miR-335 was avoided by DHA administration, and miR-335 increased the hypoxia-induced PASMC proliferation and migration. MiRNA databases and dual-luciferase reporter assay show that miR-335 directly targets Vangl2, and Vangl2 decreased the hypoxia-induced PASMC proliferation and migration. The miR-335 inhibitor failed to inhibit hypoxia-induced proliferation and migration upregulation in Vangl2 knockdown PASMCs, and the effect of DHA can be blocked by miR-335 upregulation. In hypoxic PH, MiR-335 is increased, whereas Vangl2 is decreased. MiR-335 can significantly promote the hypoxia-induced proliferation and migration of PASMCs by targeting the Vangl2 gene. DHA effectively reverses the hypoxia-induced upregulation of miR-335 expression, avoiding the miR-335-mediated downregulation of Vangl2 and thereby promoting the expression of Vangl2 to prevent PH.

Vangl2 Promotes Hematopoietic Stem Cell Expansion.

Regulation of hematopoietic stem cell (HSC) self-renewal and differentiation is essential for their maintenance, and HSC polarity has been shown to play an important role in this regulation. Vangl2, a key component of the Wnt/polarity pathway, is expressed by fetal and adult HSCs, but its role in hematopoiesis and HSC function is unknown. Here we show the deletion of Vangl2 in mouse hematopoietic cells impairs HSC expansion and hematopoietic recovery post-transplant. Old Vangl2-deficient mice showed increased expansion of myeloid-biased multipotent progenitor cells concomitant with splenomegaly. Moreover, Vangl2-deficient cells were not able to effectively reconstitute the recipient bone marrow in serial transplants, or when coming from slightly older donors, demonstrating impaired self-renewal or expansion. Aged Vangl2-deficient HSCs displayed increased levels of cell cycle inhibitor p16INK4a and active ß-catenin, which could contribute to their impaired function. Overall, our findings identify Vangl2 as a new regulator of hematopoiesis.